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+MIT License
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+[{"sourceid":"4772114","sourcedb":"","project":"","target":"","text":"Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses. Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). The PIN domain harbors the RNase catalytic center; however, it is insufficient for enzymatic activity. We found that the NTD associates with the PIN domain and significantly enhances its RNase activity. The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site. Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro. These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions. The initial sensing of infection is mediated by a set of pattern-recognition receptors (PRRs) such Toll-like receptors (TLRs) and the intracellular signaling cascades triggered by TLRs evoke transcriptional expression of inflammatory mediators that coordinate the elimination of pathogens and infected cells. Since aberrant activation of this system leads to auto immune disorders, it must be tightly regulated. Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase whose expression level is stimulated by lipopolysaccharides and prevents autoimmune diseases by directly controlling the stability of mRNAs of inflammatory genes such as interleukin (IL)-6, IL-1β, IL-2, and IL-12p40. Regnase-1 accelerates target mRNA degradation via their 3′-terminal untranslated region (3′UTR), and also degrades its own mRNA. Regnase-1 is a member of Regnase family and is composed of a PilT N-terminus like (PIN) domain followed by a CCCH-type zinc–finger (ZF) domain, which are conserved among Regnase family members. Recently, the crystal structure of the Regnase-1 PIN domain derived from Homo sapiens was reported. The structure combined with functional analyses revealed that four catalytically important Asp residues form the catalytic center and stabilize Mg2+ binding that is crucial for RNase activity. Several CCCH-type ZF motifs in RNA-binding proteins have been reported to directly bind RNA. In addition, Regnase-1 has been predicted to possess other domains in the N- and C- terminal regions. However, the structure and function of the ZF domain, N-terminal domain (NTD) and C-terminal domain (CTD) of Regnase-1 have not been solved. Here, we performed structural and functional analyses of individual domains of Regnase-1 derived from Mus musculus in order to understand the catalytic activity in vitro. Our data revealed that the catalytic activity of Regnase-1 is regulated through both intra and intermolecular domain interactions in vitro. The NTD plays a crucial role in efficient cleavage of target mRNA, through intramolecular NTD-PIN interactions. Moreover, Regnase-1 functions as a dimer through intermolecular PIN-PIN interactions during cleavage of target mRNA. Our findings suggest that Regnase-1 cleaves its target mRNA by an NTD-activated functional PIN dimer, while the ZF increases RNA affinity in the vicinity of the PIN dimer. Results Domain structures of Regnase-1 We analyzed Rengase-1 derived from Mus musculus and solved the structures of the four domains; NTD, PIN, ZF, and CTD individually by X-ray crystallography or NMR (Fig. 1a–e). X-ray crystallography was attempted for the fragment containing both the PIN and ZF domains, however, electron density was observed only for the PIN domain (Fig. 1c), consistent with a previous report on Regnase-1 derived from Homo sapiens. This suggests that the PIN and ZF domains exist independently without interacting with each other. The domain structures of NTD, ZF, and CTD were determined by NMR (Fig. 1b,d,e). The NTD and CTD are both composed of three α helices, and structurally resemble ubiquitin conjugating enzyme E2 K (PDB ID: 3K9O) and ubiquitin associated protein 1 (PDB ID: 4AE4), respectively, according to the Dali server. Contribution of each domain of Regnase-1 to the mRNA binding activity Although the PIN domain is responsible for the catalytic activity of Regnase-1, the roles of the other domains are largely unknown. First, we evaluated a role of the NTD and ZF domains for mRNA binding by an in vitro gel shift assay (Fig. 1f). Fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1—hereafter referred to as the DDNN mutant—were utilized. Upon addition of a larger amount of Regnase-1, the fluorescence of free RNA decreased, indicating that Regnase-1 bound to the RNA. Based on the decrease in the free RNA fluorescence band, we evaluated the contribution of each domain of Regnase-1 to RNA binding. While the RNA binding ability was not significantly changed in the presence of NTD, it increased in the presence of the ZF domain (Fig. 1f,g and Supplementary Fig. 1). Direct binding of the ZF domain and RNA were confirmed by NMR spectral changes. The fitting of the titration curve of Y314 resulted in an apparent dissociation constant (Kd) of 10 ± 1.1 μM (Supplementary Fig. 2). These results indicate that not only the PIN but also the ZF domain contribute to RNA binding, while the NTD is not likely to be involved in direct interaction with RNA. Contribution of each domain of Regnase-1 to RNase activity In order to characterize the role of each domain in the RNase activity of Regnase-1, we performed an in vitro cleavage assay using fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR (Fig. 1g). Regnase-1 constructs consisting of NTD-PIN-ZF completely cleaved the target mRNA and generated the cleaved products. The apparent half-life (T1/2) of the RNase activity was about 20 minutes. Regnase-1 lacking the ZF domain generated a smaller but appreciable amount of cleaved product (T1/2 ~ 70 minutes), while those lacking the NTD did not generate cleaved products (T1/2 \u003e 90 minutes). It should be noted that NTD-PIN(DDNN)-ZF, which possesses the NTD but lacks the catalytic residues in PIN, completely lost all RNase activity (Fig. 1g, right panel), as expected, confirming that the RNase catalytic center is located in the PIN domain. Taken together with the results in the previous section, we conclude that the NTD is crucial for the RNase activity of Regnase-1 in vitro, although it does not contribute to the direct mRNA binding. Dimer formation of the PIN domains During purification by gel filtration, the PIN domain exhibited extremely asymmetric elution peaks in a concentration dependent manner (Fig. 2a). By comparison with the elution volume of standard marker proteins, the PIN domain was assumed to be in equilibrium between a monomer and a dimer in solution at concentrations in the 20–200 μM range. The crystal structure of the PIN domain has been determined in three distinct crystal forms with a space group of P3121 (form I in this study and PDB ID 3V33), P3221 (form II in this study), and P41 (PDB ID 3V32 and 3V34), respectively. We found that the PIN domain formed a head-to-tail oligomer that was commonly observed in all three crystal forms in spite of the different crystallization conditions (Supplementary Fig. 3). Mutation of Arg215, whose side chain faces to the opposite side of the oligomeric surface, to Glu preserved the monomer/dimer equilibrium, similar to the wild type. On the other hand, single mutations of side chains involved in the PIN–PIN oligomeric interaction resulted in monomer formation, judging from gel filtration (Fig. 2a,b). Wild type and monomeric PIN mutants (P212A and D278R) were also analyzed by NMR. The spectra indicate that the dimer interface of the wild type PIN domain were significantly broadened compared to the monomeric mutants (Supplementary Fig. 4). These results indicate that the PIN domain forms a head-to-tail oligomer in solution similar to the crystal structure. Interestingly, the monomeric PIN mutants P212A, R214A, and D278R had no significant RNase activity for IL-6 mRNA in vitro (Fig. 2c). The side chains of these residues point away from the catalytic center on the same molecule (Fig. 2b). Therefore, we concluded that head-to-tail PIN dimerization, together with the NTD, are required for Regnase-1 RNase activity in vitro. Domain-domain interaction between the NTD and the PIN domain While the NTD does not contribute to RNA binding (Fig. 1f,g, and Supplementary Fig. 1), it increases the RNase activity of Regnase-1 (Fig. 1h). In order to gain insight into the molecular mechanism of the NTD-mediated enhancement of Regnase-1 RNase activity, we further investigated the domain-domain interaction between the NTD and the PIN domain using NMR. We used the catalytically inactive monomeric PIN mutant possessing both the DDNN and D278R mutations to avoid dimer formation of the PIN domain. The NMR signals from the PIN domain (residues V177, F210-T211, R214, F228-L232, and F234-S236) exhibited significant chemical shift changes upon addition of the NTD (Fig. 3a). Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). These results clearly indicate a direct interaction between the PIN domain and the NTD. Based on the titration curve for the chemical shift changes of L58, the apparent Kd between the isolated NTD and PIN was estimated to be 110 ± 5.8 μM. Considering the fact that the NTD and PIN domains are attached by a linker, the actual binding affinity is expected much higher in the native protein. Mapping the residues with chemical shift changes reveals the putative PIN/NTD interface, which includes a helix that harbors catalytic residues D225 and D226 on the PIN domain (Fig. 3a). Interestingly, the putative binding site for the NTD overlaps with the PIN-PIN dimer interface, implying that NTD binding can “terminate” PIN-PIN oligomerization (Fig. 2b). An in silico docking of the NTD and PIN domains using chemical shift restraints provided a model consistent with the NMR experiments (Fig. 3c). Residues critical for Regnase-1 RNase activity To gain insight into the residues critical for Regnase-1 RNase activity, each basic or aromatic residue located around the catalytic site of the PIN oligomer was mutated to alanine, and the oligomerization and RNase activity were investigated (Fig. 4). From the gel filtration assays, all mutants except R214A formed dimers, suggesting that any lack of RNase activity in the mutants, except R214A, was directly due to mutational effects of the specific residues and not to abrogation of dimer formation. The W182A, R183A, and R214A mutants markedly lost cleavage activity for IL-6 mRNA as well as for Regnase-1 mRNA. The K184A, R215A, and R220A mutants moderately but significantly decreased the cleavage activity for both target mRNAs. The importance of K219 and R247 was slightly different for IL-6 and Regnase-1 mRNA; both K219 and R247 were more important in the cleavage of IL-6 mRNA than for Regnase-1 mRNA. The other mutated residues—K152, R158, R188, R200, K204, K206, K257, and R258—were not critical for RNase activity. The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the “secondary” chain’s residue K184 is positioned near the “primary” chain’s catalytic site (Fig. 4). In contrast, R214 is important for oligomerization of the PIN domain and the “secondary” chain’s residue R214 is also positioned near the “primary” chain’s active site within the dimer interface. It should be noted that the putative-RNA binding residues K184 and R214 are unique to Regnase-1 among PIN domains. Molecular mechanism of target mRNA cleavage by the PIN dimer Our mutational experiments indicated that the observed dimer is functional and that the role of the secondary PIN domain is to position Regnase-1-unique RNA binding residues near the active site of the primary PIN domain. If this model is correct, then we reasoned that a catalytically inactive PIN and a PIN lacking the putative RNA-binding residues ought to be inactive in isolation but become active when mixed together. In order to test this hypothesis, we performed in vitro cleavage assays using combinations of Regnase-1 mutants that had no or decreased RNase activities by themselves (Fig. 5). One group consisted of catalytically active PIN domains with mutation of basic residues found in the previous section to confer decreased RNase activity (Fig. 4). These were paired with a DDNN mutant that had no RNase activity by itself. When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). When we compared the fluorescence intensity of uncleaved IL-6 mRNA, basic residue mutants W182A, K184A, R214A, and R220A were rescued upon addition of the DDNN mutant (Fig. 5b). Consistently, when we compared the fluorescence intensity of the uncleaved Regnase-1 mRNA, basic residue mutants K184A and R214A were rescued upon addition of the DDNN mutant (Fig. 5c). Rescue of K184A and R214A by the DDNN mutant was also confirmed by a significant increase in the cleaved products. This is particularly significant because the side chains of K184 and R214 in the primary PIN are oriented away from their own catalytic center, while those in the secondary PIN face toward the catalytic center of the primary PIN. R214 is an important residue for dimer formation as shown in Fig. 2, therefore, R214A in the secondary PIN cannot dimerize. According to the proposed model, an R214A PIN domain can only form a dimer when the DDNN PIN acts as the secondary PIN. Taken together, the rescue experiments above support the proposed model in which the head-to-tail dimer is functional in vitro. Discussion We determined the individual domain structures of Regnase-1 by NMR and X-ray crystallography. Although the function of the CTD remains elusive, we revealed the functions of the NTD, PIN, and ZF domains. A Regnase-1 construct consisting of PIN and ZF domains derived from Mus musculus was crystallized; however, the electron density of the ZF domain was low, indicating that the ZF domain is highly mobile in the absence of target mRNA or possibly other protein-protein interactions. Our NMR experiments confirmed direct binding of the ZF domain to IL-6 mRNA with a Kd of 10 ± 1.1 μM. Furthermore, an in vitro gel shift assay indicated that Regnase-1 containing the ZF domain enhanced target mRNA-binding, but the protein-RNA complex remained in the bottom of the well without entering into the polyacrylamide gel. These results indicate that Regnase-1 directly binds to RNA and precipitates under such experimental conditions. Due to this limitation, it is difficult to perform further structural analyses of mRNA-Regnase-1 complexes by X-ray crystallography or NMR. The previously reported crystal structure of the Regnase-1 PIN domain derived from Homo sapiens is nearly identical to the one derived from Mus musculus in this study, with a backbone RMSD of 0.2 Å. The amino acid sequences corresponding to PIN (residues 134–295) are the two non-identical residues are substituted with similar amino acids. Both the mouse and human PIN domains form head-to-tail oligomers in three distinct crystal forms. Rao and co-workers previously argued that PIN dimerization is likely to be a crystallographic artifact with no physiological significance, since monomers were dominant in their analytical ultra-centrifugation experiments. In contrast, our gel filtration data, mutational analyses, and NMR spectra all indicate that the PIN domain forms a head-to-tail dimer in solution in a manner similar to the crystal structure. This inconsistency might be due to difference in the analytical methods and/or protein concentrations used in each experiment, since the oligomer formation of PIN was dependent on the protein concentration in our study. Single mutations to residues involved in the putative oligomeric interaction of PIN monomerized as expected and these mutants lost their RNase activity as well. Since the NMR spectra of monomeric mutants overlaps with those of the oligomeric forms, it is unlikely that the tertiary structure of the monomeric mutants were affected by the mutations. (Supplementary Fig. 4b,c). Based on these observations, we concluded that PIN-PIN dimer formation is critical for Regnase-1 RNase activity in vitro. Within the crystal structure of the PIN dimer, the Regnase-1 specific basic regions in both the “primary” and “secondary” PINs are located around the catalytic site of the primary PIN (Supplementary Fig. 6). Moreover, our structure-based mutational analyses showed these two Regnase-1 specific basic regions were essential for target mRNA cleavage in vitro. The cleavage assay also showed that the NTD is crucial for efficient mRNA cleavage. Moreover, we found that the NTD associates with the oligomeric surface of the primary PIN, docking to a helix that harbors its catalytic residues (Figs 2b and 3a). Taken together, this suggests that the NTD and the PIN domain compete for a common binding site. The affinity of the domain-domain interaction between two PIN domains (Kd = ~10−4 M) is similar to that of the NTD-PIN (Kd = 110 ± 5.8 μM) interactions; however, the covalent connection corresponding to residues 90–133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1. While further analyses are necessary to prove this point, our preliminary docking and molecular dynamics simulations indicate that NTD-binding rearranges the catalytic residues of the PIN domain toward an active conformation suitable for binding Mg2+. In this context, it is interesting that, in response to TCR stimulation, Malt1 cleaves Regnase-1 at R111 to control immune responses in vivo. This result is consistent with a model in which the NTD acts as an enhancer, and cleavage of the linker lowers enzymatic activity dramatically. Based on these structural and functional analyses of Regnase-1 domain-domain interactions, we performed docking simulations of the NTD, PIN dimer, and IL-6 mRNA. We incorporated information from the cleavage site of IL-6 mRNA in vitro is indicated by denaturing polyacrylamide gel electrophoresis (Supplementary Fig. 7a,b). The docking result revealed multiple RNA binding modes that satisfied the experimental results in vitro (Supplementary Fig. 7c,d), however, it should be noted that, in vivo, there would likely be many other RNA-binding proteins that would protect loop regions from cleavage by Regnase-1. The overall model of regulation of Regnase-1 RNase activity through domain-domain interactions in vitro is summarized in Fig. 6. In the absence of target mRNA, the PIN domain forms head-to-tail oligomers at high concentration. A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). While further investigations on the domain-domain interactions of Regnase-1 in vivo are necessary, these intramolecular and intermolecular domain interactions of Regnase-1 appear to structurally constrain Regnase-1activity, which, in turn, enables tight regulation of immune responses. Methods Protein expression and purification The DNA fragment encoding Regnase-1 derived from Mus musculus was cloned into pGEX6p vector (GE Healthcare). All the mutants were generated by PCR-mediated site-directed mutagenesis and confirmed by the DNA sequence analyses. As a catalytically deficient mutant, both Asp226 and Asp244 at the catalytic center of PIN were mutated to Asn, which is referred to as DDNN mutant. Regnase-1 was expressed at 16 °C using the Escherichia coli RosettaTM(DE3)pLysS strain. After purification with a GST-affinity resin, an N-terminal GST tag was digested by HRV-3 C protease. NTD was further purified by gel filtration chromatography using a HiLoad 16/60 Superdex 75 pg (GE Healthcare). The other domains were further purified by cation exchange chromatography using Resource S (GE Healthcare), followed by gel filtration chromatography using a HiLoad 16/60 Superdex 75 pg (GE Healthcare). Uniformly 15N or 13C, 15N-double labeled proteins for NMR experiments were prepared by growing E. coli host in M9 minimal medium containing 15NH4Cl, unlabeled glucose and 15N CELTONE® Base Powder (CIL) or 15NH4Cl, 13C6-glucose, and13C, 15N CELTONE® Base Powder (CIL), respectively. X-ray crystallography Crystallization was performed using the sitting drop vapor diffusion method at 20 °C and two crystal forms (I and II) were obtained. In the case of form I crystals, drops (0.5 μl) of 6 mg/ml selenomethionine-labeled Regnase-1 PIN-ZF (residues 134–339 derived from Mus musculus) in 20 mM HEPES-NaOH (pH 6.8), 200 mM NaCl and 5 mM DTT were mixed with reservoir solution consisting of 1 M (NH4)2HPO4, 200 mM NaCl and 100 mM sodium citrate (pH 5.5) whereas in the case of form II crystals, drops (0.5 μl) of 6 mg/ml native Regnase-1 PIN-ZF (residues 134–339) in 20 mM HEPES-NaOH (pH 6.8), 200 mM NaCl and 5 mM DTT were mixed with reservoir solution consisting of 1.7 M NaCl and 100 mM HEPES-NaOH (pH 7.0). Diffraction data were collected at a Photon Factory Advanced Ring beamline NE3A (form I) or at a SPring-8 beamline BL41XU (form II), and were processed with HKL2000. The structure of the form I crystal was determined by the multiple anomalous dispersion (MAD) method. Nine Se sites were found using the program SOLVE; however, the electron density obtained by MAD phases calculated using SOLVE was not good enough to build a model even after density modification using the program RESOLVE. Then the program CNS was used to find additional three Se sites and calculate MAD phases using 12 Se sites. The electron density after density modification using CNS was good enough to build a model. Structure of the form II crystal was determined by the molecular replacement method using CNS and using the structure of the form I crystal as a search model. For all structures, further model building was performed manually with COOT, and TLS and restrained refinement using isotropic individual B factors was performed with REFMAC5 in the CCP4 program suite. Crystallographic parameters are summarized in Supplementary Table 1. NMR measurements All NMR experiments were carried out at 298 K on Inova 500-MHz, 600-MHz, and 800-MHz spectrometer (Agilent). The NMR data were processed using the NMRPipe, the Olivia (fermi.pharm.hokudai.ac.jp/olivia/), and the Sparky program (Sparky3, University of California, San Francisco). For structure calculation, NOE distance restraints were obtained from 3D 15N-NOESY-HSQC (100 ms mixing time for the NTD, 75 ms mixing time for the ZF domain and the CTD) and 13C-NOESY-HSQC spectra (100 ms mixing time for the NTD, 75 ms mixing time for the ZF domain and the CTD). The NMR structures were determined using the CANDID/CYANA2.1. Dihedral restraints were derived from backbone chemical shifts using TALOS. For the domain-domain interaction analyses between the NTD and the PIN domain, 1H-15N HSQC spectra of uniformly 15N-labeled proteins in the concentration of 100 μM were obtained in the presence of 3 or 6 molar equivalents of unlabeled proteins. Preparation of RNAs The fluorescently labeled RNAs at the 5′-end by 6-FAM were purchased from SIGMA-ALDORICH. The RNA sequences used in this study were shown below. IL-6 mRNA 3′UTR (82–106): 5′-UGUUGUUCUCUACGAAGAACUGACA-3′ (25 nts) Regnase-1 mRNA 3′UTR (191–211): 5′- CUGUUGAUACACAUUGUAUCU-3′ (21 nts) Electrophoretic mobility shift assay Catalytically deficient Regnase-1 proteins, containing DDNN mutations, and 5′-terminally 6-FAM labeled RNAs were incubated in the RNA-binding buffer (20 mM HEPES-NaOH (pH 6.8), 150 mM NaCl, 1 mM DTT, 10% glycerol (v/v), and 0.1% NP-40 (v/v)) at 4 °C for 30 minutes, then analyzed by non-denaturing polyacrylamide gel electrophoresis. The electrophoreses were performed at 4 °C using the 7.5% polyacrylamide (w/v) gel (monomer : bis = 29 : 1) in the electrophoresis buffer (25 mM Tris-HCl (pH 7.5) and 200 mM glycine). The fluorescence of 6-FAM labeled RNA was directly detected at the excitation wavelength of 460 nm with a fluorescence filter (Y515-Di) using a fluoroimaging analyzer (LAS-4000 (FUJIFILM)). The fluorescence intensity of each sample was quantified using ImageJ software. In vitro RNA cleavage assay Regnase-1 (2 μM) and 5′-terminally 6-FAM labeled RNA (1 μM) were incubated in the RNA-cleavage buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, and 1 mM DTT) at 37 °C. For the assay using combinations of Regnase-1 mutants, equimolar amounts of Regnase-1 mutants (2 μM each) were mixed with fluorescently labeled RNA (1 μM). After incubation for 30–120 minutes, the reaction was stopped by the addition of 1.5-fold volume of denaturing buffer containing 8 M urea and 100 mM EDTA, and samples were boiled. The electrophoreses were performed at room temperature using the 8 M urea containing denaturing gel with 20% polyacrylamide (w/v) (monomer : bis = 19 : 1) in 0.5 × TBE as the electrophoresis buffer. Docking calculations For docking NTD to PIN, OSCAR-star was first used to rebuild sidechains in the head-to-tail PIN dimer. Docking was carried out by surFit (http://sysimm.ifrec.osaka-u.ac.jp/docking/main/) with restraints obtained from NMR data (Fig. 3a,b) as follows. NTD: R56, L58, G59, V86, K87, H88; PIN: V177, F210, T211, R214, F228, I229, V230, K231, L232, F234, D235, S236. Top-scoring model was selected. For docking IL-6 mRNA 3′UTR to the PIN dimer, each domain of the PIN dimer structure was superimposed onto the PIN dimer of the human X-ray structure (PDB ID: 3V34) in order to graft both water molecules and Mg2+ ions to the mouse model. Each IL-6 representative structure was submitted to the HADDOCK 2.0 server, for total of 10 independent jobs. In order to be consistent with the cleavage assay, active residues consisted of all nucleotides in RNA, Mg2+ and W182, R183, K184, R188, R214, R215, K219, R220, and R247 in the protein. Docked models were selected based on the following criteria: one heavy atom within 7, 8, or 9th nucleotide from the 5′ end was \u003c5 Å from the Mg2+ ion on the primary PIN. Further classification was done manually in order to divide the selected models into two clusters. Additional Information Accession codes: The crystal structure of the Regnase-1 PIN domain has been deposited in the Protein Data Bank (accession codes: 5H9V (Form I) and 5H9W (Form II)). The chemical shift assignments of the NTD, the ZF domain, and the CTD have been deposited at Biological Magnetic Resonance Bank (accession codes: 25718, 25719, and 25720, respectively), and the coordinates for the ensemble have been deposited in the Protein Data Bank (accession codes: 2N5J, 2N5K, and 2N5L, respectively).  How to cite this article: Yokogawa, M. et al. Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions. Sci. Rep. 6, 22324; doi: 10.1038/srep22324 (2016). Supplementary Material Pathogen recognition and innate immunity. Recognition of microorganisms and activation of the immune response Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large Zc3h12a is an RNase essential for controlling immune responses by regulating mRNA decay Interleukin-1-inducible MCPIP protein has structural and functional properties of RNase and participates in degradation of IL-1beta mRNA MCPIP1 down-regulates IL-2 expression through an ARE-independent pathway mRNA degradation by the endoribonuclease Regnase-1/ZC3H12a/MCPIP-1 The IκB kinase complex regulates the stability of cytokine-encoding mRNA induced by TLR-IL-1R by controlling degradation of regnase-1 A novel CCCH-zinc finger protein family regulates proinflammatory activation of macrophages MCP-1-induced protein-1, an immune regulator Structural study of MCPIP1 N-terminal conserved domain reveals a PIN-like RNase Specificity of RNA binding by CPEB: requirement for RNA recognition motifs and a novel zinc finger Interactions of CCCH zinc finger proteins with mRNA: non-binding tristetraprolin mutants exert an inhibitory effect on degradation of AU-rich element-containing mRNAs Recognition of the mRNA AU-rich element by the zinc finger domain of TIS11d Multiple modes of RNA recognition by zinc finger proteins Monocyte chemoattractant protein-1 induces a novel transcription factor that causes cardiac myocyte apoptosis and ventricular dysfunction MCP-induced protein 1 deubiquitinates TRAF proteins and negatively regulates JNK and NF-kappaB signaling Dali server: conservation mapping in 3D Malt1-induced cleavage of regnase-1 in CD4(+) helper T cells regulates immune activation Processing of X-ray Diffraction Data Collected in Oscillation Mode Automated MAD and MIR structure solution Maximum-likelihood density modification Crystallography \u0026 NMR system: A new software suite for macromolecular structure determination Features and development of Coot Refinement of macromolecular structures by the maximum-likelihood method Overview of the CCP4 suite and current developments NMRPipe: a multidimensional spectral processing system based on UNIX pipes Automated NMR structure calculation with CYANA Protein backbone angle restraints from searching a database for chemical shift and sequence homology Fast and accurate prediction of protein side-chain conformations Author Contributions F.I. supervised the overall project. M.Y., T.T., Y.E., D.M.S., O.T., S.A. and F.I. designed the research; M.Y. and T.T. performed the research; M.Y., T.T., N.N.N., H.K., K.Y., D.M.S. and F.I. analyzed the data; and M.Y., N.N.N., H.K., K.Y., D.M.S. and F.I. wrote the paper. All authors reviewed the manuscript. Structural and functional analyses of Regnase-1. (a) Domain architecture of Regnase-1. (b) Solution structure of the NTD. (c) Crystal structure of the PIN domain. Catalytic Asp residues were shown in sticks. (d) Solution structure of the ZF domain. Three Cys residues and one His residue responsible for Zn2+-binding were shown in sticks. (e) Solution structure of the CTD. All the structures were colored in rainbow from N-terminus (blue) to C-terminus (red). (f) In vitro gel shift binding assay between Regnase-1 and IL-6 mRNA. Fluorescence intensity of the free IL-6 in each sample was indicated as the percentage against that in the absence of Regnase-1. (g) Binding of Regnase-1 and IL-6 mRNA was plotted. The percentage of the bound IL-6 was calculated based on the fluorescence intensities of the free IL-6 quantified in (f). (h) In vitro cleavage assay of Regnase-1 to IL-6 mRNA. Fluorescence intensity of the uncleaved IL-6 mRNA was indicated as the percentage against that in the absence of Regnase-1. Head-to-tail oligomer formation of the PIN domain is crucial for the RNase activity of Regnase-1. (a) Gel filtration analyses of the PIN domain. Elution volumes of the standard marker proteins were indicated by arrows at the upper part. (b) Dimer structure of the PIN domain. Two PIN molecules in the crystal were colored white and green, respectively. Catalytic residues and mutated residues were shown in sticks. Residues important for the oligomeric interaction were colored red, while R215 that was dispensable for the oligomeric interaction was colored blue. (c) RNase activity of monomeric mutants for IL-6 mRNA was analyzed. Domain-domain interaction between the NTD and the PIN domain. (a) NMR analyses of the NTD-binding to the PIN domain. The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red on the surface and ribbon structure of the PIN domain (right). Pro and the residues without analysis were colored black and gray, respectively. (b) NMR analyses of the PIN-binding to the NTD. The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red, yellow, or green on the surface and ribbon structure of the NTD. S62 was colored gray and excluded from the analysis, due to low signal intensity. (c) Docking model of the NTD and the PIN domain. The NTD and the PIN domain are shown in cyan and white, respectively. Residues in close proximity (\u003c5 Å) to each other in the docking structure were colored yellow. Catalytic residues of the PIN domain are shown in sticks, and the residues that exhibited significant chemical shift changes in (a,b) were labeled. Critical residues in the PIN domain for the RNase activity of Regnase-1. (a) In vitro cleavage assay of basic residue mutants for IL-6 mRNA. The results indicate mean ± SD of four independent experiments. (b)\nIn vitro cleavage assay of basic residue mutants for Regnase-1 mRNA. The results indicate mean ± SD of three independent experiments. The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer structure. Mutated basic residues were shown in sticks and those with significantly reduced RNase activities were colored red or yellow. Heterodimer formation by combination of the Regnase-1 basic residue mutants and the DDNN mutant restored the RNase activity. (a) Cartoon representation of the concept of the experiment. (b) In vitro cleavage assay of Regnase-1 for IL-6 mRNA. (c) In vitro cleavage assay of Regnase-1 for Regnase-1 mRNA. The results indicate mean ± SD of three independent experiments. The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer. The mutations whose RNase activities were not increased in the presence of DDNN mutant were colored in blue on the primary PIN. The mutations whose RNase activities were restored in the presence of DDNN mutant were colored in red or yellow on the primary PIN. Schematic representation of regulation of the Regnase-1 catalytic activity through the domain-domain 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diff --git a/annotated_BioC_JSON/PMC4784909_ann.json b/annotated_BioC_JSON/PMC4784909_ann.json
new file mode 100644
index 0000000000000000000000000000000000000000..5e0940e79030a4c662f709ba66cff075a55f63c0
--- /dev/null
+++ b/annotated_BioC_JSON/PMC4784909_ann.json
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+[{"sourceid":"4784909","sourcedb":"","project":"","target":"","text":"The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). Here, we report two high-resolution PduL crystal structures with bound substrates. The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen. This study describes the structure of a novel phosphotransacylase enzyme that facilitates the recycling of the essential cofactor acetyl-CoA within a bacterial organelle and discusses the properties of the enzyme's active site and how it is packaged into the organelle. Author Summary In metabolism, molecules with “high-energy” bonds (e.g., ATP and Acetyl~CoA) are critical for both catabolic and anabolic processes. Accordingly, the retention of these bonds during biochemical transformations is incredibly important. The phosphotransacylase (Pta) enzyme catalyzes the conversion between acyl-CoA and acyl-phosphate. This reaction directly links an acyl-CoA with ATP generation via substrate-level phosphorylation, producing short-chain fatty acids (e.g., acetate), and also provides a path for short-chain fatty acids to enter central metabolism. Due to this key function, Pta is conserved across the bacterial kingdom. Recently, a new type of phosphotransacylase was described that shares no evolutionary relation to Pta. This enzyme, PduL, is exclusively associated with organelles called bacterial microcompartments, which are used to catabolize various compounds. Not only does PduL facilitate substrate level phosphorylation, but it also is critical for cofactor recycling within, and product efflux from, the organelle. We solved the structure of this convergent phosphotransacylase and show that it is completely structurally different from Pta, including its active site architecture. We also discuss features of the protein important to its packaging in the organelle. Introduction Bacterial Microcompartments (BMCs) are organelles that encapsulate enzymes for sequential biochemical reactions within a protein shell. The shell is typically composed of three types of protein subunits, which form either hexagonal (BMC-H and BMC-T) or pentagonal (BMC-P) tiles that assemble into a polyhedral shell. The facets of the shell are composed primarily of hexamers that are typically perforated by pores lined with highly conserved, polar residues that presumably function as the conduits for metabolites into and out of the shell. The vitamin B12-dependent propanediol-utilizing (PDU) BMC was one of the first functionally characterized catabolic BMCs; subsequently, other types have been implicated in the degradation of ethanolamine, choline, fucose, rhamnose, and ethanol, all of which produce different aldehyde intermediates (Table 1). More recently, bioinformatic studies have demonstrated the widespread distribution of BMCs among diverse bacterial phyla and grouped them into 23 different functional types. The reactions carried out in the majority of catabolic BMCs (also known as metabolosomes) fit a generalized biochemical paradigm for the oxidation of aldehydes (Fig 1). This involves a BMC-encapsulated signature enzyme that generates a toxic and/or volatile aldehyde that the BMC shell sequesters from the cytosol. The aldehyde is subsequently converted into an acyl-CoA by aldehyde dehydrogenase, which uses NAD+ and CoA as cofactors. These two cofactors are relatively large, and their diffusion across the protein shell is thought to be restricted, necessitating their regeneration within the BMC lumen. NAD+ is recycled via alcohol dehydrogenase, and CoA is recycled via phosphotransacetylase (PTAC) (Fig 1). The final product of the BMC, an acyl-phosphate, can then be used to generate ATP via acyl kinase, or revert back to acyl-CoA by Pta for biosynthesis. Collectively, the aldehyde and alcohol dehydrogenases, as well as the PTAC, constitute the common metabolosome core. General biochemical model of aldehyde-degrading BMCs (metabolosomes) illustrating the common metabolosome core enzymes and reactions. Substrates and cofactors involving the PTAC reaction are shown in red; other substrates and enzymes are shown in black, and other cofactors are shown in gray. Characterized and predicted catabolic BMC (metabolosome) types that represent the aldehyde-degrading paradigm (for definition of types see Kerfeld and Erbilgin). Name\tPTAC Type\tSequestered Aldehyde\t \tPDU*\tPduL\tpropionaldehyde\t \tEUT1\tPTA_PTB\tacetaldehyde\t \tEUT2\tPduL\tacetaldehyde\t \tETU\tNone\tacetaldehyde\t \tGRM1/CUT\tPduL\tacetaldehyde\t \tGRM2\tPduL\tacetaldehyde\t \tGRM3*,4\tPduL\tpropionaldehyde\t \tGRM5/GRP\tPduL\tpropionaldehyde\t \tPVM*\tPduL\tlactaldehyde\t \tRMM1,2\tNone\tunknown\t \tSPU\tPduL\tunknown\t \t * PduL from these functional types of metabolosomes were purified in this study. The activities of core enzymes are not confined to BMC-associated functions: aldehyde and alcohol dehydrogenases are utilized in diverse metabolic reactions, and PTAC catalyzes a key biochemical reaction in the process of obtaining energy during fermentation. The concerted functioning of a PTAC and an acetate kinase (Ack) is crucial for ATP generation in the fermentation of pyruvate to acetate (see Reactions 1 and 2). Both enzymes are, however, not restricted to fermentative organisms. They can also work in the reverse direction to activate acetate to the CoA-thioester. This occurs, for example, during acetoclastic methanogenesis in the archaeal Methanosarcina species.  Reaction 1: acetyl-S-CoA + Pi ←→ acetyl phosphate + CoA-SH (PTAC)  Reaction 2: acetyl phosphate + ADP ←→ acetate + ATP (Ack) The canonical PTAC, Pta, is an ancient enzyme found in some eukaryotes and archaea, and widespread among the bacteria; 90% of the bacterial genomes in the Integrated Microbial Genomes database contain a gene encoding the PTA_PTB phosphotransacylase (Pfam domain PF01515). Pta has been extensively characterized due to its key role in fermentation. More recently, a second type of PTAC without any sequence homology to Pta was identified. This protein, PduL (Pfam domain PF06130), was shown to catalyze the conversion of propionyl-CoA to propionyl-phosphate and is associated with a BMC involved in propanediol utilization, the PDU BMC. Both pduL and pta genes can be found in genetic loci of functionally distinct BMCs, although the PduL type is much more prevalent, being found in all but one type of metabolosome locus: EUT1 (Table 1). Furthermore, in the Integrated Microbial Genomes Database, 91% of genomes that encode PF06130 also encode genes for shell proteins. As a member of the core biochemical machinery of functionally diverse aldehyde-oxidizing metabolosomes, PduL must have a certain level of substrate plasticity (see Table 1) that is not required of Pta, which has generally been observed to prefer acetyl-CoA. PduL from the PDU BMC of Salmonella enterica favors propionyl-CoA over acetyl-CoA, and it is likely that PduL orthologs in functionally diverse BMCs would have substrate preferences for other CoA derivatives. Another distinctive feature of BMC-associated PduL homologs is an N-terminal encapsulation peptide (EP) that is thought to “target” proteins for encapsulation by the BMC shell. EPs are frequently found on BMC-associated proteins and have been shown to interact with shell proteins. EPs have also been observed to cause proteins to aggregate, and this has recently been suggested to be functionally relevant as an initial step in metabolosome assembly, in which a multifunctional protein core is formed, around which the shell assembles. Of the three common metabolosome core enzymes, crystal structures are available for both the alcohol and aldehyde dehydrogenases. In contrast, the structure of PduL, the PTAC found in the vast majority of catabolic BMCs, has not been determined. This is a major gap in our understanding of metabolosome-encapsulated biochemistry and cofactor recycling. Structural information will be essential to working out how the core enzymes and their cofactors assemble and organize within the organelle lumen to enhance catalysis. Moreover, it will be useful for guiding efforts to engineer novel BMC cores for biotechnological applications. The primary structure of PduL homologs is subdivided into two PF06130 domains, each roughly 80 residues in length. No available protein structures contain the PF06130 domain, and homology searches using the primary structure of PduL do not return any significant results that would allow prediction of the structure. Moreover, the evident novelty of PduL makes its structure interesting in the context of convergent evolution of PTAC function; to-date, only the Pta active site and catalytic mechanism is known. Here we report high-resolution crystal structures of a PduL-type PTAC in both CoA- and phosphate-bound forms, completing our understanding of the structural basis of catalysis by the metabolosome common core enzymes. We propose a catalytic mechanism analogous but yet distinct from the ubiquitous Pta enzyme, highlighting the functional convergence of two enzymes with completely different structures and metal requirements. We also investigate the quaternary structures of three different PduL homologs and situate our findings in the context of organelle biogenesis in functionally diverse BMCs. Results Structure Determination of PduL We cloned, expressed, and purified three different PduL homologs from functionally distinct BMCs (Table 1): from the well-studied pdu locus in S. enterica Typhimurium LT2 (sPduL), from the recently characterized pvm locus in Planctomyces limnophilus (pPduL), and from the grm3 locus in Rhodopseudomonas palustris BisB18 (rPduL). While purifying full-length sPduL, we observed a tendency to aggregation as described previously, with a large fraction of the expressed protein found in the insoluble fraction in a white, cake-like pellet. Remarkably, after removing the N-terminal putative EP (27 amino acids), most of the sPduLΔEP protein was in the soluble fraction upon cell lysis. Similar differences in solubility were observed for pPduL and rPduL when comparing EP-truncated forms to the full-length protein, but none were quite as dramatic as for sPduL. We confirmed that all homologs were active (S1a and S1b Fig). Among these, we were only able to obtain diffraction-quality crystals of rPduL after removing the N-terminal putative EP (33 amino acids, also see Fig 2a) (rPduLΔEP). Truncated rPduLΔEP had comparable enzymatic activity to the full-length enzyme (S1a Fig). Structural overview of R. palustris PduL from the grm3 locus. (a) Primary and secondary structure of rPduL (tubes represent α-helices, arrows β-sheets and dashed line residues disordered in the structure. Blocks of ten residues are shaded alternatively black/dark gray. The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). Metal coordination residues are highlighted in light blue and CoA contacting residues in magenta, residues contacting the CoA of the other chain are also outlined. (b) Cartoon representation of the structure colored by domains and including secondary structure numbering. The N-and C-termini are in close proximity. Coenzyme A is shown in magenta sticks and Zinc (grey) as spheres. We collected a native dataset from rPduLΔEP crystals diffracting to a resolution of 1.54 Å (Table 2). Using a mercury-derivative crystal form diffracting to 1.99 Å (Table 2), we obtained high quality electron density for model building and used the initial model to refine against the native data to Rwork/Rfree values of 18.9/22.1%. There are two PduL molecules in the asymmetric unit of the P212121 unit cell. We were able to fit all of the primary structure of PduLΔEP into the electron density with the exception of three amino acids at the N-terminus and two amino acids at the C-terminus (Fig 2a); the model is of excellent quality (Table 2). A CoA cofactor as well as two metal ions are clearly resolved in the density (for omit maps of CoA see S2 Fig). Data collection and refinement statistics \tPduL native\tPduL mercury derivative\tPduL phosphate soaked\t \tData collection\t\t\t\t \tSpace group\tP 21 21 21\tP 21 21 21\tP 21 21 21\t \tCell dimensions\t\t\t\t \ta, b, c (Å)\t57.7, 56.4, 150.4\t55.6, 57.7, 150.2\t57.1, 58.8, 136.7\t \tα, β, γ (°)\t90, 90, 90\t90, 90, 90\t90, 90, 90\t \tResolution (Å)\t31.4 − 1.54 (1.60 − 1.54)*\t35.3 − 1.99 (2.07 − 1.99)\t39.2 − 2.10 (2.21 − 2.10)\t \tRmerge\t0.169 (1.223)\t0.084 (0.299)\t0.122 (0.856)\t \tI/σ(I)\t12.9 (1.7)\t22.1 (7.1)\t12.6 (2.0)\t \tCompleteness (%)\t99.4 (94.4)\t99.3 (93.3)\t100 (99.9)\t \tRedundancy\t13.9 (12.1)\t7.2 (7.0)\t6.5 (6.1)\t \tRefinement\t\t\t\t \tResolution (Å)\t31.4 − 1.54 (1.60 − 1.54)*\t\t39.2 − 2.10 (2.18 − 2.1)\t \tNo. reflections\t72,698\t\t27,554\t \tRwork/Rfree (%)\t18.9 (30.7) / 22.1 (34.7)\t\t17.5 (24.2) / 22.6 (30.0)\t \tNo. atoms\t3,453\t\t3,127\t \tProtein\t2,841\t\t2,838\t \tLigand/ion\t100\t\t24\t \tWater\t512\t\t265\t \tB-factors\t22.8\t\t34.7\t \tProtein\t21.5\t\t24.3\t \tLigand/ion\t21.9\t\t40.6\t \tWater\t30.3\t\t37.9\t \tR.m.s deviations\t\t\t\t \tBond lengths (Å)\t0.006\t\t0.013\t \tBond angles (°)\t1.26\t\t1.30\t \tRamachandran Plot\t\t\t\t \tfavored (%)\t99\t\t99\t \tallowed (%)\t1\t\t1\t \tdisallowed (%)\t0\t\t0\t \t *Highest resolution shell is shown in parentheses. Structurally, PduL consists of two domains (Fig 2, blue/red), each a beta-barrel that is capped on both ends by short α-helices. β-Barrel 1 consists of the N-terminal β strand and β strands from the C-terminal half of the polypeptide chain (β1, β10-β14; residues 37–46 and 155–224). β-Barrel 2 consists mainly of the central segment of primary structure (β2, β5–β9; residues 47–60 and 82–154) (Fig 2, red), but is interrupted by a short two-strand beta sheet (β3-β4, residues 61–81). This β-sheet is involved in contacts between the two domains and forms a lid over the active site. Residues in this region (Gln42, Pro43, Gly44), covering the active site, are strongly conserved (Fig 3). This structural arrangement is completely different from the functionally related Pta, which is composed of two domains, each consisting of a central flat beta sheet with alpha-helices on the top and bottom. Primary structure conservation of the PduL protein family. Sequence logo calculated from the multiple sequence alignment of PduL homologs (see Materials and Methods), but not including putative EP sequences. Residues 100% conserved across all PduL homologs in our dataset are noted with an asterisk, and residues conserved in over 90% of sequences are noted with a colon. The sequences aligning to the PF06130 domain (determined by BLAST) are highlighted in red and blue. The position numbers shown correspond to the residue numbering of rPduL; note that some positions in the logo represent gaps in the rPduL sequence. There are two PduL molecules in the asymmetric unit forming a butterfly-shaped dimer (Fig 4c). Consistent with this, results from size exclusion chromatography of rPduLΔEP suggest that it is a dimer in solution (Fig 5e). The interface between the two chains buries 882 Å2 per monomer and is mainly formed by α-helices 2 and 4 and parts of β-sheets 12 and 14, as well as a π–π stacking of the adenine moiety of CoA with Phe116 of the adjacent chain (Fig 4c). The folds of the two chains in the asymmetric unit are very similar, superimposing with a rmsd of 0.16 Å over 2,306 aligned atom pairs. The peripheral helices and the short antiparallel β3–4 sheet mediate most of the crystal contacts. Details of active site, dimeric assembly, and sequence conservation of PduL. (a,b) Proposed active site of PduL with relevant residues shown as sticks in atom coloring (nitrogen blue, oxygen red, sulfur yellow), zinc as grey colored spheres and coordinating ordered water molecules in red. Distances between atom centers are indicated in Å. (a) Coenzyme A containing, (b) phosphate-bound structure. (c) View of the dimer in the asymmetric unit from the side, domains 1 and 2 colored as in Fig 2 and the two chains differentiated by blue/red versus slate/firebrick. The bottom panel shows a top view down the 2-fold axis as indicated by the arrow in the top panel. The asterisk and double arrow marks the location of the π–π interaction between F116 and the CoA base of the other dimer chain. (d) Surface representation of the structure with indicated conservation (red: high, white: intermediate, yellow: low). Size exclusion chromatography of PduL homologs. (a)–(c): Chromatograms of sPduL (a), rPduL (b), and pPduL (c) with (orange) or without (blue) the predicted EP, post-nickel affinity purification, applied over a preparative size exclusion column (see Materials and Methods). (d)–(f): Chromatograms of sPduL (d), rPduL (e), and pPduL (f) post-preparative size exclusion chromatography with different size fractions separated, applied over an analytical size exclusion column (see Materials and Methods). All chromatograms are cropped to show only the linear range of separation based on standard runs, shown in black squares with a dashed linear trend line. All y-axes are arbitrary absorbance units except the right-hand axes for panels (a) and (d), which is the log10(molecular weight) of the standards. Active Site Properties CoA and the metal ions bind between the two domains, presumably in the active site (Figs 2b and 4a). To identify the bound metals, we performed an X-ray fluorescence scan on the crystals at various wavelengths (corresponding to the K-edges of Mn, Fe, Co, Ni, Cu, and Zn). There was a large signal at the zinc edge, and we tested for the presence of zinc by collecting full data sets before and after the Zn K-edge (1.2861 and 1.2822 Å, respectively). The large differences between the anomalous signals confirm the presence of zinc at both metal sites (S3 Fig). The first zinc ion (Zn1) is in a tetrahedral coordination state with His48, His50, Glu109, and the CoA sulfur (Fig 4a). The second (Zn2) is in octahedral coordination by three conserved histidine residues (His157, His159 and His204) as well as three water molecules (Fig 4a). The nitrogen atom coordinating the zinc is the Nε in each histidine residue, as is typical for this interaction. When the crystals were soaked in a sodium phosphate solution for 2 d prior to data collection, the CoA dissociates, and density for a phosphate molecule is visible at the active site (Table 2, Fig 4b). The phosphate-bound structure aligns well with the CoA-bound structure (0.43 Å rmsd over 2,361 atoms for the monomer, 0.83 Å over 5,259 aligned atoms for the dimer). The phosphate contacts both zinc atoms (Fig 4b) and replaces the coordination by CoA at Zn1; the coordination for Zn2 changes from octahedral with three bound waters to tetrahedral with a phosphate ion as one of the ligands (Fig 4b). Conserved Arg103 seems to be involved in maintaining the phosphate in that position. The two zinc atoms are slightly closer together in the phosphate-bound form (5.8 Å vs 6.3 Å), possibly due to the bridging effect of the phosphate. An additional phosphate molecule is bound at a crystal contact interface, perhaps accounting for the 14 Å shorter c-axis in the phosphate-bound crystal form (Table 2). Oligomeric States of PduL Orthologs Are Influenced by the EP Interestingly, some of the residues important for dimerization of rPduL, particularly Phe116, are poorly conserved across PduL homologs associated with functionally diverse BMCs (Figs 4c and 3), suggesting that they may have alternative oligomeric states. We tested this hypothesis by performing size exclusion chromatography on both full-length and truncated variants (lacking the EP, ΔEP) of sPduL, rPduL, and pPduL. These three homologs are found in functionally distinct BMCs (Table 1). Therefore, they are packaged with different signature enzymes and different ancillary proteins. It has been proposed that the catabolic BMCs may assemble in a core-first manner, with the luminal enzymes (signature enzyme, aldehyde, and alcohol dehydrogenases and the BMC PTAC) forming an initial bolus, or prometabolosome, around which a shell assembles. Given the diversity of signature enzymes (Table 1), it is plausible that PduL orthologs may adopt different oligomeric states that reflect the differences in the proteins being packaged with them in the organelle lumen. We found that not only did the different orthologs appear to assemble into different oligomeric states, but that quaternary structure was dependent on whether or not the EP was present. Full-length sPduL was unstable in solution—precipitating over time—and eluted throughout the entire volume of a size exclusion column, indicating it was nonspecifically aggregating. However, when the putative EP (residues 1–27) was removed (sPduL ΔEP), the truncated protein was stable and eluted as a single peak (Fig 5a) consistent with the size of a monomer (Fig 5d, blue curve). In contrast, both full-length rPduL and pPduL appeared to exist in two distinct oligomeric states (Fig 5b and 5c respectively, orange curves), one form of the approximate size of a dimer and the second, a higher molecular weight oligomer (~150 kDa). Upon deletion of the putative EP (residues 1–47 for rPduL, and 1–20 for pPduL), there was a distinct change in the elution profiles (Fig 5b and 5c respectively, blue curves). pPduLΔEP eluted as two smaller forms, possibly corresponding to a trimer and a monomer. In contrast, rPduLΔEP eluted as one smaller oligomer, possibly a dimer. We also analyzed purified rPduL and rPduLΔEP by size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) for a complementary approach to assessing oligomeric state. SEC-MALS analysis of rPdulΔEP is consistent with a dimer (as observed in the crystal structure) with a weighted average (Mw) and number average (Mn) of the molar mass of 58.4 kDa +/− 11.2% and 58.8 kDa +/− 10.9%, respectively (S4a Fig). rPduL full length runs as Mw = 140.3 kDa +/− 1.2% and Mn = 140.5 kDa +/− 1.2%. This corresponds to an oligomeric state of six subunits (calculated molecular weight of 144 kDa). Collectively, these data strongly suggest that the N-terminal EP of PduL plays a role in defining the quaternary structure of the protein. Discussion The hallmark attribute of an organelle is that it serves as a discrete subcellular compartment functioning as an isolated microenvironment distinct from the cytosol. In order to create and preserve this microenvironment, the defining barrier (i.e., lipid bilayer membrane or microcompartment shell) must be selectively permeable. The BMC shell not only sequesters specific enzymes but also their cofactors, thereby establishing a private cofactor pool dedicated to the encapsulated reactions. In catabolic BMCs, CoA and NAD+ must be continually recycled within the organelle (Fig 1). Homologs of the predominant cofactor utilizer (aldehyde dehydrogenase) and NAD+ regenerator (alcohol dehydrogenase) have been structurally characterized, but until now structural information was lacking for PduL, which recycles CoA in the organelle lumen. Curiously, while the housekeeping Pta could provide this function, and indeed does so in the case of one type of ethanolamine-utilizing (EUT) BMC, the evolutionarily unrelated PduL fulfills this function for the majority of metabolosomes using a novel structure and active site for convergent evolution of function. The Tertiary Structure of PduL Is Formed by Discontinuous Segments of Primary Structure The structure of PduL consists of two β-barrel domains capped by short alpha helical segments (Fig 2b). The two domains are structurally very similar (superimposing with a rmsd of 1.34 Å (over 123 out of 320/348 aligned backbone atoms, S5a Fig). However, the amino acid sequences of the two domains are only 16% identical (mainly the RHxH motif, β2 and β10), and 34% similar. Our structure reveals that the two assigned PF06130 domains (Fig 3) do not form structurally discrete units; this reduces the apparent sequence conservation at the level of primary structure. One strand of the domain 1 beta barrel (shown in blue in Fig 2) is contributed by the N-terminus, while the rest of the domain is formed by the residues from the C-terminal half of the protein. When aligned by structure, the β1 strand of the first domain (Fig 2a and 2b, blue) corresponds to the final strand of the second domain (β9), effectively making the domains continuous if the first strand was transplanted to the C-terminus. Refined domain assignment based on our structure should be able to predict domains of PF06130 homologs much more accurately. The closest structural homolog of the PduL barrel domain is a subdomain of a multienzyme complex, the alpha subunit of ethylbenzene dehydrogenase (S5b Fig, rmsd of 2.26 Å over 226 aligned atoms consisting of one beta barrel and one capping helix). In contrast to PduL, there is only one barrel present in ethylbenzene dehydrogenase, and there is no comparable active site arrangement. The PduL signature primary structure, two PF06130 domains, occurs in some multidomain proteins, most of them annotated as Acks, suggesting that PduL may also replace Pta in variants of the phosphotransacetylase-Ack pathway. These PduL homologs lack EPs, and their fusion to Ack may have evolved as a way to facilitate substrate channeling between the two enzymes. Implications for Metabolosome Core Assembly For BMC-encapsulated proteins to properly function together, they must be targeted to the lumen and assemble into an organization that facilitates substrate/product channeling among the different catalytic sites of the signature and core enzymes. The N-terminal extension on PduL homologs may serve both of these functions. The extension shares many features with previously characterized EPs: it is present only in homologs associated with BMC loci, and it is predicted to form an amphipathic α-helix. Moreover, its removal affects the oligomeric state of the protein. EP-mediated oligomerization has been observed for the signature and core BMC enzymes; for example, full-length propanediol dehydratase and ethanolamine ammonia-lyase (signature enzymes for PDU and EUT BMCs) subunits are also insoluble, but become soluble upon removal of the predicted EP. sPduL has also previously been reported to localize to inclusion bodies when overexpressed; we show here that this is dependent on the presence of the EP. This propensity of the EP to cause proteins to form complexes (Fig 5) might not be a coincidence, but could be a necessary step in the assembly of BMCs. Structured aggregation of the core enzymes has been proposed to be the initial step in metabolosome assembly and is known to be the first step of β-carboxysome biogenesis, where the core enzyme Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO) is aggregated by the CcmM protein. Likewise, CsoS2, a protein in the α-carboxysome core, also aggregates when purified and is proposed to facilitate the nucleation and encapsulation of RuBisCO molecules in the lumen of the organelle. Coupled with protein–protein interactions with other luminal components, the aggregation of these enzymes could lead to a densely packed organelle core. This role for EPs in BMC assembly is in addition to their interaction with shell proteins. Moreover, the PduL crystal structures offer a clue as to how required cofactors enter the BMC lumen during assembly. Free CoA and NAD+/H could potentially be bound to the enzymes as the core assembles and is encapsulated. However, this raises an issue of stoichiometry: if the ratio of cofactors to core enzymes is too low, then the sequestered metabolism would proceed at suboptimal rates. Our PduL crystals contained CoA that was captured from the Escherichia coli cytosol, indicating that the “ground state” of PduL is in the CoA-bound form; this could provide an elegantly simple means of guaranteeing a 1:1 ratio of CoA:PduL within the metabolosome lumen. Active Site Identification and Structural Insights into Catalysis The active site of PduL is formed at the interface of the two structural domains (Fig 2b). As expected, the amino acid sequence conservation is highest in the region around the proposed active site (Fig 4d); highly conserved residues are also involved in CoA binding (Figs 2a and 3, residues Ser45, Lys70, Arg97, Leu99, His204, Asn211). All of the metal-coordinating residues (Fig 2a) are absolutely conserved, implicating them in catalysis or the correct spatial orientation of the substrates. Arg103, which contacts the phosphate (Fig 4b), is present in all PduL homologs. The close resemblance between the structures binding CoA and phosphate likely indicates that no large changes in protein conformation are involved in catalysis, and that our crystal structures are representative of the active form. The native substrate for the forward reaction of rPduL and pPduL, propionyl-CoA, most likely binds to the enzyme in the same way at the observed nucleotide and pantothenic acid moiety, but the propionyl group in the CoA-thioester might point in a different direction. There is a pocket nearby the active site between the well-conserved residues Ser45 and Ala154, which could accommodate the propionyl group (S6 Fig). A homology model of sPduL indicates that the residues making up this pocket and the surrounding active site region are identical to that of rPduL, which is not surprising, because these two homologs presumably have the same propionyl-CoA substrate. The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus. Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms. The catalytic mechanism of Pta involves the abstraction of a thiol hydrogen by an aspartate residue, resulting in the nucleophilic attack of thiolate upon the carbonyl carbon of acetyl-phosphate, oriented by an arginine and stabilized by a serine —there are no metals involved. In contrast, in the rPduL structure, there are no conserved aspartate residues in or around the active site, and the only well-conserved glutamate residue in the active site is involved in coordinating one of the metal ions. These observations strongly suggest that an acidic residue is not directly involved in catalysis by PduL. Instead, the dimetal active site of PduL may create a nucleophile from one of the hydroxyl groups on free phosphate to attack the carbonyl carbon of the thioester bond of an acyl-CoA. In the reverse direction, the metal ion(s) could stabilize the thiolate anion that would attack the carbonyl carbon of an acyl-phosphate; a similar mechanism has been described for phosphatases where hydroxyl groups or hydroxide ions can act as a base when coordinated by a dimetal active site. Our structures provide the foundation for studies to elucidate the details of the catalytic mechanism of PduL. Conserved residues in the active site that may contribute to substrate binding and/or transition state stabilization include Ser127, Arg103, Arg194, Gln107, Gln74, and Gln/Glu77. In the phosphate-bound crystal structure, Ser127 and Arg103 appear to position the phosphate (Fig 4b). Alternatively, Arg103 might act as a base to render the phosphate more nucleophilic. The functional groups of Gln74, Gln/Glu77, and Arg194 are directed away from the active site in both CoA and phosphate-bound crystal structures and do not appear to be involved in hydrogen bonding with these substrates, although they could be important for positioning an acyl-phosphate. The free CoA-bound form is presumably poised for attack upon an acyl-phosphate, indicating that the enzyme initially binds CoA as opposed to acyl-phosphate. This hypothesis is strengthened by the fact that the CoA-bound crystals were obtained without added CoA, indicating that the protein bound CoA from the E. coli expression strain and retained it throughout purification and crystallization. The phosphate-bound structure indicates that in the opposite reaction direction phosphate is bound first, and then an acyl-CoA enters. The two high-resolution crystal structures presented here will serve as the foundation for mechanistic studies on this noncanonical PTAC enzyme to determine how the dimetal active site functions to catalyze both forward and reverse reactions. Functional, but Not Structural, Convergence of PduL and Pta PduL and Pta are mechanistically and structurally distinct enzymes that catalyze the same reaction, a prime example of evolutionary convergence upon a function. There are several examples of such functional convergence of enzymes, although typically the enzymes have independently evolved similar, or even identical active sites; for example, the carbonic anhydrase family. However, apparently less frequent is functional convergence that is supported by distinctly different active sites and accordingly catalytic mechanism, as revealed by comparison of the structures of Pta and PduL. One well-studied example of this is the β-lactamase family of enzymes, in which the active site of Class A and Class C enzymes involve serine-based catalysis, but Class B enzymes are metalloproteins. This is not surprising, as β-lactamases are not so widespread among bacteria and therefore would be expected to have evolved independently several times as a defense mechanism against β-lactam antibiotics. However, nearly all bacteria encode Pta, and it is not immediately clear why the Pta/PduL functional convergence should have evolved: it would seem to be evolutionarily more resourceful for the Pta-encoding gene to be duplicated and repurposed for BMCs, as is apparently the case in one type of BMC—EUT1 (Table 1). There could be some intrinsic biochemical difference between the two enzymes that renders PduL a more attractive candidate for encapsulation in a BMC—for example, PduL might be more amenable to tight packaging, or is better suited for the chemical microenvironment formed within the lumen of the BMC, which can be quite different from the cytosol. Further biochemical comparison between the two PTACs will likely yield exciting results that could answer this evolutionary question. Implications BMCs are now known to be widespread among the bacteria and are involved in critical segments of both autotrophic and heterotrophic biochemical pathways that confer to the host organism a competitive (metabolic) advantage in select niches. As one of the three common metabolosome core enzymes, the structure of PduL provides a key missing piece to our structural picture of the shared core biochemistry (Fig 1) of functionally diverse catabolic BMCs. We have observed the oligomeric state differences of PduL to correlate with the presence of an EP, providing new insight into the function of this sequence extension in BMC assembly. Moreover, our results suggest a means for Coenzyme A incorporation during metabolosome biogenesis. A detailed understanding of the underlying principles governing the assembly and internal structural organization of BMCs is a requisite for synthetic biologists to design custom nanoreactors that use BMC architectures as a template. Furthermore, given the growing number of metabolosomes implicated in pathogenesis, the PduL structure will be useful in the development of therapeutics. It is gradually being realized that the metabolic capabilities of a pathogen are also important for virulence, along with the more traditionally cited factors like secretion systems and effector proteins. The fact that PduL is confined almost exclusively to metabolosomes can be used to develop an inhibitor that blocks only PduL and not Pta as a way to selectively disrupt BMC-based metabolism, while not affecting most commensal organisms that require PTAC activity. Materials and Methods Molecular Cloning Genes for PduL homologs with and without the EP were amplified via PCR using the primers listed in S1 Table. sPduL was amplified using S. enterica Typhimurium LT2 genomic DNA, and pPduL and rPduL sequences were codon optimized and synthesized by GenScript with the 6xHis tag. All 5’ primers included EcoRI and BglII restriction sites, and all 3’ primers included a BamHI restriction site to facilitate cloning using the BglBricks strategy. 5’ primers also included the sequence TTTAAGAAGGAGATATACCATG downstream of the restriction sites, serving as a strong ribosome binding site. The 6x polyhistidine tag sequence was added to the 3’ end of the gene using the BglBricks strategy and was subcloned into the pETBb3 vector, a pET21b-based vector modified to be BglBricks compatible. Protein Purification, Size Exclusion Chromatography, and Protein Crystallization  E. coli BL21(DE3) expression strains containing the relevant PduL construct in the pETBb3 vector were grown overnight at 37°C in standard LB medium and then used to inoculate 1L of standard LB medium in 2.8 L Fernbach flasks at a 1:100 dilution, which were then incubated at 37°C shaking at 150 rpm, until the culture reached an OD600 of 0.8–1.0, at which point cultures were induced with 200 μM IPTG (isopropylthio-β-D-galactoside) and incubated at 20°C for 18 h, shaking at 150 rpm. Cells were centrifuged at 5,000 xg for 15 min, and cell pellets were frozen at –20°C. For protein purifications, cell pellets from 1–3 L cultures were resuspended in 20–30 ml buffer A (50 mM Tris-HCl pH 7.4, 300 mM NaCl) and lysed using a French pressure cell at 20,000 lb/in2. The resulting cell lysate was centrifuged at 15,000 xg. 30 mM imidazole was added to the supernatant that was then applied to a 5 mL HisTrap column (GE Healthcare Bio-Sciences, Pittsburgh, PA). Protein was eluted off the column using a gradient of buffer A from 0 mM to 500 mM imidazole over 20 column volumes. Fractions corresponding to PduL were pooled and concentrated using Amicon Ultra Centrifugal filters (EMD Millipore, Billerica, MA) to a volume of no more than 2.5 mL. The protein sample was then applied to a HiLoad 26/60 Superdex 200 preparative size exclusion column (GE Healthcare Bio-Sciences, Pittsburgh, PA) and eluted with buffer B (20 mM Tris pH 7.4, 50 mM NaCl). Where applicable, fractions corresponding to different oligomeric states were pooled separately, leaving one or two fractions in between to prevent cross contamination. Pooled fractions were concentrated to 1–20 mg/mL protein as determined by the Bradford method prior to applying on a Superdex 200 10/300 GL analytical size exclusion column (GE Healthcare Bio-Sciences, Pittsburgh, PA). Size standards used were Thyroglobulin 670 kDa, γ-globulin 158 kDa, Ovalbumin 44 kDa, and Myoglobin 17 kDa. For light scattering, the proteins were measured in a Protein Solutions Dynapro dynamic light scattering instrument with an acquisition time of 5 s, averaging 10 acquisitions at a constant temperature of 25°C. The radii were calculated assuming a globular particle shape. Size exclusion chromatography coupled with SEC-MALS was performed on full-length rPduL and rPduL-ΔEP similar to Luzi et al. 2015. A Wyatt DAWN Heleos-II 18-angle light scattering instrument was used in tandem with a GE AKTA pure FPLC with built in UV detector, and a Wyatt Optilab T-Rex refractive index detector. Detector 16 of the DAWN Heleos-II was replaced with a Wyatt Dynapro Nanostar QELS detector for dynamic light scattering. A GE Superdex S200 10/300 GL column was used, with 125–100 μl of protein sample at 1 mg/ml concentration injected, and the column run at 0.5 ml/min in 20 mM Tris, 50 mM NaCl, pH 7.4. Each detector of the DAWN-Heleos-II was plotted with the Zimm model in the Wyatt ASTRA software to calculate the molar mass. The molar mass was measured at each collected data point across the peaks at ~1 point per 8 μl eluent. Both the Mw and Mn of the molar mass calculations, as well as percent deviations, were also determined using Wyatt software program ASTRA. For preparing protein for crystallography, expression cells were grown as above, except were induced with 50 μM IPTG. Harvested cells were resuspended in buffer B and lysed using a French Press. Cleared lysate was applied on a 5 ml HisTrap HP column (GE Healthcare) and washed with buffer A containing 20 mM imidazole. Pdul-His was eluted with 2 CV buffer B containing 300 mM imidazole, concentrated and then applied on a HiLoad 26/60 Superdex 200 (GE Healthcare) column equilibrated in buffer B for final cleanup. Protein was then concentrated to 20–30 mg/ml for crystallization. Crystals were obtained from sitting drop experiments at 22°C, mixing 3 μl of protein solution with 3 μl of reservoir solution containing 39%–35% MPD. Crystals were flash frozen in liquid nitrogen after being adding 5 μl of a reservoir solution. For heavy atom derivatives, 0.2 μl of 100 mM Thiomerosal (Hampton Research) was added to the crystallization drop 36 h prior to freezing. For phosphate soaks, 5 μl reservoir and 1.5 μl 200 mM sodium phosphate solution (pH 7.0) were added 2 d prior to flash freezing. PTAC Activity Assay Enzyme reactions were performed in a 2 mL cuvette containing 50 mM Tris-HCl pH 7.5, 0.2 mM 5,5'-dithiobis-2-nitrobenzoic acid (DTNB; Ellman’s reagent), 0.1 mM acyl-CoA, and 0.5 μg purified PTAC, unless otherwise noted. To initiate the reaction, 5 mM NaH2PO4 was added, the cuvette was inverted to mix, and the absorbance at 412 nm was measured every 2 s over the course of four minutes in a Nanodrop 2000c, in the cuvette holder. 14,150 M-1cm-1 was used as the extinction coefficient of DTNB to determine the specific activity. PduL Sequence Analysis A multiple sequence alignment of 228 PduL sequences associated with BMCs and 20 PduL sequences not associated with BMCs was constructed using MUSCLE. PduL sequences associated with BMCs were determined from Dataset S1 of Reference, and those not associated with BMCs were determined by searching for genomes that encoded PF06130 but not PF03319 nor PF00936 in the IMG database. The multiple sequence alignment was visualized in Jalview, and the nonconserved N- and C-terminal amino acids were deleted. This trimmed alignment was used to build the sequence logo using WebLogo. Diffraction Data Collection, Structure Determination and Visualization Diffraction data were collected at the Advanced Light Source at Lawrence Berkeley National Laboratory beamline 5.0.2 (100 K, 1.0000 Å wavelength for native data, 1.0093 Å for mercury derivative, 1.2861 Å for Zn pre-edge and 1.2822 Å for Zn peak). Diffraction data were integrated with XDS and scaled with SCALA (CCP4). The structure of PduL was solved using phenix.autosol, which found 11 heavy atom sites and produced density suitable for automatic model building. The model was refined with phenix.refine, with refinement alternating with model building using 2Fo-Fc and Fo-Fc maps visualized in COOT. Statistics for diffraction data collection, structure determination and refinement are summarized in Table 2. Figures were prepared using pymol (www.pymol.org) and Raster3D. Homology Modeling Models of S. enterica Typhimurium LT2 and P. limnophilus PduL were generated with Modeller using the align2d and model-default scripts. Supporting Information Abbreviations Ack acetate kinase BMC Bacterial Microcompartment EP encapsulation peptide EUT ethanolamine-utilizing Mn number average Mw weighted average PDU propanediol-utilizing Pta phosphotransacylase PTAC phosphotransacylase RuBisCO Ribulose Bisphosphate Carboxylase/Oxygenase SEC-MALS multiangle light scattering References Bacterial microcompartments and the modular construction of microbial metabolism A taxonomy of bacterial microcompartment loci constructed by a novel scoring method The PduL phosphotransacylase is used to recycle coenzyme A within the Pdu microcompartment PduL is an Evolutionarily Distinct Phosphotransacylase Involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar typhimurium LT2 Protein structures forming the shell of primitive bacterial organelles Substrate channels revealed in the trimeric Lactobacillus reuteri bacterial microcompartment shell protein PduB The Propanediol Utilization (pdu) Operon of Salmonella enterica serovar Typhimurium LT2 Includes Genes Necessary for Formation of Polyhedral Organelles Involved in coenzyme B12-Dependent 1, 2-Propanediol Degradation The Distribution of Polyhedral Bacterial Microcompartments Suggests Frequent Horizontal Transfer and Operon Reassembly Using comparative genomics to uncover new kinds of protein-based metabolic organelles in bacteria Ethanolamine utilization in Salmonella typhimurium  PduP is a coenzyme-a-acylating propionaldehyde dehydrogenase associated with the polyhedral bodies involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar Typhimurium LT2 Evidence that a metabolic microcompartment contains and recycles private cofactor pools The PduQ enzyme is an alcohol dehydrogenase used to recycle NAD+ internally within the Pdu microcompartment of Salmonella enterica  Activation of acetate by Methanosarcina thermophila. Purification and characterization of phosphotransacetylase Enzymology of the fermentation of acetate to methane by Methanosarcina thermophila Acetate formation in the energy metabolism of parasitic helminths and protists IMG: the Integrated Microbial Genomes database and comparative analysis system Pfam: the protein families database The Pfam protein families database The purification and properties of phosphotransacetylase Phosphotransacetylase from Bacillus subtilis: purification and physiological studies Fluorometric determination of glycolytic intermediates and adenylates during sequential changes in replacement culture of Aspergillus niger Bacterial Microcompartment Assembly: The Key Role of Encapsulation Peptides Interactions between the termini of lumen enzymes and shell proteins mediate enzyme encapsulation into bacterial microcompartments Elucidating the essential role of the conserved carboxysomal protein CcmN reveals a common feature of bacterial microcompartment assembly The N-Terminal Regions of β and γ Subunits Lower the Solubility of Adenosylcobalamin-Dependent Diol Dehydratase Crystal structures of ethanolamine ammonia-lyase complexed with coenzyme B12 analogs and substrates Bacterial microcompartments moving into a synthetic biological world Streamlined construction of the cyanobacterial CO2-fixing organelle via protein domain fusions Structural and functional studies suggest a catalytic mechanism for the phosphotransacetylase from Methanosarcina thermophila Characterization of a planctomycetal organelle: a novel bacterial microcompartment for the aerobic degradation of plant saccharides Geometry of interaction of metal ions with histidine residues in protein structures The PduL Phosphotransacylase Is Used To Recycle Coenzyme A within the Pdu Microcompartment Crystal structure of ethylbenzene dehydrogenase from Aromatoleum aromaticum Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component Biogenesis of a Bacterial Organelle: The Carboxysome Assembly Pathway Solution structure of a bacterial microcompartment targeting peptide and its application in the construction of an ethanol bioreactor Enzymatic mechanisms of phosphate and sulfate transfer The active site architecture of Pisum sativum beta-carbonic anhydrase is a mirror image of that of alpha-carbonic anhydrases Carbonic anhydrase: new insights for an ancient enzyme The 3-D structure of a zinc metallo-beta-lactamase from Bacillus cereus reveals a new type of protein fold Three decades of beta-lactamase inhibitors Structural basis of the oxidative activation of the carboxysomal gamma-carbonic anhydrase, CcmM The bacterial carbon-fixing organelle is formed by shell envelopment of preassembled cargo  Salmonella enterica typhimurium colonizing the lumen of the chicken intestine grows slowly and upregulates a unique set of virulence and metabolism genes Identification of Listeria monocytogenes Genes Contributing to Intracellular Replication by Expression Profiling and Mutant Screening Ethanolamine controls expression of genes encoding components involved in interkingdom signaling and virulence in enterohemorrhagic Escherichia coli O157:H7 Identification of novel genes in genomic islands that contribute to Salmonella typhimurium replication in macrophages  Enterococcus faecalis mutations affecting virulence in the Caenorhabditis elegans model host Redefining Virulence of Bacterial Pathogens A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Subunit disassembly and inhibition of TNFalpha by a semi-synthetic bicyclic peptide MUSCLE: multiple sequence alignment with high accuracy and high throughput Jalview Version 2—a multiple sequence alignment editor and analysis workbench WebLogo: a sequence logo generator XDS Overview of the CCP4 suite and current developments Towards automated crystallographic structure refinement with phenix.refine Coot: model-building tools for molecular graphics Raster3D: photorealistic molecular graphics Comparative protein structure modeling of genes and 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diff --git a/annotated_BioC_JSON/PMC4786784_ann.json b/annotated_BioC_JSON/PMC4786784_ann.json
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+[{"sourceid":"4786784","sourcedb":"","project":"","target":"","text":"An extended U2AF65–RNA-binding domain recognizes the 3′ splice site signal How the essential pre-mRNA splicing factor U2AF65 recognizes the polypyrimidine (Py) signals of the major class of 3′ splice sites in human gene transcripts remains incompletely understood. We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3′ terminus and third nucleotide. Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF65 RRMs are complementary mechanisms for Py-tract association. Altogether, these results advance the mechanistic understanding of molecular recognition for a major class of splice site signals.  The pre-mRNA splicing factor U2AF65 recognizes 3′ splice sites in human gene transcripts, but the details are not fully understood. Here, the authors report U2AF65 structures and single molecule FRET that reveal mechanistic insights into splice site recognition. The differential skipping or inclusion of alternatively spliced pre-mRNA regions is a major source of diversity for nearly all human gene transcripts. The splice sites are marked by relatively short consensus sequences and are regulated by additional pre-mRNA motifs (reviewed in ref.). At the 3′ splice site of the major intron class, these include a polypyrimidine (Py) tract comprising primarily Us or Cs, which is preceded by a branch point sequence (BPS) that ultimately serves as the nucleophile in the splicing reaction and an AG-dinucleotide at the 3′ splice site junction. Disease-causing mutations often compromise pre-mRNA splicing (reviewed in refs), yet a priori predictions of splice sites and the consequences of their mutations are challenged by the brevity and degeneracy of known splice site sequences. High-resolution structures of intact splicing factor–RNA complexes would offer key insights regarding the juxtaposition of the distinct splice site consensus sequences and their relationship to disease-causing point mutations. The early-stage pre-mRNA splicing factor U2AF65 is essential for viability in vertebrates and other model organisms (for example, ref.). A tightly controlled assembly among U2AF65, the pre-mRNA, and partner proteins sequentially identifies the 3′ splice site and promotes association of the spliceosome, which ultimately accomplishes the task of splicing. Initially U2AF65 recognizes the Py-tract splice site signal. In turn, the ternary complex of U2AF65 with SF1 and U2AF35 identifies the surrounding BPS and 3′ splice site junctions. Subsequently U2AF65 recruits the U2 small nuclear ribonucleoprotein particle (snRNP) and ultimately dissociates from the active spliceosome. Biochemical characterizations of U2AF65 demonstrated that tandem RNA recognition motifs (RRM1 and RRM2) recognize the Py tract (Fig. 1a). Milestone crystal structures of the core U2AF65 RRM1 and RRM2 connected by a shortened inter-RRM linker (dU2AF651,2) detailed a subset of nucleotide interactions with the individual U2AF65 RRMs. A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation. As such, the molecular mechanisms for Py-tract recognition by the intact U2AF65–RNA-binding domain remained unknown. Here, we use X-ray crystallography and biochemical studies to reveal new roles in Py-tract recognition for the inter-RRM linker and key residues surrounding the core U2AF65 RRMs. We use single-molecule Förster resonance energy transfer (smFRET) to characterize the conformational dynamics of this extended U2AF65–RNA-binding domain during Py-tract recognition. Results Cognate U2AF65–Py-tract recognition requires RRM extensions The RNA affinity of the minimal U2AF651,2 domain comprising the core RRM1–RRM2 folds (U2AF651,2, residues 148–336) is relatively weak compared with full-length U2AF65 (Fig. 1a,b; Supplementary Fig. 1). Historically, this difference was attributed to the U2AF65 arginine–serine rich domain, which contacts pre-mRNA–U2 snRNA duplexes outside of the Py tract. We noticed that the RNA-binding affinity of the U2AF651,2 domain was greatly enhanced by the addition of seven and six residues at the respective N and C termini of the minimal RRM1 and RRM2 (U2AF651,2L, residues 141–342; Fig. 1a). In a fluorescence anisotropy assay for binding a representative Py tract derived from the well-characterized splice site of the adenovirus major late promoter (AdML), the RNA affinity of U2AF651,2L increased by 100-fold relative to U2AF651,2 to comparable levels as full-length U2AF65 (Fig. 1b; Supplementary Fig. 1a–d). Likewise, both U2AF651,2L and full-length U2AF65 showed similar sequence specificity for U-rich stretches in the 5′-region of the Py tract and promiscuity for C-rich regions in the 3′-region (Fig. 1c, Supplementary Fig. 1e–h). U2AF65-bound Py tract comprises nine contiguous nucleotides To investigate the structural basis for cognate U2AF65 recognition of a contiguous Py tract, we determined four crystal structures of U2AF651,2L bound to Py-tract oligonucleotides (Fig. 2a; Table 1). By sequential boot strapping (Methods), we optimized the oligonucleotide length, the position of a Br-dU, and the identity of the terminal nucleotide (rU, dU and rC) to achieve full views of U2AF651,2L bound to contiguous Py tracts at up to 1.5 Å resolution. The protein and oligonucleotide conformations are nearly identical among the four new U2AF651,2L structures (Supplementary Fig. 2a). The U2AF651,2L RRM1 and RRM2 associate with the Py tract in a parallel, side-by-side arrangement (shown for representative structure iv in Fig. 2b,c; Supplementary Movie 1). An extended conformation of the U2AF65 inter-RRM linker traverses across the α-helical surface of RRM1 and the central β-strands of RRM2 and is well defined in the electron density (Fig. 2b). The extensions at the N terminus of RRM1 and C terminus of RRM2 adopt well-ordered α-helices. Both RRM1/RRM2 extensions and the inter-RRM linker of U2AF651,2L directly recognize the bound oligonucleotide. We compare the global conformation of the U2AF651,2L structures with the prior dU2AF651,2 crystal structure and U2AF651,2 NMR structure in the Supplementary Discussion and Supplementary Fig. 2. The discovery of nine U2AF65-binding sites for contiguous Py-tract nucleotides was unexpected. Based on dU2AF651,2 structures, we originally hypothesized that the U2AF65 RRMs would bind the minimal seven nucleotides observed in these structures. Surprisingly, the RRM2 extension/inter-RRM linker contribute new central nucleotide-binding sites near the RRM1/RRM2 junction and the RRM1 extension recognizes the 3′-terminal nucleotide (Fig. 2c; Supplementary Movie 1). The U2AF651,2L structures characterize ribose (r) nucleotides at all of the binding sites except the seventh and eighth deoxy-(d)U, which are likely to lack 2′-hydroxyl contacts based on the RNA-bound dU2AF651,2 structure. Qualitatively, a subset of the U2AF651,2L-nucleotide-binding sites (sites 1–3 and 7–9) share similar locations to those of the dU2AF651,2 structures (Supplementary Figs 2c,d and 3). Yet, only the U2AF651,2L interactions at sites 1 and 7 are nearly identical to those of the dU2AF651,2 structures (Supplementary Fig. 3a,f). In striking departures from prior partial views, the U2AF651,2L structures reveal three unanticipated nucleotide-binding sites at the centre of the Py tract, as well as numerous new interactions that underlie cognate recognition of the Py tract (Fig. 3a–h). U2AF65 inter-RRM linker interacts with the Py tract The U2AF651,2L RRM2, the inter-RRM linker and RRM1 concomitantly recognize the three central nucleotides of the Py tract, which are likely to coordinate the conformational arrangement of these disparate portions of the protein. Residues in the C-terminal region of the U2AF65 inter-RRM linker comprise a centrally located binding site for the fifth nucleotide on the RRM2 surface and abutting the RRM1/RRM2 interface (Fig. 3d). The backbone amide of the linker V254 and the carbonyl of T252 engage in hydrogen bonds with the rU5-O4 and -N3H atoms. In the C-terminal β-strand of RRM1, the side chains of K225 and R227 donate additional hydrogen bonds to the rU5-O2 lone pair electrons. The C-terminal region of the inter-RRM linker also participates in the preceding rU4-binding site, where the V254 backbone carbonyl and D256 carboxylate position the K260 side chain to hydrogen bond with the rU4-O4 (Fig. 3c). Otherwise, the rU4 nucleotide packs against F304 in the signature ribonucleoprotein consensus motif (RNP)-2 of RRM2. At the opposite side of the central fifth nucleotide, the sixth rU6 nucleotide is located at the inter-RRM1/RRM2 interface (Fig. 3e; Supplementary Movie 1). This nucleotide twists to face away from the U2AF65 linker and instead inserts the rU6-uracil into a sandwich between the β2/β3 loops of RRM1 and RRM2. The rU6 base edge is relatively solvent exposed; accordingly, the rU6 hydrogen bonds with U2AF65 are water mediated apart from a single direct interaction by the RRM1-N196 side chain. We tested the contribution of the U2AF651,2L interactions with the new central nucleotide to Py-tract affinity (Fig. 3i; Supplementary Fig. 4a,b). Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1–R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively. The energetic penalties due to these mutations (ΔΔG 0.8–0.9 kcal mol−1) are consistent with the loss of each hydrogen bond with the rU5 base and support the relevance of the central nucleotide interactions observed in the U2AF651,2L structures. U2AF65 RRM extensions interact with the Py tract The N- and C-terminal extensions of the U2AF65 RRM1 and RRM2 directly contact the bound Py tract. Rather than interacting with a new 5′-terminal nucleotide as we had hypothesized, the C-terminal α-helix of RRM2 instead folds across one surface of rU3 in the third binding site (Fig. 3b). There, a salt bridge between the K340 side chain and nucleotide phosphate, as well as G338-base stacking and a hydrogen bond between the backbone amide of G338 and the rU3-O4, secure the RRM2 extension. Indirectly, the additional contacts with the third nucleotide shift the rU2 nucleotide in the second binding site closer to the C-terminal β-strand of RRM2. Consequently, the U2AF651,2L-bound rU2-O4 and -N3H form dual hydrogen bonds with the K329 backbone atoms (Fig. 3a), rather than a single hydrogen bond with the K329 side chain as in the prior dU2AF651,2 structure (Supplementary Fig. 3b). At the N terminus, the α-helical extension of U2AF65 RRM1 positions the Q147 side chain to bridge the eighth and ninth nucleotides at the 3′ terminus of the Py tract (Fig. 3f–h). The Q147 residue participates in hydrogen bonds with the -N3H of the eighth uracil and -O2 of the ninth pyrimidine. The adjacent R146 guanidinium group donates hydrogen bonds to the 3′-terminal ribose-O2′ and O3′ atoms, where it could form a salt bridge with a phospho-diester group in the context of a longer pre-mRNA. Consistent with loss of a hydrogen bond with the ninth pyrimidine-O2 (ΔΔG 1.0 kcal mol−1), mutation of the Q147 to an alanine reduced U2AF651,2L affinity for the AdML Py tract by five-fold (Fig. 3i; Supplementary Fig. 4c). We compare U2AF65 interactions with uracil relative to cytosine pyrimidines at the ninth binding site in Fig. 3g,h and the Supplementary Discussion. Versatile primary sequence of the U2AF65 inter-RRM linker The U2AF651,2L structures reveal that the inter-RRM linker mediates an extensive interface with the second α-helix of RRM1, the β2/β3 strands of RRM2 and the N-terminal α-helical extension of RRM1. Altogether, the U2AF65 inter-RRM linker residues (R228–K260) bury 2,800 Å2 of surface area in the U2AF651,2L holo-protein, suggestive of a cognate interface compared with 1,900 Å2 for a typical protein–protein complex. The path of the linker initiates at P229 following the core RRM1 β-strand, in a kink that is positioned by intra-molecular stacking among the consecutive R228, Y232 and P234 side chains (Fig. 4a, lower right). A second kink at P236, coupled with respective packing of the L235 and M238 side chains on the N-terminal α-helical RRM1 extension and the core RRM1 α2-helix, reverses the direction of the inter-RRM linker towards the RRM1/RRM2 interface and away from the RNA-binding site. In the neighbouring apical region of the linker, the V244 and V246 side chains pack in a hydrophobic pocket between two α-helices of the core RRM1. The adjacent V249 and V250 are notable for their respective interactions that connect RRM1 and RRM2 at this distal interface from the RNA-binding site (Fig. 4a, top). A third kink stacks P247 and G248 with Y245 and re-orients the C-terminal region of the linker towards the RRM2 and bound RNA. At the RNA surface, the key V254 that recognizes the fifth uracil is secured via hydrophobic contacts between its side chain and the β-sheet surface of RRM2, chiefly the consensus RNP1-F304 residue that stacks with the fourth uracil (Fig. 4a, lower left). Few direct contacts are made between the remaining residues of the linker and the U2AF65 RRM2; instead, the C-terminal conformation of the linker appears primarily RNA mediated (Fig. 3c,d). We investigated whether the observed contacts between the RRMs and linker were critical for RNA binding by structure-guided mutagenesis (Fig. 4b). We titrated these mutant U2AF651,2L proteins into fluorescein-labelled AdML Py-tract RNA and fit the fluorescence anisotropy changes to obtain the apparent equilibrium affinities (Supplementary Fig. 4d–h). We introduced glycine substitutions to maximally reduce the buried surface area without directly interfering with its hydrogen bonds between backbone atoms and the base. First, we replaced V249 and V250 at the RRM1/RRM2 interface and V254 at the bound RNA site with glycine (3Gly). However, the resulting decrease in the AdML RNA affinity of the U2AF651,2L-3Gly mutant relative to wild-type protein was not significant (Fig. 4b). In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein. A more conservative substitution of these five residues (251–255) with an unrelated sequence capable of backbone-mediated hydrogen bonds (STVVP\u003eNLALA) confirmed the subtle impact of this versatile inter-RRM sequence on affinity for the AdML Py tract. Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). Despite 12 concurrent mutations, the AdML RNA affinity of the U2AF651,2L-12Gly variant was reduced by only three-fold relative to the unmodified protein (Fig. 4b), which is less than the penalty of the V254P mutation that disrupts the rU5 hydrogen bond (Fig. 3d,i). To test the interplay of the U2AF65 inter-RRM linker with its N- and C-terminal RRM extensions, we constructed an internal linker deletion of 20-residues within the extended RNA-binding domain (dU2AF651,2L). We found that the affinity of dU2AF651,2L for the AdML RNA was significantly reduced relative to U2AF651,2L (four-fold, Figs 1b and 4b; Supplementary Fig. 4i). Yet, it is well known that the linker deletion in the context of the minimal RRM1–RRM2 boundaries has no detectable effect on the RNA affinities of dU2AF651,2 compared with U2AF651,2 (refs; Figs 1b and 4b; Supplementary Fig. 4j). The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. However, stretching of the truncated dU2AF651,2L linker to connect the RRM termini is expected to disrupt its nucleotide interactions. Likewise, deletion of the N-terminal RRM1 extension in the shortened constructs would remove packing interactions that position the linker in a kinked turn following P229 (Fig. 4a), consistent with the lower RNA affinities of dU2AF651,2L, dU2AF651,2 and U2AF651,2 compared with U2AF651,2L. To further test cooperation among the U2AF65 RRM extensions and inter-RRM linker for RNA recognition, we tested the impact of a triple Q147A/V254P/R227A mutation (U2AF651,2L-3Mut) for RNA binding (Fig. 4b; Supplementary Fig. 4d). Notably, the Q147A/V254P/R227A mutation reduced the RNA affinity of the U2AF651,2L-3Mut protein by 30-fold more than would be expected based on simple addition of the ΔΔG's for the single mutations. This difference indicates that the linearly distant regions of the U2AF65 primary sequence, including Q147 in the N-terminal RRM1 extension and R227/V254 in the N-/C-terminal linker regions at the fifth nucleotide site, cooperatively recognize the Py tract. Altogether, we conclude that the conformation of the U2AF65 inter-RRM linker is key for recognizing RNA and is positioned by the RRM extension but otherwise relatively independent of the side chain composition. The non-additive effects of the Q147A/V254P/R227A triple mutation, coupled with the context-dependent penalties of an internal U2AF65 linker deletion, highlights the importance of the structural interplay among the U2AF65 linker and the N- and C-terminal extensions flanking the core RRMs. Importance of U2AF65–RNA contacts for pre-mRNA splicing We proceeded to test the importance of new U2AF65–Py-tract interactions for splicing of a model pre-mRNA substrate in a human cell line (Fig. 5; Supplementary Fig. 5). As a representative splicing substrate, we utilized a well-characterized minigene splicing reporter (called pyPY) comprising a weak (that is, degenerate, py) and strong (that is, U-rich, PY) polypyrimidine tracts preceding two alternative splice sites (Fig. 5a). When transfected into HEK293T cells containing only endogenous U2AF65, the PY splice site is used and the remaining transcript remains unspliced. When co-transfected with an expression plasmid for wild-type U2AF65, use of the py splice site significantly increases (by more than five-fold) and as documented converts a fraction of the unspliced to spliced transcript. The strong PY splice site is insensitive to added U2AF65, suggesting that endogenous U2AF65 levels are sufficient to saturate this site (Supplementary Fig. 5b). We introduced the triple mutation (V254P/R227A/Q147A) that significantly reduced U2AF651,2L association with the Py tract (Fig. 4b) in the context of full-length U2AF65 (U2AF65-3Mut). Co-transfection of the U2AF65-3Mut with the pyPY splicing substrate significantly reduced splicing of the weak ‘py' splice site relative to wild-type U2AF65 (Fig. 5b,c). We conclude that the Py-tract interactions with these residues of the U2AF65 inter-RRM linker and RRM extensions are important for splicing as well as for binding a representative of the major U2-class of splice sites. Sparse inter-RRM contacts underlie apo-U2AF65 dynamics The direct interface between U2AF651,2L RRM1 and RRM2 is minor, burying 265 Å2 of solvent accessible surface area compared with 570 Å2 on average for a crystal packing interface. A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c). This minor U2AF65 RRM1/RRM2 interface, coupled with the versatile sequence of the inter-RRM linker, highlighted the potential role for inter-RRM conformational dynamics in U2AF65-splice site recognition. Paramagnetic resonance enhancement (PRE) measurements previously had suggested a predominant back-to-back, or ‘closed' conformation of the apo-U2AF651,2 RRM1 and RRM2 in equilibrium with a minor ‘open' conformation resembling the RNA-bound inter-RRM arrangement. Yet, small-angle X-ray scattering (SAXS) data indicated that both the minimal U2AF651,2 and longer constructs comprise a highly diverse continuum of conformations in the absence of RNA that includes the ‘closed' and ‘open' conformations. To complement the static portraits of U2AF651,2L structure that we had determined by X-ray crystallography, we used smFRET to characterize the probability distribution functions and time dependence of U2AF65 inter-RRM conformational dynamics in solution. The inter-RRM dynamics of U2AF65 were followed using FRET between fluorophores attached to RRM1 and RRM2 (Fig. 6a,b, Methods). The positions of single cysteine mutations for fluorophore attachment (A181C in RRM1 and Q324C in RRM2) were chosen based on inspection of the U2AF651,2L structures and the ‘closed' model of apo-U2AF651,2. Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50 Å for U2AF651,2L–Py tract and 30 Å for the closed apo-model) that are expected to be near the Förster radius (Ro) for the Cy3/Cy5 pair (56 Å), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the ‘closed' apo-model as opposed to the ‘open' RNA-bound structures (by ∼30%). The FRET efficiencies of either of these structurally characterized conformations also are expected to be significantly greater than elongated U2AF65 conformations that lack inter-RRM contacts. Double-cysteine variant of U2AF651,2 was modified with equimolar amount of Cy3 and Cy5. Only traces that showed single photobleaching events for both donor and acceptor dyes and anti-correlated changes in acceptor and donor fluorescence were included in smFRET data analysis. Hence, molecules that were conjugated to two donor or two acceptor fluorophores were excluded from analysis. We first characterized the conformational dynamics spectrum of U2AF65 in the absence of RNA (Fig. 6c,d; Supplementary Fig. 7a,b). The double-labelled U2AF651,2LFRET(Cy3/Cy5) protein was tethered to a slide via biotin-NTA/Ni+2 resin. Virtually no fluorescent molecules were detected in the absence of biotin-NTA/Ni+2, which demonstrates the absence of detectable non-specific binding of U2AF651,2LFRET to the slide. The FRET distribution histogram built from more than a thousand traces of U2AF651,2LFRET(Cy3/Cy5) in the absence of ligand showed an extremely broad distribution centred at a FRET efficiency of ∼0.4 (Fig. 6d). Approximately 40% of the smFRET traces showed apparent transitions between multiple FRET values (for example, Fig. 6c). Despite the large width of the FRET-distribution histogram, the majority (80%) of traces that showed fluctuations sampled only two distinct FRET states (for example, Supplementary Fig. 7a). Approximately 70% of observed fluctuations were interchanges between the ∼0.65 and ∼0.45 FRET values (Supplementary Fig. 7b). We cannot exclude a possibility that tethering of U2AF651,2LFRET(Cy3/Cy5) to the microscope slide introduces structural heterogeneity into the protein and, thus, contributes to the breadth of the FRET distribution histogram. However, the presence of repetitive fluctuations between particular FRET values supports the hypothesis that RNA-free U2AF65 samples several distinct conformations. This result is consistent with the broad ensembles of extended solution conformations that best fit the SAXS data collected for U2AF651,2 as well as for a longer construct (residues 136–347). We conclude that weak contacts between the U2AF65 RRM1 and RRM2 permit dissociation of these RRMs in the absence of RNA. U2AF65 conformational selection and induced fit by bound RNA We next used smFRET to probe the conformational selection of distinct inter-RRM arrangements following association of U2AF65 with the AdML Py-tract prototype. Addition of the AdML RNA to tethered U2AF651,2LFRET(Cy3/Cy5) selectively increases a fraction of molecules showing an ∼0.45 apparent FRET efficiency, suggesting that RNA binding stabilizes a single conformation, which corresponds to the 0.45 FRET state (Fig. 6e,f). To assess the possible contributions of RNA-free conformations of U2AF65 and/or structural heterogeneity introduced by tethering of U2AF651,2LFRET(Cy3/Cy5) to the slide to the observed distribution of FRET values, we reversed the immobilization scheme. We tethered the AdML RNA to the slide via a biotinylated oligonucleotide DNA handle and added U2AF651,2LFRET(Cy3/Cy5) in the absence of biotin-NTA resin (Fig. 6g,h; Supplementary Fig. 7c–g). A 0.45 FRET value was again predominant, indicating a similar RNA-bound conformation and structural dynamics for the untethered and tethered U2AF651,2LFRET(Cy3/Cy5). We examined the effect on U2AF651,2L conformations of purine interruptions that often occur in relatively degenerate human Py tracts. We introduced an rArA purine dinucleotide within a variant of the AdML Py tract (detailed in Methods). Insertion of adenine nucleotides decreased binding affinity of U2AF65 to RNA by approximately five-fold. Nevertheless, in the presence of saturating concentrations of rArA-interrupted RNA slide-tethered U2AF651,2LFRET(Cy3/Cy5) showed a prevalent ∼0.45 apparent FRET value (Fig. 6i,j), which was also predominant in the presence of continuous Py tract. Therefore, RRM1-to-RRM2 distance remains similar regardless of whether U2AF65 is bound to interrupted or continuous Py tract. The inter-fluorophore distances derived from the observed 0.45 FRET state agree with the distances between the α-carbon atoms of the respective residues in the crystal structures of U2AF651,2L bound to Py-tract oligonucleotides. It should be noted that inferring distances from FRET values is prone to significant error because of uncertainties in the determination of fluorophore orientation factor κ2 and Förster radius R0, the parameters used in distance calculations. Nevertheless, the predominant 0.45 FRET state in the presence of RNA agrees with the Py-tract-bound crystal structure of U2AF651,2L. Importantly, the majority of traces (∼70%) of U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA lacked FRET fluctuations and predominately exhibited a ∼0.45 FRET value (for example, Fig. 6g). The remaining ∼30% of traces for U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA showed fluctuations between distinct FRET values. The majority of traces that show fluctuations began at high (0.65–0.8) FRET value and transitioned to a ∼0.45 FRET value (Supplementary Fig. 7c–g). Hidden Markov modelling analysis of smFRET traces suggests that RNA-bound U2AF651,2L can sample at least two other conformations corresponding to ∼0.7–0.8 and ∼0.3 FRET values in addition to the predominant conformation corresponding to the 0.45 FRET state. Although a compact conformation (or multiple conformations) of U2AF651,2L corresponding to ∼0.7–0.8 FRET values can bind RNA, on RNA binding, these compact conformations of U2AF651,2L transition into a more stable structural state that corresponds to ∼0.45 FRET value and is likely similar to the side-by-side inter-RRM-arrangement of the U2AF651,2L crystal structures. Thus, the sequence of structural rearrangements of U2AF65 observed in smFRET traces (Supplementary Fig. 7c–g) suggests that a ‘conformational selection' mechanism of Py-tract recognition (that is, RNA ligand stabilization of a pre-configured U2AF65 conformation) is complemented by ‘induced fit' (that is, RNA-induced rearrangement of the U2AF65 RRMs to achieve the final ‘side-by-side' conformation), as discussed below. Discussion The U2AF65 structures and analyses presented here represent a successful step towards defining a molecular map of the 3′ splice site. Several observations indicate that the numerous intramolecular contacts, here revealed among the inter-RRM linker and RRM1, RRM2, and the N-terminal RRM1 extension, synergistically coordinate U2AF65–Py-tract recognition. Truncation of U2AF65 to the core RRM1–RRM2 region reduces its RNA affinity by 100-fold. Likewise, deletion of 20 inter-RRM linker residues significantly reduces U2AF65–RNA binding only when introduced in the context of the longer U2AF651,2L construct comprising the RRM extensions, which in turn position the linker for RNA interactions. Notably, a triple mutation of three residues (V254P, Q147A and R227A) in the respective inter-RRM linker, N- and C-terminal extensions non-additively reduce RNA binding by 150-fold. Altogether, these data indicate that interactions among the U2AF65 RRM1/RRM2, inter-RRM linker, N-and C-terminal extensions are mutually inter-dependent for cognate Py-tract recognition. The implications of this finding for U2AF65 conservation and Py-tract recognition are detailed in the Supplementary Discussion. Recently, high-throughput sequencing studies have shown that somatic mutations in pre-mRNA splicing factors occur in the majority of patients with myelodysplastic syndrome (MDS). MDS-relevant mutations are common in the small U2AF subunit (U2AF35, or U2AF1), yet such mutations are rare in the large U2AF65 subunit (also called U2AF2)—possibly due to the selective versus nearly universal requirements of these factors for splicing. A confirmed somatic mutation of U2AF65 in patients with MDS, L187V, is located on a solvent-exposed surface of RRM1 that is distinct from the RNA interface (Fig. 7a). This L187 surface is oriented towards the N terminus of the U2AF651,2L construct, where it is expected to abut the U2AF35-binding site in the context of the full-length U2AF heterodimer. Likewise, an unconfirmed M144I mutation reported by the same group corresponds to the N-terminal residue of U2AF651,2L, which is separated by only ∼20 residues from the U2AF35-binding site. As such, we suggest that the MDS-relevant U2AF65 mutations contribute to MDS progression indirectly, by destabilizing a relevant conformation of the conjoined U2AF35 subunit rather than affecting U2AF65 functions in RNA binding or spliceosome recruitment per se. Our smFRET results agree with prior NMR/PRE evidence for multi-domain conformational selection as one mechanistic basis for U2AF65–RNA association (Fig. 7b). The ‘induced fit' versus ‘conformational selection' models are the prevailing views of the mechanisms underlying bio-molecular interactions (reviewed in ref.). In the former, ligand binding promotes a subsequent conformational change in the protein, whereas in the latter, the ligand selects a protein conformation from a pre-existing ensemble and thereby shifts the population towards that state. An ∼0.45 FRET value is likely to correspond to the U2AF65 conformation visualized in our U2AF651,2L crystal structures, in which the RRM1 and RRM2 bind side-by-side to the Py-tract oligonucleotide. The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. An increased prevalence of the ∼0.45 FRET value following U2AF65–RNA binding, coupled with the apparent absence of transitions in many ∼0.45-value single molecule traces (for example, Fig. 6e), suggests a population shift in which RNA binds to (and draws the equilibrium towards) a pre-configured inter-RRM proximity that most often corresponds to the ∼0.45 FRET value. Notably, our smFRET results reveal that U2AF65–Py-tract recognition can be characterized by an ‘extended conformational selection' model (Fig. 7b). In this recent model for macromolecular interactions, the pure ‘conformational selection' and ‘induced fit' scenarios represent the limits of a mechanistic spectrum and may compete or occur sequentially. Examples of ‘extended conformational selection' during ligand binding have been characterized for a growing number of macromolecules (for example, adenylate kinase, LAO-binding protein, poly-ubiquitin, maltose-binding protein and the preQ1 riboswitch, among others). Here, the majority of changes in smFRET traces for U2AF651,2LFRET(Cy3/Cy5) bound to slide-tethered RNA began at high (0.65–0.8) FRET value and transition to the predominant 0.45 FRET value (Supplementary Fig. 7c–g). These transitions could correspond to rearrangement from the ‘closed' NMR/PRE-based U2AF65 conformation in which the RNA-binding surface of only a single RRM is exposed and available for RNA binding, to the structural state seen in the side-by-side, RNA-bound crystal structure. As such, the smFRET approach reconciles prior inconsistencies between two major conformations that were detected by NMR/PRE experiments and a broad ensemble of diverse inter-RRM arrangements that fit the SAXS data for the apo-protein. Similar interdisciplinary structural approaches are likely to illuminate whether similar mechanistic bases for RNA binding are widespread among other members of the vast multi-RRM family. The finding that U2AF65 recognizes a nine base pair Py tract contributes to an elusive ‘code' for predicting splicing patterns from primary sequences in the post-genomic era (reviewed in ref.). Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. Further research will be needed to understand the roles of SF1 and U2AF35 subunits in the conformational equilibria underlying U2AF65 association with Py tracts. Moreover, structural differences among U2AF65 homologues and paralogues may regulate splice site selection. Ultimately, these guidelines will assist the identification of 3′ splice sites and the relationship of disease-causing mutations to penalties for U2AF65 association. Methods Protein expression and purification For crystallization and RNA-binding experiments, human U2AF651,2L (residues 141–342 of NCBI RefSeq NP_009210) was expressed in Escherichia coli strain BL21 Rosetta-2 as a GST-fusion protein in the vector pGEX6P-2 and purified by glutathione affinity, followed by anion exchange and gel filtration chromatography. The GST-tagged protein was bound to a GSTrap column (GE Healthcare) in 1 M NaCl, 25 mM HEPES, pH 7.4 and eluted using 150 mM NaCl, 100 mM Tris, pH 8 containing 10 mM glutathione. The GST tag was cleaved from the protein by treatment with PreScission Protease during dialysis against a buffer containing 100 mM NaCl, 25 mM HEPES, pH 8, 5% (v/v) glycerol, 5 mM DTT, 0.25 mM EDTA and 0.1 mM PMSF. Cleaved GST was separated from the U2AF651,2L by subtractive glutathione affinity chromatography in 100 mM NaCl, 25 mM Tris, pH 8, 0.2 mM TCEP followed by subtractive anion-exchange chromatography with a HiTrap Q column (GE Healthcare). The final purification step was size-exclusion chromatography on a Superdex-75 prep-grade column (GE Healthcare) that had been previously equilibrated with 100 mM NaCl, 15 mM HEPES, pH 6.8, 0.2 mM tris(2-carboxy-ethyl)phosphine (TCEP). The purified U2AF651,2L was concentrated using a Vivaspin 15 R (Sartorius) centrifugal concentrator with 10 kDa MWCO, and the protein concentration was estimated using the calculated extinction coefficient of 8,940 M−1cm−1 and absorbance at 280 nm. Shorter constructs (U2AF651,2, residues 148–336; dU2AF651,2, residues 148–237, 258–336; dU2AF651,2L, residues 141–237, 258–342) (Fig. 1a) and individual U2AF651,2L Q147A, R227A, V254P mutants used for RNA-binding experiments were purified similarly. For comparative RNA-binding experiments, full-length human U2AF65 (residues 1–475) and the U2AF35-UHM (U2AF homology motif; residues 43–146, NCBI RefSeq NP_006749) initially were expressed and purified separately as GST fusion proteins. Following GST cleavage and ion-exchange chromatography (SP-HiTrap and Q-HiTrap, respectively), U2AF65 was combined with slight excess U2AF35-UHM (in stoichiometric ratio of 1:1.2) and dialysed overnight. The final U2AF heterodimer was purified by size-exclusion chromatography using a Superdex-200 prep-grade column (GE Healthcare) pre-equilibrated with 150 mM NaCl, 25 mM HEPES, pH 6.8, 0.2 mM TCEP. Representative purified U2AF651,2L and U2AF65–U2AF35-UHM proteins are shown in Supplementary Fig. 1a. Oligonucleotide preparation High-performance liquid chromatography-purified oligonucleotides (sequences shown in Supplementary Fig. 2a) were purchased for crystallization (Integrated DNA Technologies, Inc.). The lyophilized oligonucleotides were diluted in gel filtration buffer for crystallization experiments. The 5′-fluorescein (Fl)-labelled RNAs (AdML: 5′-Fl-CCCUUUUUUUUCC-3′, Py tract of the AdML splicing substrate; 5′-4rU: 5′-Fl-CCUUUUCCCCCCC-3′; 3′-4rU: 5′-Fl-CCCCCCCUUUUCC-3′) for RNA-binding experiments (Dharmacon Research, Inc., Thermo Scientific) was deprotected according to the manufacturer's protocol, vacuum dried and resuspended in nuclease-free water. RNA and RNA–DNA concentrations were calculated using the calculated molar extinction coefficients and absorbance at 260 nm. Fluorescence anisotropy RNA-binding experiments For RNA-binding experiments, purified proteins and RNA were diluted separately \u003e100-fold in binding buffer (100 mM NaCl, 15 mM HEPES, pH 6.8, 0.2 mM TCEP, 0.1 U μl−1 Superase-In (Ambion Life Technologies)). The final RNA concentration in the cuvette was 30 nM. Volume changes during addition of the protein were \u003c10% to minimize dilution effects. The fluorescence anisotropy changes during titration were measured using a FluoroMax-3 spectrophotometer temperature controlled by a circulating water bath at 23 °C. Samples were excited at 490 nm and emission intensities recorded at 520 nm with a slit width of 5 nm. The titrations were repeated three times in succession. Each titration was fit with Graphpad Prism v4.0 to obtain the apparent equilibrium dissociation constant (KD). The apparent equilibrium affinities (KA) are the reciprocal of the KD. The average KD's or KA's and s.e.m. among the three replicates were calculated using Excel and are reported in Figs 3 and 4; Supplementary Figs 1 and 4. The P values from a two-tailed unpaired t-test were calculated using Graphpad Prism v4.0. Transfection, immunoblotting and RT-PCR analyses For transfection experiments, the full-length human U2AF65 cDNA in pCMV6-XL5 (Origene Tech. Inc., clone ID BC008740) was used (WT U2AF65) and in parallel mutated to encode the Q147A/R227A/V254P triple-mutant protein (Mut U2AF65). The pyPY minigene was a gift from M. Carmo-Fonseca (University of Lisbon, Portugal). HEK293T cells (kindly provided by Dr Lata Balakrishnan, originally purchased from ATCC, cat. no. CRL3216) were seeded into 12-well plates (2–4 × 105 cells per well) and grown as monolayers in MEM (Gibco Life Technologies) supplemented with 10% (v/v) of heat-inactivated fetal bovine serum, 1% (v/v) L-glutamine and 1% (v/v) penicillin–streptomycin. After 1 day, the cells were transiently transfected with either 0.5 μg of pyPY plasmid or a mixture of 0.5 μg of U2AF65 variant and 0.5 μg of pyPY plasmid per well using appropriately adjusted Lipofectamine 2000 (Invitrogen Life Technologies) ratio according to the manufacturer's instructions. For immunoblots of WT U2AF65 and Mut U2AF65 expression levels (Supplementary Fig. 5a), transfected or control cells were lysed in radioimmunoprecipitation assay buffer with proteinase and kinase inhibitors. Total protein (20 μg) was separated by SDS–PAGE, and transferred onto polyvinylidene difluoride membranes (Millipore Corp., Billerica, MA, USA) and immunoblotted using mouse monoclonal antibodies directed against U2AF65 (ref.) (MC3, cat. no. U4758 Sigma-Aldrich at 1:500 dilution) or as a control for comparison, GAPDH (glyceraldehyde-3-phosphate dehydrogenase; monoclonal clone 71.1, cat. no. G8795 Sigma-Aldrich at 1:5,000 dilution). Immunoblots were developed using anti-mouse horseradish peroxidase-conjugates (cat. no. U4758 Sigma-Aldrich, Co. at 1:2,500 or 1:10,000 dilutions for GAPDH and U2AF65, respectively) and detected using SuperSignal WestPico chemi-luminescent substrate (Pierce Thermo Scientific Inc.). Blots were imaged using a IS4000MM system (Carestream, Rochester, NY, USA). For size analysis, fluorescent images of the BioRad Precision Plus Dual Color Standards were overlaid directly. For reverse transcription PCR (RT-PCR), the total RNA was isolated 2 days post transfection using the Cells-to-cDNA II kit (Ambion Life Technologies). The RT-PCR reaction comprised 35 cycles (94 °C per 60 s—60 °C per 50 s—72 °C per 60 s) with forward (5′-TGAGGGGAGGTGAATGAGGAG-3′) and reverse (5′-TCCACTGGAAAGACCGCGAAG-3′) primers for the pyPY product or forward (5′-CATGTTCGTCATGGGTGTGAACCA-3′) and reverse (5′-ATGGCATGGACTGTGGTCATGAGT-3′) primers for a GAPDH control. The RT-PCR products were separated by 2% agarose gel electrophoresis and stained with ethidium bromide. The percentages of splice site use were calculated from the background corrected intensities I using the formula 100% × I(py)/[I(py)+I(PY)+I(unspliced)] for py spliced (Fig. 5b,c) or 100% × I(PY)/[I(py)+I(PY)+I(unspliced)] for PY spliced (Supplementary Fig. 5b). The band intensities of four independent biological replicates were measured using ImageQuant software. Crystallization, data collection and structure determination Before crystallization, the purified U2AF651,2L and given oligonucleotide were mixed to achieve respective final concentrations of 1.0 and 1.1 mM and incubated on ice for 20–30 min. For each oligonucleotide, sparse matrix screens of the Jancarik and Kim Crystal Screen(in hanging drop format; Hampton Research, Corp.) and JCSG-Plus (in sitting drop format; Molecular Dimensions) were used to identify initial crystallization conditions, which were obtained from the latter screen and further optimized in hanging drop format. In optimized crystallization experiments, a mixture of sample and reservoir solution (1.2:1 μl) was equilibrated against 700 μl reservoir solution at 4 °C. The oligonucleotide sequences were optimized and the structures were determined as follows: in addition to the previously characterized dU2AF651,2-binding sites for seven nucleotides, the new terminal residues of the U2AF651,2L construct were presumed to contact an additional nucleotide and the crystal packing of a central nucleotide between the RRM1/RRM2 of dU2AF651,2 was presumed to represent one nucleotide. Also considering the known proclivity for deoxy(d)U to co-crystallize with dU2AF651,2 (ref.) and for 5-bromo-dU (5BrdU) to bind a given site of dU2AF651,2 (ref.), we initially designed two 9-mer oligonucleotides (5′-ribose (r)UrUrUrUrU(5BrdU)dUrUrU and 5′-rUrUrUdUdU(5BrdU)dUrUrU) and screened for co-crystallization with U2AF651,2L. The former oligonucleotide failed to produce crystals in these screens. The latter oligonucleotide comprising central dU nucleotides produced diffracting crystals, which were frozen directly from a reservoir comprising 100 mM phosphate–citrate buffer pH 4.2, 40% Peg 300. The structure determined by molecular replacement using Phenix with a data set collected at beamline (BL) 12-2 of the Stanford Synchrotron Radiation Lightsource (SSRL; Menlo Park, CA, USA) (Table 1). The search models comprising each of the individual RRMs bound to two nucleotides were derived from the dU2AF651,2 structure (PDB ID 2G4B) (translation function Z-score equivalent 12.9, log-likelihood gain 528). For comparison, searches with the NMR structure (PDB ID 2YH1) as a search model failed to find a solution. The initial structure revealed a greater number of central nucleotide-binding sites than expected. The oligonucleotide binding register had slipped to place the BrdU in the preferred site, leave the 5′ terminal-binding sites empty, and the terminal nucleotide unbound and disordered. Subsequent oligonucleotides were designed to place BrdU in the preferred site, fill the unoccupied 5′ terminal sites, capture rU at the central sites, and compare rC at the terminal site. The U2AF651,2L protein co-crystals with oligonucleotide 5′-phosphorylated (P)-rUrUdUdUrUdU(BrdU)dU were obtained using a reservoir of 200 mM LiCl, 100 mM sodium citrate pH 4.0, 8% (w/v) polyethylene glycol (PEG) 6,000, 10% (v/v) PEG 300, 10% (v/v) dioxane with 0.1 μl of N,N-bis[3-(D-gluconamido)propyl]deoxy-cholamide (deoxy-BigCHAP) (14 mM) added to the hanging drop and cryoprotected by sequential layering with reservoir solution supplemented with increasing PEG 300 to a final concentration of 26%. Co-crystals with either 5′-(P)rUrUdUrUrU(BrdU)dUdU or 5′-(P)rUrUrUdUrUrU(BrdU)dUrC were obtained from 1 M succinate, 100 mM HEPES, pH 7.0, 1–3% (w/v) PEG monomethylether 2,000. The former was cryoprotected by coating with a 1:1 (v/v) mixture of silicon oil and Paratone-N and the latter by sequential transfer to 21% (v/v) glycerol. Data sets for flash-cooled crystals were collected at 100 K using remote access to SSRL BL12-2. Structures were determined by molecular replacement using the initial U2AF651,2L/rUrUrUdUdU(BrdU)dUrUrU structure as a search model. Consistent sets of free-R reflections were maintained (6% of the total reflections). Models were built using COOT and refined with PHENIX. No non-glycine/non-proline residues were found in the disallowed regions of the Ramachandran plots. Clash scores and Molprobity scores calculated using the program Molprobity were above average. Structure illustrations were prepared using PYMOL. Crystallographic data and refinement statistics are given in Table 1. Sample preparation for single-molecule FRET The U2AF651,2LFRET construct used for smFRET comprises the six histidine and T7 tags from the pET28a vector (Merck), a GGGS linker and U2AF65 residues 113–343. The single cysteine of human U2AF65 was replaced by alanine (C305A), which is a natural amino-acid variation among U2AF65 homologues. Single A181C and Q324C mutations were introduced in each RRM for fluorophore attachment at residues that were carefully selected to meet experimental criteria described in the Results. The U2AF651,2LFRET was purified by the same method as described above for U2AF651,2L protein and binds RNA with similar affinity as U2AF651,2L (Supplementary Fig. 6a). Before labelling, the purified U2AF651,2LFRET protein was incubated with 10 mM DTT on ice for 30 min and then buffer exchanged into Labelling Buffer (100 mM NaCl, 25 mM HEPES pH 7.0, 5 mM EDTA, 0.5 mM tris(2-carboxy-ethyl)phosphine (TCEP)) using Zeba Spin Desalting Columns 7K MWCO (Pierce, ThermoFisher Scientific). To initiate the labelling reaction, 4 μl each of cyanine (Cy)3-Maleimide and Cy5-Maleimide (Combinix, Inc.) stock solutions (10 mM in DMSO) were pre-mixed (total volume 8 μl) and then added to 200 μl of 20 μM protein (final 20:1 molar ratio of dye:protein). The labelling reaction was incubated at room temperature in the dark for 2 h and then quenched by the addition of 10 mM DTT. The labelled protein was separated from excess dye using a Zeba Spin Desalting Column followed by size exclusion chromatography using a pre-packed Superdex-75 10/300 GL (GE Healthcare) column in Labelling Buffer. Our previous experience of conjugating cysteines with maleimide derivatives of fluorophores and suggests that nonspecific modification of aminogroups of proteins with fluorescent dyes under the employed experimental conditions is negligible. Consistent with specific labelling of A181C and Q324C, the labelling efficiencies were ∼60% each for Cy3 and Cy5 as estimated using the dye extinction coefficients (ɛCy3=150,000 M−1 cm−1 at 550 nm, ɛCy5=170,000 M−1 cm−1 at 650 nm) and the calculated extinction coefficient of the U2AF651,2LFRET protein (ɛprot=8,940 M−1 cm−1 at 280 nm), and correcting for the absorbance (A) of the dyes at 280 nm (GE Healthcare, Amersham CyDye Maleimide product booklet): For smFRET experiments with a ‘strong', homogeneous Py tract, we used the prototypical AdML sequence (5′-CCUUUUUUUUCC-3′). To investigate the inter-RRM separation in the presence of a ‘weak' Py tract interrupted by purines, we compared the U2AF651,2L affinity for a purine-interrupted Py tract comprising an rUrUrUrUrU tract that is expected to bind U2AF65 RRM2/inter-RRM linker, a central rArA and an rUrUrUrCrC tract that is expected to bind RRM1. The tandem purines represent a compromise between significant inhibition of U2AF65 binding by longer A interruptions and an approximately five-fold penalty for the rArA mutation in the AdML Py tract (Supplementary Fig. 6b,c). To maintain avidity and provide flanking phosphoryl groups in case of inter-RRM adjustment, we included the 5′-C and 3′-A of parent AdML sequence, which are respective low-affinity nucleotides for binding RRM2 and RRM1 (ref.), in the final rArA-interrupted RNA oligonucleotide (5′-rCrUrUrUrUrUrArArUrUrUrCrCrA-3′). For the reversed immobilization of RNA via a complementary biotinyl-DNA primer experiment, the AdML Py-tract RNA was extended to include the DNA counterpart of downstream AdML intron/exon sequences that were complementary to the biotinyl-DNA primer. To increase separation from the slide surface, a hexaethylene glycol linker (18PEG) was inserted between the AdML Py-tract RNA and the tethered DNA duplex. The tethered oligonucleotide sequences included: 5′-rCrCrUrUrUrUrUrUrUrUrCrC/18PEG/dAdCdAdGdCdTdCdGdCdG-dGdTdTdGdAdGdGdAdCdAdA-3′ annealed to 5′-biotinyl-dTdTdGdTdCdCdTdCdAdA-dCdCdGdCdGdAdGdCdTdGdT-3' (purchased with high-performance liquid chromatography purification from Integrated DNA Technologies). Single-molecule FRET data acquisition and analysis The smFRET measurements were carried out at room temperature in 50 mM HEPES, pH 7.4, 100 mM NaCl. The imaging buffer also contained an oxygen-scavenging system (0.8 mg ml−1 glucose oxidase, 0.625% glucose, 0.02 mg ml−1 catalase), 1.5 mM Trolox (used to eliminate Cy5 blinking) and 6 mM β-mercaptoethanol. The sample chamber was assembled from quartz microscope slides and glass cover slips coated with a mixture of m-PEG and biotin-PEG and pre-treated with neutravidin (0.2 mg ml−1). Surface tethering of doubly labelled U2AF651,2LFRET(Cy3/Cy5) via its His-tag (Fig. 6c–f,i,j; Supplementary Fig. 7a,b) was achieved by pre-incubating the sample chamber with 50 nM biotinyl-NTA resin (Biotin-X NTA, Biotium), pre-loaded with three-fold excess NiSO4) for 20 min before addition of 5 nM U2AF651,2LFRET(Cy3/Cy5). After 10 min, unbound sample was removed by washing the sample chamber with imaging buffer. The AdML RNA ligand was added to the imaging buffer at a concentration of 5 μM (100-fold higher than the measured KD value), whereas the rArA-interrupted RNA was added at a concentration of 10 μM. Alternatively, to detect binding of doubly labelled U2AF651,2LFRET(Cy3/Cy5) to surface-tethered RNA ligand (Fig. 6g,h Supplementary Fig. 7c–g), 10 nM AdML RNA (pre-annealed to biotinyl-DNA primer) was incubated in the neutravidin-treated sample chamber for 20 min, and 1 nM U2AF651,2LFRET(Cy3/Cy5) was then added to the imaging buffer. Single-molecule FRET measurements were taken as previously described. An Olympus IX71 inverted microscope, equipped with a UPlanApo 60x/1.20w objective lens, a 532 nm laser (Spectra-Physics) for excitation of Cy3 dyes, and a 642 nm laser (Spectra-Physics) for excitation of Cy5 dyes was used. Total internal reflection (TIR) was obtained by a quartz prism (ESKMA Optics). Fluorescence emission was split into Cy3 and Cy5 fluorescence using a dual view imaging system DV2 (Photometrics) equipped with a 630 nm dichroic mirror and recorded via an Andor iXon+ EMCCD camera. Movies were recorded using the Single software (downloaded from Prof. Taekjip Ha's laboratory website at the University of Illinois at Urbana-Champaign, physics.illinois.edu/cplc/software), with the exposure time set at 100 ms. We typically took up to five 5-minute-long movies while imaging different sections of the slide for each sample. Before each measurement, we checked for non-specific binding by adding doubly-labeled U2Fret to the slide in the absence of neutravidin and imaging the slide. Non-specific binding was virtually absent. Collected data sets were processed with IDL and Matlab softwares, using scripts downloaded from a freely available source: physics.illinois.edu/cplc/software. Apparent FRET efficiencies (Eapp) were calculated from the emission intensities of donor (ICy3) and acceptor (ICy5) as follows: Eapp=ICy5/(ICy5+ICy3). The FRET distribution histograms were built from traces that showed single-step photobleaching in both Cy3 and Cy5 signals using a Matlab script generously provided by Prof. Peter Cornish (University of Missouri, Columbia). Anti-correlated changes in donor and acceptor intensities with constant sum of intensities indicated the presence of an energy transfer in single molecules labelled with one donor and one acceptor dye. All histograms were smoothed with a five-point window and plotted using Origin software (Origin Lab Co). Idealization of FRET trajectories was done using the hidden Markov model algorithms via HaMMy software (http://bio.physics.illinois.edu/HaMMy.asp). Transition density plots were generated from transitions detected in idealized FRET trajectories obtained by HaMMy fit of raw FRET traces via Matlab. Frequency of transitions from starting FRET efficiency value (x-axis) to ending FRET efficiency value (y-axis) was represented by a heat map. The range of FRET efficiencies from 0 to 1 was separated in 200 bins. The resulting heat map was normalized to the most populated bin in the plot; the lower- and upper-bound thresholds were set to 20% and 100% of the most populated bin, respectively. The surface contour plots were generated as follows: the individual single-molecule FRET traces (for example, Fig. 6g of the main text and Supplementary Fig. 7e,f) were post synchronized at the first time point showing non-zero (\u003e0.15) FRET efficiency, corresponding to binding. The time range (x-axis, 0–10 s) was separated into 100 bins. The FRET efficiency range (y-axis, 0–1 FRET) was separated into 100 bins. A heat map is used to represent the frequency of sampling of each FRET state over time; frequency in each bin was normalized to the most populated bin in the plot with lower- and upper-bound thresholds set at 10% and 80% of the most populated bin, respectively. Additional information Accession codes: Coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 5EV1, 5EV2, 5EV3 and 5EV4 for respective U2AF651,2L-oligonucleotide structures (i)–(iv). How to cite this article: Agrawal, A. A. et al. An extended U2AF65–RNA-binding domain recognizes the 3′ splice site signal. Nat. Commun. 7:10950 doi: 10.1038/ncomms10950 (2016). Supplementary Material Alternative isoform regulation in human tissue transcriptomes  Pre-mRNA splicing in disease and therapeutics Acquired mutations that affect pre-mRNA splicing in hematologic malignancies and solid tumors Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly The splicing factor BBP interacts specifically with the pre-mRNA branchpoint sequence UACUAAC A cooperative interaction between U2AF65 and mBBP/SF1 facilitates branchpoint region recognition Functional recognition of the 3' splice site AG by the splicing factor U2AF35 Both subunits of U2AF recognize the 3' splice site in Caenorhabditis elegans Inhibition of msl-2 splicing by Sex-lethal reveals interaction between U2AF35 and the 3' splice site AG Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins Cloning and domain structure of the mammalian splicing factor U2AF U2AF65 adapts to diverse pre-mRNA splice sites through conformational selection of specific and promiscuous RNA recognition motifs Structural basis of polypyrimidine tract recognition by the essential pre-mRNA splicing factor, U2AF65 Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF Structuring of the 3' splice site by U2AF65 Interaction of U2AF65 RS region with pre-mRNA branch point and promotion of base pairing with U2 snRNA A pathway of sequential arginine-serine-rich domain-splicing signal interactions during mammalian spliceosome assembly Structure-guided U2AF65 variant improves recognition and splicing of a defective pre-mRNA Protein-protein interaction and quaternary structure Solution conformation and thermodynamic characteristics of RNA binding by the splicing factor U2AF65 Solution structures of the first and second RNA-binding domains of human U2 small nuclear ribonucleoprotein particle auxiliary factor (U2AF65) In vivo requirement of the small subunit of U2AF for recognition of a weak 3' splice site A broad range of conformations contribute to the solution ensemble of the essential splicing factor U2AF65 Transient electrostatic interactions dominate the conformational equilibrium sampled by multidomain splicing factor U2AF65: a combined NMR and SAXS study Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single molecule data A practical guide to single-molecule FRET Landscape of genetic lesions in 944 patients with myelodysplastic syndromes Frequent pathway mutations of splicing machinery in myelodysplasia A novel peptide recognition mode revealed by the X-ray structure of a core U2AF35/U2AF65 heterodimer Induced fit, conformational selection and independent dynamic segments: an extended view of binding events Conformational selection or induced fit: a flux description of reaction mechanism Illuminating the mechanistic roles of enzyme conformational dynamics Intrinsic motions along an enzymatic reaction trajectory A role for both conformational selection and induced fit in ligand binding by the LAO protein Conformational selection and induced fit mechanism underlie specificity in noncovalent interactions with ubiquitin A single-molecule dissection of ligand binding to a protein with intrinsic dynamics Mg(2+) shifts ligand-mediated folding of a riboswitch from induced-fit to conformational selection Splicing regulation: from a parts list of regulatory elements to an integrated splicing code Revised UV extinction coefficients for nucleoside-5'-monophosphates and unpaired DNA and RNA Targeting of U2AF65 to sites of active splicing in the nucleus Sparse matrix sampling: a screening method for crystallization of proteins Crystallization and preliminary X-ray analysis of U2AF65 variant in complex with a polypyrimidine tract analogue by use of protein engineering PHENIX: a comprehensive Python-based system for macromolecular structure solution Coot: model-building tools for molecular graphics MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes  Following movement of domain IV of elongation factor G during ribosomal translocation Analysis of single-molecule FRET trajectories using hidden Markov modeling Author contributions A.A.A. performed crystallization, refinement, molecular biology and most RNA-binding experiments. E.S. performed smFRET experiments. R.C. labelled protein and S.H. completed a subset of RNA-binding experiments. C.L.K. cryoprotected crystals, collected crystallographic data and built structures. J.L.J. performed molecular replacement and completed structure refinements. M.R.G. and C.L.K. conceived the study. C.L.K. and D.N.E. designed the experiments. C.L.K., D.N.E. and E.S. wrote the paper with input from J.L.J. and A.A.A. The intact U2AF65 RRM1/RRM2-containing domain and flanking residues are required for binding contiguous Py tracts. (a) Domain organization of full-length (fl) U2AF65 and constructs used for RNA binding and structural experiments. The N- and C-terminal residue numbers are indicated. An internal deletion (d, Δ) of residues 238–257 removes a portion of the inter-RRM linker from the dU2AF651,2 and dU2AF651,2L constructs. (b) Comparison of the apparent equilibrium affinities of various U2AF65 constructs for binding the prototypical AdML Py tract (5′-CCCUUUUUUUUCC-3′). The flU2AF65 protein includes a heterodimerization domain of the U2AF35 subunit to promote solubility and folding. The apparent equilibrium dissociation constants (KD) for binding the AdML 13mer are as follows: flU2AF65, 30±3 nM; U2AF651,2L, 35±6 nM; U2AF651,2, 3,600±300 nM. (c) Comparison of the RNA sequence specificities of flU2AF65 and U2AF651,2L constructs binding C-rich Py tracts with 4U's embedded in either the 5′- (light grey fill) or 3′- (dark grey fill) regions. The KD's for binding 5′-CCUUUUCCCCCCC-3′ are: flU2AF65, 41±2 nM; U2AF651,2L, 31±3 nM. The KD's for binding 5′-CCCCCCCUUUUCC-3′ are: flU2AF65, 414±12 nM; U2AF651,2L, 417±10 nM. Bar graphs are hatched to match the constructs shown in a. The average apparent equilibrium affinity (KA) and s.e.m. for three independent titrations are plotted. The P values from two-tailed unpaired t-tests with Welch's correction are indicated as follows: **P\u003c0.01; NS, not significant, P\u003e0.05. The purified protein and average fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 1. RRM, RNA recognition motif; RS, arginine-serine rich; UHM, U2AF homology motif; ULM, U2AF ligand motif. Structures of U2AF651,2L recognizing a contiguous Py tract. (a) Alignment of oligonucleotide sequences that were co-crystallized in the indicated U2AF651,2L structures. The regions of RRM1, RRM2 and linker contacts are indicated above by respective black and blue arrows from N- to C-terminus. For clarity, we consistently number the U2AF651,2L nucleotide-binding sites from one to nine, although in some cases the co-crystallized oligonucleotide comprises eight nucleotides and as such leaves the first binding site empty. The prior dU2AF651,2 nucleotide-binding sites are given in parentheses (site 4' interacts with dU2AF65 RRM1 and RRM2 by crystallographic symmetry). Italics, disordered in the structure. (b) Stereo views of a ‘kicked' 2|Fo|−|Fc| electron density map contoured at 1σ for the inter-RRM linker, N- and C-terminal residues (blue) or bound oligonucleotide of a representative U2AF651,2L structure (structure iv, bound to 5′-(P)rUrUrUdUrUrU(BrdU)dUrC) (magenta). (c) Cartoon diagram of this structure. Crystallographic statistics are given in Table 1 and the overall conformations of U2AF651,2L and prior dU2AF651,2/U2AF651,2 structures are compared in Supplementary Fig. 2. BrdU, 5-bromo-deoxy-uridine; d, deoxy-ribose; P-, 5′-phosphorylation; r, ribose. Representative views of the U2AF651,2L interactions with each new nucleotide of the bound Py tract. New residues of the U2AF651,2L structures are coloured a darker shade of blue, apart from residues that were tested by site-directed mutagenesis, which are coloured yellow. The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. (i) Bar graph of apparent equilibrium affinities (KA) of the wild type (blue) and the indicated mutant (yellow) U2AF651,2L proteins binding the AdML Py tract (5′-CCCUUUUUUUUCC-3′). The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; R227A, 166±2 nM; V254P, 137±10 nM; Q147A, 171±21 nM. The average KA and s.e.m. for three independent titrations are plotted. The P values from two-tailed unpaired t-tests with Welch's correction are indicated as follows: **P\u003c0.01; *P\u003c0.05; NS, not significant, P\u003e0.05. The average fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 4a–c. The U2AF65 linker/RRM and inter-RRM interactions. (a) Contacts of the U2AF65 inter-RRM linker with the RRMs. A semi-transparent space-filling surface is shown for the RRM1 (green) and RRM2 (light blue). Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. Other linker residues are coloured either dark blue for new residues in the U2AF651,2L structure or light blue for the remaining inter-RRM residues. The central panel shows an overall view with stick diagrams for mutated residues; boxed regions are expanded to show the C-terminal (bottom left) and central linker regions (top) at the inter-RRM interfaces, and N-terminal linker region contacts with RRM1 (bottom right). (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; 3Gly, 47±4 nM; 5Gly, 61±3 nM; 12Gly, 88±21 nM; NLALA, 45±3 nM; dU2AF651,2L, 123±5 nM; dU2AF651,2, 5000±100 nM; 3Mut, 5630±70 nM. The average KA and s.e.m. for three independent titrations are plotted. The P values from two-tailed unpaired t-tests with Welch's correction are indicated as follows: **P\u003c0.01; *P\u003c0.05; NS, not significant, P\u003e0.05. The fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 4d–j. (c) Close view of the U2AF65 RRM1/RRM2 interface following a two-fold rotation about the x-axis relative to a. U2AF65 inter-domain residues are important for splicing a representative pre-mRNA substrate in human cells. (a) Schematic diagram of the pyPY reporter minigene construct comprising two alternative splice sites preceded by either the weak IgM Py tract (py) or the strong AdML Py tract (PY) (sequences inset). (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). The average percentages and s.d.'s are given among four independent biological replicates. ****P\u003c0.0001 for two-tailed unpaired t-test with Welch's correction. Protein overexpression and qRT-PCR results are shown in Supplementary Fig. 5. RNA binding stabilizes the side-by-side conformation of U2AF65 RRMs. (a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of ‘side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or ‘closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2. The U2AF651,2LFRET proteins were doubly labelled at A181C/Q324C such that a mixture of Cy3/Cy5 fluorophores are expected to be present at each site. (c–f,i,j) The U2AF651,2LFRET(Cy3/Cy5) protein was immobilized on the microscope slide via biotin-NTA/Ni+2 (orange line) on a neutravidin (black X)-biotin-PEG (orange triangle)-treated surface and imaged either in the absence of ligands (c,d), in the presence of 5 μM AdML Py-tract RNA (5′-CCUUUUUUUUCC-3′) (e,f), or in the presence of 10 μM adenosine-interrupted variant RNA (5′-CUUUUUAAUUUCCA-3′) (i,j). In g and h, the immobilization protocol was reversed. The untethered U2AF651,2LFRET(Cy3/Cy5) protein (1 nM) was added to AdML RNA–polyethylene-glycol-linker–DNA oligonucleotide (10 nM), which was immobilized on the microscope slide by annealing with a complementary biotinyl-DNA oligonucleotide (black vertical line). Typical single-molecule FRET traces (c,e,g,i) show fluorescence intensities from Cy3 (green) and Cy5 (red) and the calculated apparent FRET efficiency (blue). Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). N is the number of single-molecule traces compiled for each histogram. Schematic models of U2AF65 recognizing the Py tract. (a) Diagram of the U2AF65, SF1 and U2AF35 splicing factors bound to the consensus elements of the 3′ splice site. A surface representation of U2AF651,2L is shown bound to nine nucleotides (nt); the relative distances and juxtaposition of the branch point sequence (BPS) and consensus AG dinucleotide at the 3′ splice site are unknown. MDS-relevant mutated residues of U2AF65 are shown as yellow spheres (L187 and M144). (b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the ‘closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to ∼0.45 FRET value, which is consistent with ‘open', side-by-side RRMs such as the U2AF651,2L crystal structures. Alternatively, a conformation of U2AF65 corresponding to ∼0.45 FRET value can directly bind to RNA; RNA binding stabilizes the ‘open', side-by-side conformation and thus shifts the U2AF65 population towards the ∼0.45 FRET value. RRM1, green; RRM2, pale blue; RRM extensions/linker, blue. Crystallographic data and refinement statistics*. Structure\tU2AF651,2L with rUrUrUdUdU(BrdU)dUrUrU\tU2AF651,2L with (P)rUrUdUdUrUdU(BrdU)dU\tU2AF651,2L with (P)rUrUdUrUrU(BrdU)dUdU\tU2AF651,2L with (P)rUrUrUdUrUrU(BrdU)dUrC\t \tData collection\t(i)\t(ii)\t(iii)\t(iv)\t \tSpace group\tC2221\tC2221\tP212121\tP212121\t \tUnit cell (Å) a,b,c\t62.1, 114.2, 59.4\t61.9, 115.1, 59.5\t43.4, 62.2, 77.4\t43.5, 63.4, 77.7\t \tResolution limits (Å)\t32.46–2.04\t32.57–1.86\t38.71–1.50\t38.83–1.57\t \tCompleteness (%)\t95.5 (78.3)\t98.7 (95.9)\t98.2 (69.8)\t98.3 (71.7)\t \tRedundancy\t4.6 (4.1)\t4.3 (4.2)\t6.1 (3.0)\t6.2 (3.1)\t \tI/σ(I)\t21.2 (4.2)\t24.6 (4.6)\t38.0 (6.5)\t42.9 (6.9)\t \tRsym (%)\t3.9 (32.1)\t3.9 (30.3)\t2.4 (14.8)\t2.2 (14.9)\t \tRefinement\t \t No. reflections (work/test)\t12,124/1,055\t17,870/1,456\t31,802/1,996\t28,162/2,000\t \t Rwork/Rfree (%)\t17.3/22.8\t15.1/18.8\t15.3/18.6\t15.4/17.6\t \tNo. atoms\t \t Protein\t2,982\t3,052\t2,986\t2,978\t \t Oligonucleotide\t214\t209\t198\t255\t \t Water\t118\t203\t263\t177\t \tBond r.m.s.d.\t \t Bond lengths (Å)\t0.013\t0.010\t0.008\t0.009\t \t Bond angles (°)\t1.32\t1.1\t1.05\t1.05\t \t\u003cB\u003e factors (Å2)\t \t Protein\t46.4\t27.4\t26.3\t26.7\t \t Oligonucleotide\t61.8\t35.2\t24.5\t30.5\t \t Water\t45.2\t35.2\t30.7\t29.8\t \t All available crystallographic data was used for refinement. *A single crystal was used for each structure. Values from the highest resolution shell are given in parentheses: 2.15–2.04; 1.90–1.86; 1.53–1.50; 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+[{"sourceid":"4792962","sourcedb":"","project":"","target":"","text":"A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome Biogenesis of the 20S proteasome is tightly regulated. The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic. Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease.  The proteasome, an essential molecular machine, is a threonine protease, but the evolution and the components of its proteolytic centre are unclear. Here, the authors use structural biology and biochemistry to investigate the role of proteasome active site residues on maturation and activity. The 20S proteasome core particle (CP) is the key non-lysosomal protease of eukaryotic cells. Its seven different α and seven different β subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder. While the inactive α subunits build the two outer rings, the β subunits form the inner rings. Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides. In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1). Release of the propeptides creates a functionally active CP that cleaves proteins into short peptides. Although the chemical nature of the substrate-binding channel and hence substrate preferences are unique to each of the distinct active β subunits, all active sites employ an identical reaction mechanism to hydrolyse peptide bonds. Nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of the scissile peptide bond creates a first cleavage product and a covalent acyl-enzyme intermediate. Hydrolysis of this complex by the addition of a nucleophilic water molecule regenerates the enzyme and releases the second peptide fragment. The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage. This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed. An alternative candidate for deprotonating the Thr1 hydroxyl group is the side chain of Lys33 as it is within hydrogen-bonding distance to Thr1OH (2.7 Å). In principle it could function as the general base during both autocatalytic removal of the propeptide and protein substrate cleavage. Here we provide experimental evidences for this distinct view of the proteasome active-site mechanism. Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome. Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays. Results Inactivation of proteasome subunits by T1A mutations Proteasome-mediated degradation of cell-cycle regulators and potentially toxic misfolded proteins is required for the viability of eukaryotic cells. Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites. Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1). These results indicate that the β1 and β2 proteolytic activities are not essential for cell survival. By contrast, the T1A mutation in subunit β5 has been reported to be lethal or nearly so. Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1). Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). The extremely weak growth of the β5-T1A mutant pp cis described by Chen and Hochstrasser compared with the inviability reported by Heinemeyer et al. prompted us to analyse this discrepancy. Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods). We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts. The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1). Propeptide conformation and triggering of autolysis In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature β-subunit domains. For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2). Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.). Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1). In subunit β1, we found that Gly(-1) indeed forms a sharp turn, which relaxes on prosegment cleavage (Fig. 1a and Supplementary Fig. 2a). However, the γ-turn conformation and the associated hydrogen bond initially proposed is for geometric and chemical reasons inappropriate and would not perfectly position the carbonyl carbon atom of Gly(-1) for nucleophilic attack by Thr1. Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. Thr(-2) positions Gly(-1)O via hydrogen bonding (∼2.8 Å) in a perfect trajectory for the nucleophilic attack by Thr1Oγ (Fig. 1b and Supplementary Fig. 2b). Next, we examined the position of the β5 propeptide in the β5-T1A-K81R mutant. Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet. Nonetheless, the carbonyl carbon of Gly(-1) would be ideally placed for nucleophilic attack by Thr1Oγ (Fig. 1c and Supplementary Fig. 2c,d). As the K81R mutation is located far from the active site (Thr1Cα–Arg81Cα: 24 Å), any influence on propeptide conformation can be excluded. Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis. Processing of β-subunit precursors requires deprotonation of Thr1OH; however, the general base initiating autolysis is unknown. Remarkably, eukaryotic proteasomal β5 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a). As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). In agreement, the chymotrypsin-like (ChT-L) activity of H(-2)N and H(-2)F mutant yCPs was impaired in situ and in vitro (Supplementary Fig. 3c). Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO–FC electron densities. Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned. This observation indicates a mixture of processed and unprocessed β5 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base. Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. As expected from β5-T1A mutants, the yeasts show severe growth phenotypes, with minor variations (Supplementary Fig. 4a and Table 1). We determined crystal structures of the β5-H(-2)L-T1A, β5-H(-2)T-T1A and the β5-H(-2)A-T1A-K81R mutants (Supplementary Table 1). For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d). By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable. Nevertheless, both Leu(-2) and Thr(-2) were found to occupy the S1 specificity pocket formed by Met45 (Fig. 2a,b and Supplementary Fig. 4f–h). This result proves that the naturally occurring His(-2) of the β5 propeptide does not stably fit into the S1 site. Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. Although we observed fragments of 2FO–FC electron density in the β1 active site, the data were not interpretable. Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). Notably, the 2FO–FC electron-density map displays a different orientation for the β2 propeptide than has been observed for the β2-T1A proteasome. In particular, Val(-2) is displaced from the S1 site and Gly(-1) is severely shifted (movement of the carbonyl oxygen atom of 3.8 Å), thereby preventing nucleophilic attack of Thr1 (Fig. 2d and Supplementary Fig. 4j,k). These results further confirm that correct positioning of the active-site residues and Gly(-1) is decisive for the maturation of the proteasome. The active site of the proteasome Proton shuttling from the proteasomal active site Thr1OH to Thr1NH2 via a nucleophilic water molecule was suggested to initiate peptide-bond hydrolysis. However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1Oγ. Instead, Lys33NH2, which is in hydrogen-bonding distance to Thr1Oγ (2.7 Å) in all catalytically active β subunits (Fig. 3a,b), was proposed to serve as the proton acceptor. Owing to its likely protonation at neutral pH, however, it was assumed not to act as the general base. A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site. Twenty years later, with a plethora of yCP X-ray structures in hand, we decided to re-analyse the active site of the proteasome and to resolve the uncertainty regarding the nature of the general base. Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1). The phenotype of the β5-K33A mutant was however less pronounced than for the β5-T1A-K81R yeast (Fig. 4a). This discrepancy in growth was traced to an additional point mutation L(-49)S in the β5-propeptide of the β5-K33A mutant (see also Supplementary Note 1). Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. This structural alteration destroys active-site integrity and abolishes catalytic activity of the β5 active site (Supplementary Fig. 5a). Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans). The Thr1 N terminus of this mutant is not blocked by the propeptide, yet its catalytic activity is reduced by ∼83% (Supplementary Fig. 6b). Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). This water hydrogen bonds also to Arg19O (∼3.0 Å) and Asp17Oδ (∼3.0 Å), and thereby presumably enables residual activity of the mutant. Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis. Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1). While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e). The conservative mutation of Asp17 to Asn in subunit β5 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1). Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP. As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. Autolysis and residual catalytic activity of the β5-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1. In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis. Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b). On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d). This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2. To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit. Asp166Oδ is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis. The β5-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1). X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a). Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing. The hydrogen bonds involving Ser169OH are intact and may account for residual substrate turnover. Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c). In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation. In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166. Whereas Asn can to some degree replace Asp166 due to its carbonyl group in the side chain, Ala at this position was found to prevent both autolysis and catalysis. These results suggest that Asp166 and Ser129 function as a proton shuttle and affect the protonation state of Thr1N during autolysis and catalysis. Substitution of the active-site Thr1 by Cys Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum. In yeast, this mutation causes a strong growth defect (Fig. 4a and Table 1), although the propeptide is hydrolysed, as shown here by its X-ray structure. In one of the two β5 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c). His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization. Owing to the unequal positions of the two β5 subunits within the CP in the crystal lattice, maturation and propeptide displacement may occur at different timescales in the two subunits. Despite propeptide hydrolysis, the β5-T1C active site is catalytically inactive (Fig. 4b and Supplementary Fig. 9a). In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. Moreover, the structural data reveal that the thiol group of Cys1 is rotated by 74° with respect to the hydroxyl side chain of Thr1 (Fig. 4f and Supplementary Fig. 9b). This presumably results from the larger radius of the sulfur atom compared with oxygen. Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1. Notably, the 2FO–FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered. Together, these observations suggest that efficient peptide-bond hydrolysis requires that Lys33NH2 hydrogen bonds to the active site nucleophile. The benefit of Thr over Ser as the active-site nucleophile All proteasomes strictly employ threonine as the active-site residue instead of serine. To investigate the reason for this singularity, we analysed a β5-T1S mutant, which is viable but suffers from growth defects (Fig. 4a and Table 1). Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c). By contrast, turnover of the substrate Z-GGL-pNA, used to monitor ChT-L activity in situ but in a less quantitative fashion, is not detectably impaired (Supplementary Fig. 9a). Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). However, the apo crystal structure revealed that Ser1Oγ is turned away from the substrate-binding channel (Fig. 4g). Compared with Thr1Oγ in WT CP structures, Ser1Oγ is rotated by 60°. This renders it unavailable for direct nucleophilic attack onto incoming substrates and first requires its reorientation, which is expected to delay substrate turnover. Because both conformations of Ser1Oγ are hydrogen-bonded to Lys33NH2 (Fig. 4h), the relay system is capable of hydrolysing peptide substrates, albeit at lower rates compared with Thr1. The active-site residue Thr1 is fixed in its position, as its methyl group is engaged in hydrophobic interactions with Thr3 and Ala46 (Fig. 4h). Consequently, the hydroxyl group of Thr1 requires no reorientation before substrate cleavage and is thus more catalytically efficient than Ser1. In agreement, at an elevated growing temperature of 37 °C the T1S mutant is unable to grow (Fig. 4a). In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5). Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors. Notably, the affinity of the tetrapeptide carfilzomib is less impaired, as it is better stabilized in the substrate-binding channel than the dipeptide bortezomib, which lacks a defined P3 site and has only a few interactions with the surrounding protein. Hence, the mean residence time of carfilzomib at the active site is prolonged and the probability to covalently react with Ser1 is increased. Considered together, these results provide a plausible explanation for the invariance of threonine as the active-site nucleophile in proteasomes in all three domains of life, as well as in proteasome-like proteases such as HslV (ref.). Discussion The 20S proteasome CP is the major non-lysosomal protease in eukaryotic cells, and its assembly is highly organized. The β-subunit propeptides, particularly that of β5, are key factors that help drive proper assembly of the CP complex. In addition, they prevent irreversible inactivation of the Thr1 N terminus by N-acetylation. By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli. In eukaryotes, deletion of or failure to cleave the β1 and β2 propeptides is well tolerated. However, removal of the β5 prosegment or any interference with its cleavage causes severe phenotypic defects. These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP. Here we have described the atomic structures of various β5-T1A mutants, which allowed for the first time visualization of the residual β5 propeptide. Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1. From these data we conclude that only the positioning of Gly(-1) and Thr1 as well as the integrity of the proteasomal active site are required for autolysis. In this regard, inappropriate N-acetylation of the Thr1 N terminus cannot be removed by Thr1Oγ due to the rotational freedom and flexibility of the acetyl group. The propeptide needs some anchoring in the substrate-binding channel to properly position Gly(-1), but this seems to be independent of the orientation of residue (-2). Autolytic activation of the CP constitutes one of the final steps of proteasome biogenesis, but the trigger for propeptide cleavage had remained enigmatic. On the basis of the numerous CP:ligand complexes solved during the past 18 years and in the current study, we provide a revised interpretation of proteasome active-site architecture. We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å). Analogously to the proteasome, a Thr–Lys–Asp triad is also found in L-asparaginase. Thus, specific protein surroundings can significantly alter the chemical properties of amino acids such as Lys to function as an acid–base catalyst. In this new view of the proteasomal active site, the positively charged Thr1NH3+-terminus hydrogen bonds to the amide nitrogen of incoming peptide substrates and stabilizes as well as activates them for the endoproteolytic cleavage by Thr1Oγ (Fig. 3d). Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.). Furthermore, opening of the β-lactone compound omuralide by Thr1 creates a C3-hydroxyl group, whose proton originates from Thr1NH3+. The resulting uncharged Thr1NH2 is hydrogen-bridged to the C3-OH group. In agreement, acetylation of the Thr1 N terminus irreversibly blocks hydrolytic activity, and binding of substrates is prevented for steric reasons. By acting as a proton donor during catalysis, the Thr1 N terminus may also favour cleavage of substrate peptide bonds (Fig. 3d). In all proteases, collapse of the tetrahedral transition state results in selective breakage of the substrate amide bond, while the covalent interaction between the substrate and the enzyme persists. Cleavage of the scissile peptide bond requires protonation of the emerging free amine, and in the proteasome, the Thr1 amine group is likely to assume this function. Analogously, Thr1NH3+ might promote the bivalent reaction mode of epoxyketone inhibitors by protonating the epoxide moiety to create a positively charged trivalent oxygen atom that is subsequently nucleophilically attacked by Thr1NH2. During autolysis the Thr1 N terminus is engaged in a hydroxyoxazolidine ring intermediate (Fig. 3d), which is unstable and short-lived. Breakdown of this tetrahedral transition state releases the Thr1 N terminus that is protonated by aspartic acid 166 via Ser129OH to yield Thr1NH3+. The residues Ser129 and Asp166 are expected to increase the pKa value of Thr1N, thereby favouring its charged state. Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. The mutation D166N lowers the pKa of Thr1N, which is thus more likely to exist in the uncharged deprotonated state (Thr1NH2). This renders the N terminus less suitable to stabilize substrates and to protonate the first cleavage product during catalysis, although it favours its ability to act as a nucleophile. This interpretation agrees with the strongly reduced catalytic activity of the β5-D166N mutant on the one hand, and the ability to react readily with carfilzomib on the other. Hence, the proteasome can be viewed as having a second triad that is essential for efficient proteolysis. While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1. In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. Consequently, efficient substrate turnover or covalent modification by ligands is prevented. However, owing to Cys being a strong nucleophile, the propeptide can still be cleaved off over time. While only one single turnover is necessary for autolysis, continuous enzymatic activity is required for significant and detectable substrate hydrolysis. Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally. Activity assays with the β5-T1S mutant revealed reduced turnover of Suc-LLVY-AMC. We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib. Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes. Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ). Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h). Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity. Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site. The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes. Methods Yeast mutagenesis Site-directed mutagenesis was performed by standard techniques using oligonucleotides listed in Supplementary Table 2. The pre2/doa3 (β5) mutant alleles in the centromeric, TRP1- or LEU2-marked shuttle vectors YCplac22 and pRS315, respectively, were verified by sequencing and subsequently introduced into the yeast strains MHY784 (ref.) or YWH20 (ref.), which express WT PRE2 from a URA3-marked plasmid. Counter-selection against the URA3 marker with 5-fluoroorotic acid yielded strains expressing only the mutant forms of β5. The strain producing a processed β5-T1A variant and the β5 propeptide in trans is a derivative of YWH212 (ref.). It carries an additional deletion of the NAT1 gene to avoid N-acetylation of Ala1; this strain exhibits extremely slow growth rates and served for crystallographic analysis only. All strains used in this study are listed in Supplementary Table 3. Purification of yeast proteasomes Yeast strains were grown in 18-l cultures at 30 °C in YPD into early stationary phase, and the yCPs were purified according to published procedures. In brief, 120 g yeast cells were solubilized in 150 ml of 50 mM KH2PO4/K2HPO4 buffer (pH 7.5) and disrupted with a French press. Cell debris were removed by centrifugation for 30 min at 21,000 r.p.m. (4 °C). The resulting supernatant was filtered and ammonium sulfate (saturated solution) was added to a final concentration of 30% (v/v). This solution was loaded on a Phenyl Sepharose 6 Fast Flow column (GE Healthcare) pre-equilibrated with 1 M ammonium sulfate in 20 mM KH2PO4/K2HPO4 (pH 7.5). CPs were eluted by applying a linear gradient from 1 to 0 M ammonium sulfate. Proteasome-containing fractions were pooled and loaded onto a hydroxyapatite column (Bio-Rad) equilibrated with 20 mM KH2PO4/K2HPO4 (pH 7.5). Elution of the CPs was achieved by increasing the phosphate buffer concentration from 20 to 500 mM. Anion-exchange chromatogaphy (Resource Q column (GE Healthcare), elution gradient from 0 to 500 mM sodium chloride in 20 mM Tris-HCl (pH 7.5)) and subsequent size-exclusion chromatography (Superose 6 10/300 GL (GE Healthcare), 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl) resulted in pure CPs for crystallization and activity assays. Fluorescence-based activity assay ChT-L (β5) activity of CPs was monitored by fluorescence spectroscopy using the model substrate Suc-LLVY-AMC. Purified yCPs (66 nM in 100 mM Tris-HCl, pH 7.5) were incubated with 300 μM substrate for 1 h at room temperature or 37 °C. The reactions were stopped by diluting samples 1:10 in 20 mM Tris-HCl, pH 7.5. AMC fluorophores released by proteasomal activity were measured in triplicate with a Varian Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies) at λexc=360 nm and λem=460 nm. Inhibition assays Purified yCPs were mixed with dimethylsulfoxide as a control or serial dilutions of inhibitor and incubated for 45 min at room temperature. A final concentration of yCP of 66 nM was used. After addition of the peptide substrate Suc-LLVY-AMC to a final concentration of 200 μM and incubation for 1 h at room temperature, the reaction was stopped by diluting the samples 1:10 in 20 mM Tris-HCl, pH 7.5. AMC fluorophores released by residual proteasomal activity were measured in triplicate at λexc=360 nm and λem=460 nm. Relative fluorescence units were normalized to the dimethylsulfoxide-treated control. The calculated residual activities were plotted against the logarithm of the applied inhibitor concentration and fitted with GraphPad Prism 5. The IC50 value, the ligand concentration that leads to 50% inhibition of the enzymatic activity, was deduced from the fitted data. Crystallization and structure determination Mutant yCPs were crystallized as previously described for WT 20S proteasomes. Crystals were grown at 20 °C using the hanging drop vapour diffusion method. Drops contained a 1:1 mixture of protein (40 mg ml−1) and reservoir solution (25 mM magnesium acetate, 100 mM 2-(N-morpholino)ethanesulfonic acid (MES; pH 6.8) and 9–13% (v/v) 2-methyl-2,4-pentanediol (MPD)). Crystals were cryoprotected by addition of 5 μl cryobuffer (20 mM magnesium acetate, 100 mM MES, pH 6.8, and 30% (v/v) MPD). Inhibitor complex structures were obtained by incubating crystals in 5 μl cryobuffer supplemented with bortezomib or carfilzomib at a final concentration of 1.5 mM for at least 8 h. Diffraction data were collected at the beamline X06SA at the Paul Scherrer Institute, SLS, Villigen, Switzerland (λ=1.0 Å). Evaluation of reflection intensities and data reduction were performed with the programme package XDS. Molecular replacement was carried out with the coordinates of the yeast 20S proteasome (PDB entry code: 5CZ4) by rigid body refinements (REFMAC5; ref.). MAIN and COOT were used to build models. TLS (Translation/Libration/Screw) refinements finally yielded excellent Rwork and Rfree, as well as root mean squared deviation bond and angle values. The coordinates, proven to have good stereochemistry from the Ramachandran plots, were deposited in the RCSB Protein Data Bank (Supplementary Table 1). The coordinates for the yeast 20S proteasome deposited under the entry code 1RYP do not represent the WT yCP but the double-mutant β5-K33R β1-T1A. At the time of deposition (in 1997), these data were the best available on the yCP. As yCP structure determination has become routine today, and structure refinement procedures have significantly improved, we here provide coordinates for the WT yCP at 2.3 Å resolution (PDB entry code: 5CZ4). Furthermore, the structures of most mutant yCPs described in this work were determined in their apo and ligand-bound states. For mutants with proteolytically inactive β5 subunits, the best crystallographic data obtained are given. For ligands or propeptide segments that were only partially defined in the 2FO–FC electron-density map the occupancy was reduced (for details see Supplementary Table 1). Additional information Accession codes: Coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (for PDB entry codes see Supplementary Table 1). How to cite this article: Huber, E. M. et al. A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome. Nat. Commun. 7:10900 doi: 10.1038/ncomms10900 (2016). Supplementary Material Autocatalytic subunit processing couples active site formation in the 20S proteasome to completion of assembly Structure of 20S proteasome from yeast at 2.4 Å resolution 20S proteasome biogenesis The catalytic sites of 20S proteasomes and their role in subunit maturation: a mutational and crystallographic study Conformational constraints for protein self-cleavage in the proteasome Immuno- and constitutive proteasome crystal structures reveal differences in substrate and inhibitor specificity Systematic analyses of substrate preferences of 20S proteasomes using peptidic epoxyketone inhibitors Catalytic mechanism and assembly of the proteasome Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 Å resolution A protein catalytic framework with an N-terminal nucleophile is capable of self-activation Proteasome from Thermoplasma acidophilum: a threonine protease Contribution of proteasomal beta-subunits to the cleavage of peptide substrates analyzed with yeast mutants The active sites of the eukaryotic 20S proteasome and their involvement in subunit precursor processing Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation Proteasome beta-type subunits: unequal roles of propeptides in core particle maturation and a hierarchy of active site function Eukaryotic 20S proteasome catalytic subunit propeptides prevent active site inactivation by N-terminal acetylation and promote particle assembly The catalytic triad of serine peptidases Distinct elements in the proteasomal beta5 subunit propeptide required for autocatalytic processing and proteasome assembly Analysis of mammalian 20S proteasome biogenesis: the maturation of beta-subunits is an ordered two-step mechanism involving autocatalysis Proteasome: a complex protease with a new fold and a distinct mechanism Autocatalytic processing of the 20S proteasome Crystal structure of the boronic acid-based proteasome inhibitor bortezomib in complex with the yeast 20S proteasome Bortezomib-resistant mutant proteasomes: structural and biochemical evaluation with carfilzomib and ONX 0914 Crystal structure of heat shock locus V (HslV) from Escherichia coli Critical elements in proteasome assembly Investigations on the maturation and regulation of archaebacterial proteasomes Atomic resolution structure of Erwinia chrysanthemi L-asparaginase Understanding nature's catalytic toolkit Proteasome inhibition by fellutamide B induces nerve growth factor synthesis Systematic comparison of peptidic proteasome inhibitors highlights the alpha-ketoamide electrophile as an auspicious reversible lead motif Inhibitor-binding mode of homobelactosin C to proteasomes: new insights into class I MHC ligand generation Crystal structures of Salinosporamide A (NPI-0052) and B (NPI-0047) in complex with the 20S proteasome reveal important consequences of beta-lactone ring opening and a mechanism for irreversible binding Effects of site-directed mutations on processing and activities of penicillin G acylase from Escherichia coli ATCC 11105 Crystal structure of human Taspase1, a crucial protease regulating the function of MLL Why does threonine, and not serine, function as the active site nucleophile in proteasomes? Analysing properties of proteasome inhibitors using kinetic and X-ray crystallographic studies Purification, crystallization, and X-ray analysis of the yeast 20S proteasome XDS REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use MAIN software for density averaging, model building, structure refinement and validation Features and development of Coot Author contributions E.M.H., W.H., X.L., C.S.A. and M.H. created yeast mutants; E.M.H. and W.H. performed activity and growth assays; E.M.H. and M.G. collected and analysed X-ray data; E.M.H., M.H. and M.G. wrote the manuscript. Conformation of proteasomal propeptides. (a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. Only the residues (-5) to (-1) of the β1-T1A propeptide are displayed. The major determinant of the S1 specificity pocket, residue 45, is depicted. Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow). The black arrow indicates the attack of Thr1Oγ onto the carbonyl carbon atom of Gly(-1). (b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well. Thr(-2)OH is hydrogen-bonded to Gly(-1)O (∼2.8 Å; black dashed line). The major determinant of the S1 specificity pocket, residue 45, is depicted. (c) Structural superposition of the β1-T1A, the β2-T1A and the β5-T1A-K81R propeptide remnants depict their differences in conformation. While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. Nonetheless, in all mutants the carbonyl carbon atom of Gly(-1) is ideally placed for the nucleophilic attack by Thr1Oγ. The hydrogen bond between Thr(-2)OH and Gly(-1)O (∼2.8 Å) is indicated by a black dashed line. Mutations of residue (-2) and their influence on propeptide conformation and autolysis. (a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide. The (-2) residues of both prosegments point into the S1 pocket. (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. While the residues (-2) to (-4) vary in their conformation, Gly(-1) and Ala1 are located in all structures at the same positions. (c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide. The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line). (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of Gly(-1). For all panels stereo views are provided in Supplementary Fig. 4g–j. Architecture and proposed reaction mechanism of the proteasomal active site. (a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines). Thr1OH is hydrogen-bonded to Lys33NH2 (2.7 Å), which in turn interacts with Asp17Oδ. The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. In the latter, a water molecule (red sphere) is found at the position where in the WT structure the side chain amine group of Lys33 is located. Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH. Note, the strong interaction with the water molecule causes a minor shift of Thr1, while all other active-site residues remain in place. (d) Proposed chemical reaction mechanism for autocatalytic precursor processing and proteolysis in the proteasome. The active-site Thr1 is depicted in blue, the propeptide segment and the peptide substrate are coloured in green, whereas the scissile peptide bond is highlighted in red. Autolysis (left set of structures) is initiated by deprotonation of Thr1OH via Lys33NH2 and the formation of a tetrahedral transition state. The strictly conserved oxyanion hole Gly47NH stabilizing the negatively charged intermediate is illustrated as a semicircle. Collapse of the transition state frees the Thr1 N terminus (by completing an N-to-O acyl shift of the propeptide), which is subsequently protonated by Asp166OH via Ser129OH. Next, Thr1NH2 polarizes a water molecule for the nucleophilic attack of the acyl-enzyme intermediate. On hydrolysis of the latter, the active-site Thr1 is ready for catalysis (right set of structures). Substrate processing starts with nucleophilic attack of the carbonyl carbon atom of the scissile peptide bond. The charged Thr1 N terminus may engage in the orientation of the amide moiety and donate a proton to the emerging N terminus of the C-terminal cleavage product. The resulting deprotonated Thr1NH2 finally activates a water molecule for hydrolysis of the acyl-enzyme. The proteasome favours threonine as the active-site nucleophile. (a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation. (b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (β5) using the substrate Suc-LLVY-AMC. Relative fluorescence units were measured in triplicate after 1 h of incubation at room temperature and are given as mean values. S.d.'s are indicated by error bars. (c) Illustration of the 2FO–FC electron-density map (blue mesh contoured at 1σ) for the β5-T1C propeptide fragment. The prosegment is cleaved but still bound in the substrate-binding channel. Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the β5-T1A-K81R mutant. (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). The inhibitor as well as the propeptides adopt similar conformations in the substrate-binding channel. (f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1. The SH group is rotated by 74° compared with the OH group. (g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively. The 2FO–FC electron-density map for Ser1 (blue mesh contoured at 1σ) is illustrated. (h) The methyl group of Thr1 is anchored by hydrophobic interactions with Ala46Cβ and Thr3Cγ. Ser1 lacks this stabilization and is therefore rotated by 60°. Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib. Inhibition assays (left panel). Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC. IC50 values were determined in triplicate; s.d.'s are indicated by error bars. Note that IC50 values depend on time and enzyme concentration. Proteasomes (final concentration: 66 nM) were incubated with inhibitor for 45 min before substrate addition (final concentration: 200 μM). Structures of the β5-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel). The 2FO–FC electron-density maps (blue mesh) for Ser1 (brown) and the covalently bound ligands (green; only the P1 site (Leu1) is shown) are contoured at 1σ. The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib.  Growth phenotypes and status of autolysis and catalysis of mutants. Mutant\tViability\tTemperature sensitivity\tAutolysis state of the mutant subunit*\tActivity of the mutant subunit\t \tWT\t++++\t+\t+\t+++\t \tβ1-T1A (ref.)\t++++\t+\t−\t−\t \tβ2-T1A (ref.)\t+++\t++\t−\t−\t \tβ1-T1A β2-T1A (ref.)\t+++\t++\t−\t−\t \tβ5-T1A\t+/−\t++++\t−\t−\t \tβ5-H(-2)A-T1A\t+/−\tND\t−\t−\t \tβ5-H(-2)T-T1A\t+/−\tND\t−\t−\t \tβ5-H(-2)L-T1A\t++\tND\t−\t−\t \tβ5-T1A, pp trans, nat1Δ\t+/−\t++++\tpp trans\t−\t \tβ5-T1A-K81R\t+\t++++\t−\t−\t \tβ5-H(-2)A-T1A-K81R\t+/−\tND\t−\t−\t \tβ5-H(-2)T-T1A-K81R\t+/−\tND\t−\t−\t \tβ5-H(-2)L-T1A-K81R\t++\tND\t−\t−\t \tβ5-H(-2)A\t++++\t++\t+\t+++\t \tβ5-H(-2)K\t++++\t++\t+\t+++\t \tβ5-H(-2)F\t++++\t+++\t+\t++\t \tβ5-H(-2)N\t++++\t+++\t+\t++\t \tβ5pp-β1 (ref. 18)\t++++\t+\t+/−\t+/−\t \tβ2-T(-2)V\t++++\t+\t−\t−\t \tβ5-L(-49S)-K33A (ref.)\t+\t++++\t−\t−\t \tβ5-K33A, pp trans\t+\t++++\tpp trans\t+/−\t \tβ5-F(-45)S-K33R (ref.)\t++\t++++\t+\t−\t \tβ5-D17N\t+/−\t++++\tND†\tND†\t \tβ5-L(-49)S-D17N\t+\t++++\t+/−\t+/−\t \tβ5-D17N, pp trans\t+\t++++\tpp trans\t+/−\t \tβ5-D166N\t++\t++++\t+\t+/−\t \tβ5-D166N, pp trans\t+++\t++++\tpp trans\t+/−\t \tβ5-T1S\t+++\t++++\t+\t++\t \tβ5-T1C\t++\t++++\t+\t−\t \t ND, not determined. *The autolysis state was assessed by purification and crystallization of the mutant proteasomes. †Purification of this mutant proteasome was not 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diff --git a/annotated_BioC_JSON/PMC4832331_ann.json b/annotated_BioC_JSON/PMC4832331_ann.json
new file mode 100644
index 0000000000000000000000000000000000000000..0468191d0f718f87e28d03ea95d7570ddd309e80
--- /dev/null
+++ b/annotated_BioC_JSON/PMC4832331_ann.json
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+[{"sourceid":"4832331","sourcedb":"","project":"","target":"","text":"Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions. Enterobacterial inducible decarboxylases of basic amino acids lysine, arginine and ornithine have a common evolutionary origin and belong to the α-family of pyridoxal-5′-phosphate (PLP)-dependent enzymes. They counteract acid stress experienced by the bacterium in the host digestive and urinary tract, and in particular in the extremely acidic stomach. Each decarboxylase is induced by an excess of the target amino acid and a specific range of extracellular pH, and works in conjunction with a cognate inner membrane antiporter. Decarboxylation of the amino acid into a polyamine is catalysed by a PLP cofactor in a multistep reaction that consumes a cytoplasmic proton and produces a CO2 molecule passively diffusing out of the cell, while the polyamine is excreted by the antiporter in exchange for a new amino acid substrate. Consequently, these enzymes buffer both the bacterial cytoplasm and the local extracellular environment. These amino acid decarboxylases are therefore called acid stress inducible or biodegradative to distinguish them from their biosynthetic lysine and ornithine decarboxylase paralogs catalysing the same reaction but responsible for the polyamine production at neutral pH. Inducible enterobacterial amino acid decarboxylases have been intensively studied since the early 1940 because the ability of bacteria to withstand acid stress can be linked to their pathogenicity in humans. In particular, the inducible lysine decarboxylase LdcI (or CadA) attracts attention due to its broad pH range of activity and its capacity to promote survival and growth of pathogenic enterobacteria such as Salmonella enterica serovar Typhimurium, Vibrio cholerae and Vibrio vulnificus under acidic conditions. Furthermore, both LdcI and the biosynthetic lysine decarboxylase LdcC of uropathogenic Escherichia coli (UPEC) appear to play an important role in increased resistance of this pathogen to nitrosative stress produced by nitric oxide and other damaging reactive nitrogen intermediates accumulating during the course of urinary tract infections (UTI). This effect is attributed to cadaverine, the diamine produced by decarboxylation of lysine by LdcI and LdcC, that was shown to enhance UPEC colonisation of the bladder. In addition, the biosynthetic E. coli lysine decarboxylase LdcC, long thought to be constitutively expressed in low amounts, was demonstrated to be strongly upregulated by fluoroquinolones via their induction of RpoS. A direct correlation between the level of cadaverine and the resistance of E. coli to these antibiotics commonly used as a first-line treatment of UTI could be established. Both acid pH and cadaverine induce closure of outer membrane porins thereby contributing to bacterial protection from acid stress, but also from certain antibiotics, by reduction in membrane permeability. The crystal structure of the E. coli LdcI as well as its low resolution characterisation by electron microscopy (EM) showed that it is a decamer made of two pentameric rings. Each monomer is composed of three domains – an N-terminal wing domain (residues 1–129), a PLP-binding core domain (residues 130–563), and a C-terminal domain (CTD, residues 564–715). Monomers tightly associate via their core domains into 2-fold symmetrical dimers with two complete active sites, and further build a toroidal D5-symmetrical structure held by the wing and core domain interactions around the central pore, with the CTDs at the periphery. Ten years ago we showed that the E. coli AAA+ ATPase RavA, involved in multiple stress response pathways, tightly interacted with LdcI but was not capable of binding to LdcC. We described how two double pentameric rings of the LdcI tightly associate with five hexameric rings of RavA to form a unique cage-like architecture that enables the bacterium to withstand acid stress even under conditions of nutrient deprivation eliciting stringent response. Furthermore, we recently solved the structure of the E. coli LdcI-RavA complex by cryo-electron microscopy (cryoEM) and combined it with the crystal structures of the individual proteins. This allowed us to make a pseudoatomic model of the whole assembly, underpinned by a cryoEM map of the LdcI-LARA complex (with LARA standing for LdcI associating domain of RavA), and to identify conformational rearrangements and specific elements essential for complex formation. The main determinants of the LdcI-RavA cage assembly appeared to be the N-terminal loop of the LARA domain of RavA and the C-terminal β-sheet of LdcI. In spite of this wealth of structural information, the fact that LdcC does not interact with RavA, although the two lysine decarboxylases are 69% identical and 84% similar, and the physiological significance of the absence of this interaction remained unexplored. To solve this discrepancy, in the present work we provided a three-dimensional (3D) cryoEM reconstruction of LdcC and compared it with the available LdcI and LdcI-RavA structures. Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). This comparison pinpointed differences between the biodegradative and the biosynthetic lysine decarboxylases and brought to light interdomain movements associated to pH-dependent enzyme activation and RavA binding, notably at the predicted RavA binding site at the level of the C-terminal β-sheet of LdcI. Consequently, we tested the capacity of cage formation by LdcI-LdcC chimeras where we interchanged the C-terminal β-sheets in question. Finally, we performed multiple sequence alignment of 22 lysine decarboxylases from Enterobacteriaceae containing the ravA-viaA operon in their genome. Remarkably, this analysis revealed that several specific residues in the above-mentioned β-sheet, independently of the rest of the protein sequence, are sufficient to define if a particular lysine decarboxylase should be classified as an “LdcC-like” or an “LdcI-like”. Moreover, this classification perfectly agrees with the genetic environment of the lysine decarboxylase genes. This fascinating parallelism between the propensity for RavA binding and the genetic environment of an enterobacterial lysine decarboxylase, as well as the high degree of conservation of this small structural motif, emphasize the functional importance of the interaction between biodegradative enterobacterial lysine decarboxylases and the AAA+ ATPase RavA. Results and Discussion CryoEM 3D reconstructions of LdcC, LdcIa and LdcI-LARA In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity – a 5.5 Å resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1 Å resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2). In addition, we improved our earlier cryoEM map of the LdcI-LARA complex from 7.5 Å to 6.2 Å resolution (Figs 1E,F and S3). Based on these reconstructions, reliable pseudoatomic models of the three assemblies were obtained by flexible fitting of either the crystal structure of LdcIi or a derived structural homology model of LdcC (Table S1). Significant differences between these pseudoatomic models can be interpreted as movements between specific biological states of the proteins as described below. The wing domains as a stable anchor at the center of the double-ring As a first step of a comparative analysis, we superimposed the three cryoEM reconstructions (LdcIa, LdcI-LARA and LdcC) and the crystal structure of the LdcIi decamer (Fig. 2 and Movie S1). This superposition reveals that the densities lining the central hole of the toroid are roughly at the same location, while the rest of the structure exhibits noticeable changes. Specifically, at the center of the double-ring the wing domains of the subunits provide the conserved basis for the assembly with the lowest root mean square deviation (RMSD) (between 1.4 and 2 Å for the Cα atoms only), whereas the peripheral CTDs containing the RavA binding interface manifest the highest RMSD (up to 4.2 Å) (Table S2). In addition, the wing domains of all structures are very similar, with the RMSD after optimal rigid body alignment (RMSDmin) less than 1.1 Å. Thus, taking the limited resolution of the cryoEM maps into account, we consider that the wing domains of all the four structures are essentially identical and that in the present study the RMSD of less than 2 Å can serve as a baseline below which differences may be assumed as insignificant. This preservation of the central part of the double-ring assembly may help the enzymes to maintain their decameric state upon activation and incorporation into the LdcI-RavA cage. The core domain and the active site rearrangements upon pH-dependent enzyme activation and LARA binding Both visual inspection (Fig. 2) and RMSD calculations (Table S2) show that globally the three structures at active pH (LdcIa, LdcI-LARA and LdcC) are more similar to each other than to the structure determined at high pH conditions (LdcIi). The decameric enzyme is built of five dimers associating into a 5-fold symmetrical double-ring (two monomers making a dimer are delineated in Fig. 1). As common for the α family of the PLP-dependent decarboxylases, dimerization is required for the enzymatic activity because the active site is buried in the dimer interface (Fig. 3A,B). This interface is formed essentially by the core domains with some contribution of the CTDs. The core domain is built by the PLP-binding subdomain (PLP-SD, residues 184–417) flanked by two smaller subdomains rich in partly disordered loops – the linker region (residues 130–183) and the subdomain 4 (residues 418–563). Zooming in the variations in the PLP-SD shows that most of the structural changes concern displacements in the active site (Fig. 3C–F). The most conspicuous differences between the PLP-SDs can be linked to the pH-dependent activation of the enzymes. The resolution of the cryoEM maps does not allow modeling the position of the PLP moiety and calls for caution in detailed mechanistic interpretations in terms of individual amino acids. Therefore we restrict our analysis to secondary structure elements. In particular, transition from LdcIi to LdcI-LARA involves ~3.5 Å and ~4.5 Å shifts away from the 5-fold axis in the active site α-helices spanning residues 218–232 and 246–254 respectively (Fig. 3C–E). Between these two extremes, the PLP-SDs of LdcIa and LdcC are similar both in the context of the decamer (Fig. 3F) and in terms of RMSDmin = 0.9 Å, which probably reflects the fact that, at the optimal pH, these lysine decarboxylases have a similar enzymatic activity. In addition, our earlier biochemical observation that the enzymatic activity of LdcIa is unaffected by RavA binding is consistent with the relatively small changes undergone by the active site upon transition from LdcIa to LdcI-LARA. Worthy of note, our previous comparison of the crystal structure of LdcIi with that of the inducible arginine decarboxylase AdiA revealed high conservation of the PLP-coordinating residues and identified a patch of negatively charged residues lining the active site channel as a potential binding site for the target amino acid substrate (Figs S3 and S4 in ref.). Rearrangements of the ppGpp binding pocket upon pH-dependent enzyme activation and LARA binding An inhibitor of the LdcI and LdcC activity, the stringent response alarmone ppGpp, is known to bind at the interface between neighboring monomers within each ring (Fig. S4). The ppGpp binding pocket is made up by residues from all domains and is located approximately 30 Å away from the PLP moiety. Whereas the crystal structure of the ppGpp-LdcIi was solved to 2 Å resolution, only a 4.1 Å resolution structure of the ppGpp-free LdcIi could be obtained. At this resolution, the apo-LdcIi and ppGpp-LdcIi structures (both solved at pH 8.5) appeared indistinguishable except for the presence of ppGpp (Fig. S11 in ref. ). Thus, we speculated that inhibition of LdcI by ppGpp would be accompanied by a transduction of subtle structural changes at the level of individual amino acid side chains between the ppGpp binding pocket and the active site of the enzyme. All our current cryoEM reconstructions of the lysine decarboxylases were obtained in the absence of ppGpp in order to be closer to the active state of the enzymes under study. While differences in the ppGpp binding site could indeed be visualized (Fig. S4), the level of resolution warns against speculations about their significance. The fact that interaction with RavA reduces the ppGpp affinity for LdcI despite the long distance of ~30 Å between the LARA domain binding site and the closest ppGpp binding pocket (Fig. S5) seems to favor an allosteric regulation mechanism. Interestingly, although a number of ppGpp binding residues are strictly conserved between LdcI and AdiA that also forms decamers at low pH optimal for its arginine decarboxylase activity, no ppGpp regulation of AdiA could be demonstrated. Swinging and stretching of the CTDs upon pH-dependent LdcI activation and LARA binding Inspection of the superimposed decameric structures (Figs 2 and S6) suggests a depiction of the wing domains as an anchor around which the peripheral CTDs swing. This swinging movement seems to be mediated by the core domains and is accompanied by a stretching of the whole LdcI subunits attracted by the RavA magnets. Indeed, all CTDs have very similar structures (RMSDmin \u003c1 Å). Yet the superposition of the decamers lays bare a progressive movement of the CTD as a whole upon enzyme activation by pH and the binding of LARA. The LdcIi monomer is the most compact, whereas LdcIa and especially LdcI-LARA gradually extend their CTDs towards the LARA domain of RavA (Figs 2 and 4). These small but noticeable swinging and stretching (up to ~4 Å) may be related to the incorporation of the LdcI decamer into the LdcI-RavA cage. The C-terminal β-sheet of a lysine decarboxylase as a major determinant of the interaction with RavA In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. However, at the level of this structural element the two proteins are actually surpisingly similar. Therefore, we wanted to check the influence of the primary sequence of the two proteins in this region on their ability to interact with RavA. To this end, we swapped the relevant β-sheets of the two proteins and produced their chimeras, namely LdcIC (i.e. LdcI with the C-terminal β-sheet of LdcC) and LdcCI (i.e. LdcC with the C-terminal β-sheet of LdcI) (Fig. 5A–C). Both constructs could be purified and could form decamers visually indistinguishable from the wild-type proteins. As expected, binding of LdcI to RavA was completely abolished by this procedure and no LdcIC-RavA complex could be detected. On the contrary, introduction of the C-terminal β-sheet of LdcI into LdcC led to an assembly of the LdcCI-RavA complex. On the negative stain EM grid, the chimeric cages appeared less rigid than the native LdcI-RavA, which probably means that the environment of the β-sheet contributes to the efficiency of the interaction and the stability of the entire architecture (Fig. 5D–F). The C-terminal β-sheet of a lysine decarboxylase is a highly conserved signature allowing to distinguish between LdcI and LdcC Alignment of the primary sequences of the E. coli LdcI and LdcC shows that some amino acid residues of the C-terminal β-sheet are the same in the two proteins, whereas others are notably different in chemical nature. Importantly, most of the amino acid differences between the two enzymes are located in this very region. Thus, to advance beyond our experimental confirmation of the C-terminal β-sheet as a major determinant of the capacity of a particular lysine decarboxylase to form a cage with RavA, we set out to investigate whether certain residues in this β-sheet are conserved in lysine decarboxylases of different enterobacteria that have the ravA-viaA operon in their genome. We inspected the genetic environment of lysine decarboxylases from 22 enterobacterial species referenced in the NCBI database, corrected the gene annotation where necessary (Tables S3 and S4), and performed multiple sequence alignment coupled to a phylogenetic analysis (see Methods). This procedure yielded several unexpected and exciting results. First of all, consensus sequence for the entire lysine decarboxylase family was derived. Second, the phylogenetic analysis clearly split the lysine decarboxylases into two groups (Fig. 6A). All lysine decarboxylases predicted to be “LdcI-like” or biodegradable based on their genetic environment, as for example their organization in an operon with a gene encoding the CadB antiporter (see Methods), were found in one group, whereas all enzymes predicted as “LdcC-like” or biosynthetic partitioned into another group. Thus, consensus sequences could also be determined for each of the two groups (Figs 6B,C and S7). Inspection of these consensus sequences revealed important differences between the groups regarding charge, size and hydrophobicity of several residues precisely at the level of the C-terminal β-sheet that is responsible for the interaction with RavA (Fig. 6B–D). For example, in our previous study, site-directed mutations identified Y697 as critically required for the RavA binding. Our current analysis shows that Y697 is strictly conserved in the “LdcI-like” group whereas the “LdcC-like” enzymes always have a lysine in this position; it also uncovers several other residues potentially essential for the interaction with RavA which can now be addressed by site-directed mutagenesis. The third and most remarkable finding was that exactly the same separation into “LdcI-like” and “LdcC”-like groups can be obtained based on a comparison of the C-terminal β-sheets only, without taking the rest of the primary sequence into account. Therefore the C-terminal β-sheet emerges as being a highly conserved signature sequence, sufficient to unambiguously discriminate between the “LdcI-like” and “LdcC-like” enterobacterial lysine decarboxylases independently of any other information (Figs 6 and S7). Our structures show that this motif is not involved in the enzymatic activity or the oligomeric state of the proteins. Thus, enterobacteria identified here (Fig. 6, Table S4) appear to exert evolutionary pressure on the biodegradative lysine decarboxylase towards the RavA binding. One of the elucidated roles of the LdcI-RavA cage is to maintain LdcI activity under conditions of enterobacterial starvation by preventing LdcI inhibition by the stringent response alarmone ppGpp. Furthermore, the recently documented interaction of both LdcI and RavA with specific subunits of the respiratory complex I, together with the unanticipated link between RavA and maturation of numerous iron-sulfur proteins, tend to suggest an additional intriguing function for this 3.5 MDa assembly. The conformational rearrangements of LdcI upon enzyme activation and RavA binding revealed in this work, and our amazing finding that the molecular determinant of the LdcI-RavA interaction is the one that straightforwardly determines if a particular enterobacterial lysine decarboxylase belongs to “LdcI-like” or “LdcC-like” proteins, should give a new impetus to functional studies of the unique LdcI-RavA cage. Besides, the structures and the pseudoatomic models of the active ppGpp-free states of both the biodegradative and the biosynthetic E. coli lysine decarboxylases offer an additional tool for analysis of their role in UPEC infectivity. Together with the apo-LdcI and ppGpp-LdcIi crystal structures, our cryoEM reconstructions provide a structural framework for future studies of structure-function relationships of lysine decarboxylases from other enterobacteria and even of their homologues outside Enterobacteriaceae. For example, the lysine decarboxylase of Eikenella corrodens is thought to play a major role in the periodontal disease and its inhibitors were shown to retard gingivitis development. Finally, cadaverine being an important platform chemical for the production of industrial polymers such as nylon, structural information is valuable for optimisation of bacterial lysine decarboxylases used for its production in biotechnology. Methods Protein expression and purification LdcI and LdcC were expressed and purified as described from an E. coli strain that cannot produce ppGpp (MG1655 ΔrelA ΔspoT strain). LdcI was stored in 20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, 0.1 mM PLP, pH 6.8 (buffer A) and LdcC in 20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, 0.1 mM PLP, pH 7.5 (buffer B). Chimeric LdcIC and LdcCI were constructed, expressed and purified as follows. The chimeras were designed by exchange, between LdcI and LdcC, of residues from 631 to 640 and from 697 to the C-terminus, corresponding to the regions around the two strands of the C-terminal β-sheet (Fig. 5B,C), while leaving the rest of the sequence unaltered. The synthetic ldcIC and ldcCI genes (2148 bp and 2154 bp respectively), provided within a pUC57 vector (GenScript) were subcloned into pET-TEV vector based on pET-28a (Invitrogen) containing an N-terminal TEV-cleavable 6x-His-Tag. Proteins were expressed in Rosetta 2 (DE3) cells (Novagen) in LB medium supplemented with kanamycin and chloramphenicol at 37 °C, upon induction with 0.5 mM IPTG at 18 °C. Cells were harvested by centrifugation, the pellet resuspended in a 50 mM Tris-HCl, 150 mM NaCl, pH 8 buffer supplemented with Complete EDTA free (Roche) and 0.1 mM PMSF (Sigma), and disrupted by sonication at 4 °C. After centrifugation at 75000 g at 4 °C for 20 min, the supernatant was loaded on a Ni-NTA column. The eluted protein-containing fractions were pooled and the His-Tag removed by incubation with the TEV protease at 1/100 ratio and an extensive dialysis in a 50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 5 mM EDTA, pH 8 buffer. After a second dialysis in a 50 mM Tris-HCl, 150 mM NaCl, pH 8 buffer supplemented with 10 mM imidazole, the sample was loaded on a Ni-NTA column in the same buffer, which allowed to separate the TEV protease and LdcCI/LdcIC. Finally, the pure proteins were obtained by size exclusion chromatography on a Superdex-S200 column in buffer A. LdcIa -cryoEM data collection and 3D reconstruction LdcI was prepared at 2 mg/ml in a buffer containing 25 mM MES, 100 mM NaCl, 0.2 mM PLP, 1 mM DTT, pH 6.2. 3 μl of sample were applied to glow-discharged quantifoil grids 300 mesh 2/1 (Quantifoil Micro Tools GmbH, Germany), excess solution was blotted during 2.5 s with a Vitrobot (FEI) and the grid frozen in liquid ethane. Data collection was performed on a FEI Polara microscope operated at 300 kV under low dose conditions. Micrographs were recorded on Kodak SO-163 film at 59,000 magnification, with defocus ranging from 0.6 to 4.9 μm. Films were digitized on a Zeiss scanner (Photoscan) at a step size of 7 μm giving a pixel size of 1.186 Å. The contrast transfer function (CTF) for each micrograph was determined with CTFFIND3. Initially ~2500 particles of 256 × 256 pixels were extracted manually, binned 4 times and subjected to one round of multivariate statistical analysis and classification using IMAGIC. Representative class averages corresponding to projections in different orientations were used as input for an ab-initio 3D reconstruction by RICOserver (rico.ibs.fr/). The resulting 3D reconstruction was refined using RELION, which yielded an 18 Å resolution map. Using projections of this model as a template, particles of size 256 × 256 pixels were semi-automatically selected from all the micrographs using the Fast Projection Matching (FPM) algorithm. The resulting dataset of ~46000 particles was processed in RELION with the previous map as an initial model and with a full CTF correction after the first peak. The final map comprised 44207 particles with a resolution of 7.4 Å as per the gold-standard FSC = 0.143 criterion. It was sharpened with EMBfactor using calculated B-factor of −350 Å2 and masked with a soft mask to obtain a final map with a resolution of 6.1 Å (Fig. S3, Table S1). LdcC - cryoEM data collection and 3D reconstruction LdcC was prepared at 2 mg/ml in a buffer containing 25 mM HEPES, 100 mM NaCl, 0.2 mM PLP, 1 mM DTT, pH 7.2. Grids were prepared and sample imaged as LdcIa. Data were processed essentially as LdcIa described above. Briefly, an initial ~20 Å resolution model was generated by angular reconstitution after manual picking of circa 3000 particles from the first micrographs, filtered to 60 Å resolution, refined with RELION and used for a semi-automatic selection with FPM. The dataset was processed in RELION with a full CTF correction to yield a final reconstruction comprising 61000 particles. The map was sharpened with Relion postprocessing, using a soft mask and automated B-factor estimation (−690 Å2), yielding a map at 5.5 Å resolution (Fig. S1, Table S1). LdcI-LARA - 3D reconstruction In our first study, the dataset was processed in SPIDER and the CTF correction involved a simple phase-flipping. Therefore, for consistency with the present work, we revisited the dataset and processed it in RELION with a full CTF correction after the first peak. It was sharpened with EMBfactor using calculated B-factor of −350 Å2 and masked with a soft mask to obtain a final map with a resolution of 6.2 Å (Fig. S2). This reconstruction is of a slightly better quality in terms of the continuity of the internal density. Therefore we used this improved map for fitting of the atomic model and further analysis (Fig. S2, Table S1). Additional image processing As a crosscheck, each data set was also refined either from other initial models: a “featureless donut” with approximate dimensions of the decamer, and low pass-filtered reconstructions from the two other data sets (i.e. the LdcC reconstruction was used as a model for the LdcIa and LdcI-LARA data sets, etc). All refinements converged to the same solutions independently of the starting model. Local resolution of all maps was determined with ResMap. LdcCI and LdcIC chimeras —negative stain EM and 2D image analysis 0.4 mg/ml of RavA (in a 20 mM Tris-HCl, 500 mM NaCl, 10 mM MgCl2, 1 mM DTT, 5% glycerol, pH 6.8 buffer) was mixed with 0.3 mg/ml of either LdcI, LdcC, LdcCI or LdcIC in the presence of 2 mM ADP and 10 mM MgCl2 in a buffer containing 20 mM Hepes and 150 mM NaCl at pH 7.4. After 10 minutes incubation at room temperature, 3 μl of mixture were applied to the clear side of the carbon on a carbon-mica interface and negatively stained with 2% uranyl acetate. Images were recorded with a JEOL 1200 EX II microscope at 100 kV at a nominal magnification of 15000 on a CCD camera yielding a pixel size of 4.667 Å. No complexes between RavA and LdcC or LdcIC could be observed, whereas the LdcCI-RavA preparation manifested cage-like particles similar to the previously published LdcI-RavA, but also unbound RavA and LdcCI, which implies that the affinity of RavA to the LdcCI chimera is lower than its affinity to the native LdcI. 1260 particles of 96 × 96 pixels were extracted interactively from several micrographs. 2D centering, multivariate statistical analysis and classification were performed using IMAGIC. Class-averages similar to the cage-like LdcI-RavA complex were used as references for multi-reference alignment followed by multivariate statistical analysis and classification. Fitting of atomic models into cryoEM maps A homology model of LdcC was obtained using the atomic coordinates of the LdcI monomer (3N75) as the template in SWISS-MODEL server. The RMSD between the template and the resulting model was 0.26 Å. The LdcC model was then fitted as a rigid body into the LdcC cryoEM map using the fit-in-map module of UCSF Chimera. This rigid fit indicated movements of several parts of the protein. Therefore, the density corresponding to one LdcC monomer was extracted and flexible fitting was performed using IMODFIT at 8 Å resolution. This monomeric model was then docked into the decameric cryoEM map with URO and its graphical version VEDA that use symmetry information for fitting in Fourier space. The Cα RMSDmin between the initial model of the LdcC monomer and the final IMODFIT LdcC model is 1.2 Å. In the case of LdcIa, the density corresponding to an individual monomer was extracted and the fit performed similarly to the one described above, with the final model of the decameric particle obtained with URO and VEDA. The Cα RMSDmin between the LdcIi monomer and the final IMODFIT model is 1.4 Å. For LdcI-LARA, the density accounting for one LdcI monomer bound to a LARA domain was extracted and further separated into individual densities corresponding to LdcI and to LARA. The fit of LdcI was performed as for LdcC and LdcIa, while the crystal structure of LARA was docked into the monomeric LdcI-LARA map as a rigid body using SITUS. The resulting pseudoatomic models were used to create the final model of the LdcI-LARA decamer with URO and VEDA. The Cα RMSDmin between the LdcIi monomer and the final IMODFIT model is 1.4 Å. A brief summary of relevant parameters is provided in Table S1. Sequence analysis Out of a non-exhaustive list of 50 species of Enterobacteriaceae (Table S3), 22 were found to contain genes annotated as ldcI or ldcC and containing the ravA-viaA operon (Table S4). An analysis using MUSCLE with default parameters showed that these genes share more than 65% identity. To verify annotation of these genes, we compared their genetic environment with that of E. coli ldcI and ldcC. Indeed, in E. coli the ldcI gene is in operon with the cadB gene encoding a lysine-cadaverine antiporter, whereas the ldcC gene is present between the accA gene (encoding an acetyl-CoA carboxylase alpha subunit carboxyltransferase) and the yaeR gene (coding for an unknown protein belonging to the family of Glyoxalase/Dioxygenase/Bleomycin resistance proteins). Compared with this genetic environment, the annotation of several ldcI and ldcC genes in enterobacteria was found to be inconsistent (Table S4). For example, several strains contain genes annotated as ldcC in the genetic background of ldcI and vice versa, as in the case of Salmonella enterica and Trabulsiella guamensi. Furthermore, the gene with an “ldcC-like” environment was found to be annotated as cadA in particular strains of Citrobacter freundii, Cronobacter sakazakii, Enterobacter cloacae subsp. Cloaca, Erwinia amylovora, Pantoea agglomerans, Rahnella aquatilis, Shigella dysenteriae, and Yersinia enterocolitica subsp. enterocolitica, whereas in Hafnia alvei, Kluyvera ascorbata, and Serratia marcescens subsp. marcescens, the gene with an “ldcI-like” environment was found to be annotated as ldcC. In addition, as far as the genetic environment is concerned, Plesiomonas appears to have two ldc genes with the organization of the E. coli ldcI (operon cadA-cadB). Consequently, we corrected for gene annotation consistent with the genetic environment and made multiple sequence alignments using version 8.0.1 of the CLC Genomics Workbench software. A phylogenetic tree was generated based on Maximum Likelihood and following the Neighbor-Joining method with the WAG protein substitution model. The reliability of the generated phylogenetic tree was assessed by bootstrap analysis. The presented unrooted phylogenetic tree shows the nodes that are reliable over 95% (Fig. 6A). Remarkably, the multiple sequence alignment and the resulting phylogenetic tree clearly grouped together all sequences annotated as ldcI on the one side, and all sequences annotated as ldcC on the other side. Thus, we conclude that all modifications in gene annotations that we introduced for the sake of consistency with the genetic environment are perfectly corroborated by the multiple sequence alignment and the phylogenetic analysis. Since the regulation of the lysine decarboxylase gene (i.e. inducible or constitutive) cannot be assessed by this analysis, we call the resulting groups “ldcI-like” and “ldcC-like” as referred to the E. coli enzymes, and make a parallel between the membership in a given group and the ability of the protein to form a cage complex with RavA. Additional Information Accession codes: CryoEM maps and corresponding fitted atomic structures (main chain atoms) have been deposited in the Electron Microscopy Data Bank and Protein Data Bank, respectively, with accession codes EMD-3205 and 5FKZ for LdcC, EMD-3204 and 5FKX for LdcIa and EMD-3206 and 5FL2 for LdcI-LARA. How to cite this article: Kandiah, E. et al. Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA. Sci. Rep. 6, 24601; doi: 10.1038/srep24601 (2016). Supplementary Material From cofactor to enzymes. The molecular evolution of pyridoxal-5′-phosphate-dependent enzymes Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations Acid stress response in enteropathogenic gammaproteobacteria: an aptitude for survival Mechanisms of acid resistance in Escherichia coli Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella The effect of the pH of the medium during growth on the enzymic activities of bacteria (Escherichia coli and Micrococcus lysodeikticus) and the biological significance of the changes produced Studies on bacterial amino-acid decarboxylases: 1. l(+)-lysine decarboxylase The cadA gene of Vibrio cholerae is induced during infection and plays a role in acid tolerance Lysine decarboxylase expression by Vibrio vulnificus is induced by SoxR in response to superoxide stress Polyamine-mediated resistance of uropathogenic Escherichia coli to nitrosative stress Conditioning of uropathogenic Escherichia coli for enhanced colonization of host Lysine Decarboxylase Activity as a Factor of Fluoroquinolone Resistance in Escherichia coli RpoS-dependent expression of the second lysine decarboxylase gene in Escherichia coli Cadaverine inhibition of porin plays a role in cell survival at acidic pH The role of OmpC and OmpF in acidic resistance in Escherichia coli Role of polyamines in formation of multiple antibiotic resistance of Escherichia coli under stress conditions Linkage between the bacterial acid stress and stringent responses: the structure of the inducible lysine decarboxylase Purification and physical properties of inducible Escherichia coli lysine decarboxylase Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase Novel structural and functional insights into the MoxR family of AAA+ ATPases Structure of RavA MoxR AAA+ protein reveals the design principles of a molecular cage modulating the inducible lysine decarboxylase activity The MoxR ATPase RavA and its cofactor ViaA interact with the NADH:ubiquinone oxidoreductase I in Escherichia coli Assembly principles of a unique cage formed by hexameric and decameric E. coli proteins The Escherichia coli ldcC gene encodes another lysine decarboxylase, probably a constitutive enzyme Crystal structure of diaminopelargonic acid synthase: evolutionary relationships between pyridoxal-5′-phosphate-dependent enzymes The enzymatic activities of the Escherichia coli basic aliphatic amino acid decarboxylases exhibit a pH zone of inhibition Disruption of individual nuo-genes leads to the formation of partially assembled NADH:ubiquinone oxidoreductase (complex I) in Escherichia coli Biofilm Lysine Decarboxylase, a New Therapeutic Target for Periodontal Inflammation Effects of immunization with natural and recombinant lysine decarboxylase on canine gingivitis development Bacterial lysine decarboxylase influences human dental biofilm lysine content, biofilm accumulation, and subclinical gingival inflammation Optimization of Direct Lysine Decarboxylase Biotransformation for Cadaverine Production with Whole-Cell Biocatalysts at High Lysine Concentration Enhanced cadaverine production from L-lysine using recombinant Escherichia coli co-overexpressing CadA and CadB Cadaverine production by heterologous expression of Klebsiella oxytoca lysine decarboxylase Cryo-electron microscopy of vitrified specimens Accurate determination of local defocus and specimen tilt in electron microscopy A new generation of the IMAGIC image processing system On the three-dimensional reconstruction of icosahedral particles RELION: implementation of a Bayesian approach to cryo-EM structure determination Ab initio high-resolution single-particle 3D reconstructions: the symmetry adapted functions way Prevention of overfitting in cryo-EM structure determination Sharpening high resolution information in single particle electron cryomicroscopy Quantifying the local resolution of cryo-EM density maps SWISS-MODEL: An automated protein homology-modeling server UCSF Chimera–a visualization system for exploratory research and analysis iMODFIT: efficient and robust flexible fitting based on vibrational analysis in internal coordinates On the fitting of model electron densities into EM reconstructions: a reciprocal-space formulation UROX 2.0: an interactive tool for fitting atomic models into electron-microscopy reconstructions MUSCLE: multiple sequence alignment with high accuracy and high throughput A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach Author Contributions E.K., H.M. and I.G. carried out EM data collection with assistance of M.B. and analyzed the data. D.C. performed cloning, multiple sequence alignment and phylogenetic analysis under the direction of S.E. and I.G., J.P. cloned and purified chimeric proteins under the direction of S.O.C., K.L. and S.W.S.C. purified LdcI, LdcC and LARA under the direction of W.A.H., I.G. conceived and directed the studies and wrote the manuscript with input from E.K. 3D cryoEM reconstructions of LdcC, LdcI-LARA and LdcIa. (A,C,E) cryoEM map of the LdcC (A), LdcIa\n(C) and LdcI-LARA (E) decamers with one protomer in light\ngrey. In the rest of the protomers, the wing, core and C-terminal domains\nare colored from light to dark in shades of green for LdcC (A), pink\nfor LdcIa (C) and blue for LdcI in LdcI-LARA (E).\nIn (E), the LARA domain density is shown in dark grey. Two monomers\nmaking a dimer are delineated. Scale bar 50 Å.\n(B,D,F) One protomer from the cryoEM map of the LdcC (B),\nLdcIa (D) and LdcI-LARA (F) in light grey with\nthe pseudoatomic model represented as cartoons and colored as the densities\nin (A,C,E). Each domain is indicated for clarity. Scale bar\n50 Å. See also Figs S1 and S3. Analysis of conformational rearrangements. Superposition of the pseudoatomic models of LdcC, LdcI from LdcI-LARA and\nLdcIa colored as in Fig. 1, and the\ncrystal structure of LdcIi in shades of yellow. Only one of the\ntwo rings of the double toroid is shown for clarity. The dashed circle\nindicates the central region that remains virtually unchanged between all\nthe structures, while the periphery undergoes visible movements. Scale bar\n50 Å. Conformational rearrangements in the enzyme active site. (A) LdcIi crystal structure, with one ring represented as a\ngrey surface and the second as a cartoon. A monomer with its PLP cofactor is\ndelineated. The PLP moieties of the cartoon ring are shown in red.\n(B) The LdcIi dimer extracted from the crystal structure\nof the decamer. One monomer is colored in shades of yellow as in Figs 1 and 2, while the monomer\nrelated by C2 symmetry is grey. The PLP is red. The active site is boxed.\n(C–F) Close-up views of the active site. The PLP\nmoiety in red is from the LdcIi crystal structure. We did not\nattempt to model it in the cryoEM maps. The dimer interface is shown as a\ndashed line and the active site α-helices mentioned in the text\nare highlighted. (C) Compares LdcIi (yellow) and\nLdcIa (pink), (D) compares LdcIa (pink) and\nLdcI-LARA (blue), and (E) compares LdcIi (yellow),\nLdcIa (pink) and LdcI-LARA (blue) simultaneously in order to\nshow the progressive shift described in the text. (F) Shows the\nsimilarity between LdcIa and LdcC at the level of the secondary\nstructure elements composing the active site. Colors are as in the other\nfigures. Stretching of the LdcI monomer upon pH-dependent enzyme activation and LARA\nbinding. (A–C) A slice through the pseudoatomic models of the LdcI\nmonomers extracted from the superimposed decamers (Fig.\n2) The rectangle indicates the regions enlarged in\n(D–F). (A) compares LdcIi (yellow)\nand LdcIa (pink), (B) compares LdcIa (pink) and\nLdcI-LARA (blue), and (C) compares LdcIi (yellow),\nLdcIa (pink) and LdcI-LARA (blue) simultaneously in order to\nshow the progressive stretching described in the text. The cryoEM density of\nthe LARA domain is represented as a grey surface to show the position of the\nbinding site and the direction of the movement. (D–F)\nInserts zooming at the CTD part in proximity of the LARA binding site. Loop\nregions are removed for a clearer visual comparison. An arrow indicates a\nswinging movement. Analysis of the LdcIC and LdcCI chimeras. (A) A slice through the pseudoatomic models of the LdcIa\n(purple) and LdcC (green) monomers extracted from the superimposed decamers\n(Fig. 2). (B) The C-terminal\nβ-sheet in LdcIa and LdcC enlarged from\n(A,C) Exchanged primary sequences (capital letters) and\ntheir immediate vicinity (lower case letters) colored as in\n(A,B), with the corresponding secondary structure elements\nand the amino acid numbering shown. (D,E) A gallery of negative stain\nEM images of (D) the wild type LdcI-RavA cage and (E) the\nLdcCI-RavA cage-like particles. (F) Some representative class\naverages of the LdcCI-RavA cage-like particles. Scale bar\n20 nm. Sequence analysis of enterobacterial lysine decarboxylases. (A) Maximum likelihood tree with the\n“LdcC-like” and the\n“LdcI-like” groups highlighted in green and pink,\nrespectively. Only nodes with higher values than 95% are shown (500\nreplicates of the original dataset, see Methods for details). Scale bar\nindicates the average number of substitutions per site. (B) Analysis\nof consensus “LdcI-like” and\n“LdcC-like” sequences around the first and second\nC-terminal β-strands. The height of the bars and the letters\nrepresenting the amino acids reflects the degree of conservation of each\nparticular position is in the alignment. Amino acids are colored according\nto a polarity color scheme with hydrophobic residues in black, hydrophilic\nin green, acidic in red and basic in blue. Numbering as in E. coli.\n(C) Signature sequences of LdcI and LdcC in the C-terminal\nβ-sheet. Polarity differences are highlighted. (D)\nPosition and nature of these differences at the surface of the respective\ncryoEM maps with the color code as in B. See also Fig. S7 and Tables S3 and 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diff --git a/annotated_BioC_JSON/PMC4833862_ann.json b/annotated_BioC_JSON/PMC4833862_ann.json
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+[{"sourceid":"4833862","sourcedb":"","project":"","target":"","text":"The dynamic organization of fungal acetyl-CoA carboxylase Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control.  Acetyl-CoA carboxylases are central regulatory hubs of fatty acid metabolism and are important targets for drug development in obesity and cancer. Here, the authors demonstrate that the regulation of these highly dynamic enzymes in fungi is governed by a mechanism based on phosphorylation-dependent conformational variability. Biotin-dependent acetyl-CoA carboxylases (ACCs) are essential enzymes that catalyse the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. This reaction provides the committed activated substrate for the biosynthesis of fatty acids via fatty-acid synthase. By catalysing this rate-limiting step in fatty-acid biosynthesis, ACC plays a key role in anabolic metabolism. ACC inhibition and knock-out studies show the potential of targeting ACC for treatment of the metabolic syndrome. Furthermore, elevated ACC activity is observed in malignant tumours. A direct link between ACC and cancer is provided by cancer-associated mutations in the breast cancer susceptibility gene 1 (BRCA1), which relieve inhibitory interactions of BRCA1 with ACC. Thus, ACC is a relevant drug target for type 2 diabetes and cancer. Microbial ACCs are also the principal target of antifungal and antibiotic compounds, such as Soraphen A. The principal functional protein components of ACCs have been described already in the late 1960s for Escherichia coli (E. coli) ACC: Biotin carboxylase (BC) catalyses the ATP-dependent carboxylation of a biotin moiety, which is covalently linked to the biotin carboxyl carrier protein (BCCP). Carboxyltransferase (CT) transfers the activated carboxyl group from carboxybiotin to acetyl-CoA to yield malonyl-CoA. Prokaryotic ACCs are transient assemblies of individual BC, CT and BCCP subunits. Eukaryotic ACCs, instead, are multienzymes, which integrate all functional components into a single polypeptide chain of ∼2,300 amino acids. Human ACC occurs in two closely related isoforms, ACC1 and 2, located in the cytosol and at the outer mitochondrial membrane, respectively. In addition to the canonical ACC components, eukaryotic ACCs contain two non-catalytic regions, the large central domain (CD) and the BC–CT interaction domain (BT). The CD comprises one-third of the protein and is a unique feature of eukaryotic ACCs without homologues in other proteins. The function of this domain remains poorly characterized, although phosphorylation of several serine residues in the CD regulates ACC activity. The BT domain has been visualized in bacterial carboxylases, where it mediates contacts between α- and β-subunits. Structural studies on the functional architecture of intact ACCs have been hindered by their huge size and pronounced dynamics, as well as the transient assembly mode of bacterial ACCs. However, crystal structures of individual components or domains from prokaryotic and eukaryotic ACCs, respectively, have been solved. The structure determination of the holoenzymes of bacterial biotin-dependent carboxylases, which lack the characteristic CD, such as the pyruvate carboxylase (PC), propionyl-CoA carboxylase, 3-methyl-crotonyl-CoA carboxylase and a long-chain acyl-CoA carboxylase revealed strikingly divergent architectures despite a general conservation of all functional components. In these structures, the BC and CT active sites are at distances between 40 and 80 Å, such that substrate transfer could be mediated solely by the mobility of the flexibly tethered BCCP. Human ACC1 is regulated allosterically, via specific protein–protein interactions, and by reversible phosphorylation. Dynamic polymerization of human ACC1 is linked to increased activity and is regulated allosterically by the activator citrate and the inhibitor palmitate, or by binding of the small protein MIG-12 (ref.). Human ACC1 is further regulated by specific phosphorylation-dependent binding of BRCA1 to Ser1263 in the CD. BRCA1 binds only to the phosphorylated form of ACC1 and prevents ACC activation by phosphatase-mediated dephosphorylation. Furthermore, phosphorylation by AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKA) leads to a decrease in ACC1 activity. AMPK phosphorylates ACC1 in vitro at Ser80, Ser1201 and Ser1216 and PKA at Ser78 and Ser1201. However, regulatory effects on ACC1 activity are mainly mediated by phosphorylation of Ser80 and Ser1201 (refs). Phosphorylated Ser80, which is highly conserved only in higher eukaryotes, presumably binds into the Soraphen A-binding pocket. The regulatory Ser1201 shows only moderate conservation across higher eukaryotes, while the phosphorylated Ser1216 is highly conserved across all eukaryotes. However, no effect of Ser1216 phosphorylation on ACC activity has been reported in higher eukaryotes. For fungal ACC, neither spontaneous nor inducible polymerization has been detected despite considerable sequence conservation to human ACC1. The BRCA1-interacting phosphoserine position is not conserved in fungal ACC, and no other phospho-dependent protein–protein interactions of fungal ACC have been described. In yeast ACC, phosphorylation sites have been identified at Ser2, Ser735, Ser1148, Ser1157 and Ser1162 (ref.). Of these, only Ser1157 is highly conserved in fungal ACC and aligns to Ser1216 in human ACC1. Its phosphorylation by the AMPK homologue SNF1 results in strongly reduced ACC activity. Despite the outstanding relevance of ACC in primary metabolism and disease, the dynamic organization and regulation of the giant eukaryotic, and in particular fungal ACC, remain poorly characterized. Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a). Integrating these data with small-angle X-ray scattering (SAXS) and electron microscopy (EM) observations yield a comprehensive representation of the dynamic structure and regulation of fungal ACC. Results The organization of the yeast ACC CD First, we focused on structure determination of the 82-kDa CD. The crystal structure of the CD of SceACC (SceCD) was determined at 3.0 Å resolution by experimental phasing and refined to Rwork/Rfree=0.20/0.24 (Table 1). The overall extent of the SceCD is 70 by 75 Å (Fig. 1b and Supplementary Fig. 1a,b), and the attachment points of the N-terminal 26-residue linker to the BCCP domain and the C-terminal CT domain are separated by 46 Å (the N- and C termini are indicated with spheres in Fig. 1b). SceCD comprises four distinct domains, an N-terminal α-helical domain (CDN), and a central four-helix bundle linker domain (CDL), followed by two α–β-fold C-terminal domains (CDC1/CDC2). CDN adopts a letter C shape, where one of the ends is a regular four-helix bundle (Nα3-6), the other end is a helical hairpin (Nα8,9) and the bridging region comprises six helices (Nα1,2,7,10–12). CDL is composed of a small, irregular four-helix bundle (Lα1–4) and tightly interacts with the open face of CDC1 via an interface of 1,300 Å2 involving helices Lα3 and Lα4. CDL does not interact with CDN apart from the covalent linkage and forms only a small contact to CDC2 via a loop between Lα2/α3 and the N-terminal end of Lα1, with an interface area of 400 Å2. CDC1/CDC2 share a common fold; they are composed of six-stranded β-sheets flanked on one side by two long, bent helices inserted between strands β3/β4 and β4/β5. CDC2 is extended at its C terminus by an additional β-strand and an irregular β-hairpin. On the basis of a root mean square deviation of main chain atom positions of 2.2 Å, CDC1/CDC2 are structurally more closely related to each other than to any other protein (Fig. 1c); they may thus have evolved by duplication. Close structural homologues could not be found for the CDN or the CDC domains. A regulatory loop mediates interdomain interactions To define the functional state of insect-cell-expressed ACC variants, we employed mass spectrometry (MS) for phosphorylation site detection. In insect-cell-expressed full-length SceACC, the highly conserved Ser1157 is the only fully occupied phosphorylation site with functional relevance in S. cerevisiae. Additional phosphorylation was detected for Ser2101 and Tyr2179; however, these sites are neither conserved across fungal ACC nor natively phosphorylated in yeast. MS analysis of dissolved crystals confirmed the phosphorylated state of Ser1157 also in SceCD crystals. The SceCD structure thus authentically represents the state of SceACC, where the enzyme is inhibited by SNF1-dependent phosphorylation. In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands β2 and β3 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here. This regulatory loop wedges between the CDC1 and CDC2 domains and provides the largest contribution to the interdomain interface. The N-terminal region of the regulatory loop also directly contacts the C-terminal region of CDC2 leading into CT. Phosphoserine 1157 is tightly bound by two highly conserved arginines (Arg1173 and Arg1260) of CDC1 (Fig. 1d). Already the binding of phosphorylated Ser1157 apparently stabilizes the regulatory loop conformation; the accessory phosphorylation sites Ser1148 and Ser1162 in the same loop may further modulate the strength of interaction between the regulatory loop and the CDC1 and CDC2 domains. Phosphorylation of the regulatory loop thus determines interdomain interactions of CDC1 and CDC2, suggesting that it may exert its regulatory function by modifying the overall structure and dynamics of the CD. The functional role of Ser1157 was confirmed by an activity assay based on the incorporation of radioactive carbonate into acid non-volatile material. Phosphorylated SceACC shows only residual activity (kcat=0.4±0.2 s−1, s.d. based on five replicate measurements), which increases 16-fold (kcat=6.5±0.3 s−1) after dephosphorylation with λ protein phosphatase. The values obtained for dephosphorylated SceACC are comparable to earlier measurements of non-phosphorylated yeast ACC expressed in E. coli. The variable CD is conserved between yeast and human To compare the organization of fungal and human ACC CD, we determined the structure of a human ACC1 fragment that comprises the BT and CD domains (HsaBT-CD), but lacks the mobile BCCP in between (Fig. 1a). An experimentally phased map was obtained at 3.7 Å resolution for a cadmium-derivatized crystal and was interpreted by a poly-alanine model (Fig. 1e and Table 1). Each of the four CD domains in HsaBT-CD individually resembles the corresponding SceCD domain; however, human and yeast CDs exhibit distinct overall structures. In agreement with their tight interaction in SceCD, the relative spatial arrangement of CDL and CDC1 is preserved in HsaBT-CD, but the human CDL/CDC1 didomain is tilted by 30° based on a superposition of human and yeast CDC2 (Supplementary Fig. 1c). As a result, the N terminus of CDL at helix Lα1, which connects to CDN, is shifted by 12 Å. Remarkably, CDN of HsaBT-CD adopts a completely different orientation compared with SceCD. With CDL/CDC1 superposed, CDN in HsaBT-CD is rotated by 160° around a hinge at the connection of CDN/CDL (Supplementary Fig. 1d). This rotation displaces the N terminus of CDN in HsaBT-CD by 51 Å compared with SceCD, resulting in a separation of the attachment points of the N-terminal linker to the BCCP domain and the C-terminal CT domain by 67 Å (the attachment points are indicated with spheres in Fig. 1e). The BT domain of HsaBT-CD consists of a helix that is surrounded at its N terminus by an antiparallel eight-stranded β-barrel. It resembles the BT of propionyl-CoA carboxylase; only the four C-terminal strands of the β-barrel are slightly tilted. On the basis of MS analysis of insect-cell-expressed human full-length ACC, Ser80 shows the highest degree of phosphorylation (90%). Ser29 and Ser1263, implicated in insulin-dependent phosphorylation and BRCA1 binding, respectively, are phosphorylated at intermediate levels (40%). The highly conserved Ser1216 (corresponding to S. cerevisiae Ser1157), as well as Ser1201, both in the regulatory loop discussed above, are not phosphorylated. However, residual phosphorylation levels were detected for Ser1204 (7%) and Ser1218 (7%) in the same loop. MS analysis of the HsaBT-CD crystallization sample reveals partial proteolytic digestion of the regulatory loop. Accordingly, most of this loop is not represented in the HsaBT-CD crystal structure. The absence of the regulatory loop might be linked to the less-restrained interface of CDL/CDC1 and CDC2 and altered relative orientations of these domains. Besides the regulatory loop, also the phosphopeptide target region for BRCA1 interaction is not resolved presumably because of pronounced flexibility. At the level of isolated yeast and human CD, the structural analysis indicates the presence of at least two hinges, one with large-scale flexibility at the CDN/CDL connection, and one with tunable plasticity between CDL/CDC1 and CDC2, plausibly affected by phosphorylation in the regulatory loop region. The integration of CD into the fungal ACC multienzyme To further obtain insights into the functional architecture of fungal ACC, we characterized larger multidomain fragments up to the intact enzymes. Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). No crystals diffracting to sufficient resolution were obtained for larger BC-containing fragments, or for full-length Cth or SceACC. To improve crystallizability, we generated ΔBCCP variants of full-length ACC, which, based on SAXS analysis, preserve properties of intact ACC (Supplementary Table 1 and Supplementary Fig. 2a–c). For CthΔBCCP, crystals diffracting to 8.4 Å resolution were obtained. However, molecular replacement did not reveal a unique positioning of the BC domain. Owing to the limited resolution the discussion of structures of CthCD-CT and CthΔBCCP is restricted to the analysis of domain localization. Still, these structures contribute considerably to the visualization of an intrinsically dynamic fungal ACC. In all these crystal structures, the CT domains build a canonical head-to-tail dimer, with active sites formed by contributions from both protomers (Fig. 2 and Supplementary Fig. 3a). The connection of CD and CT is provided by a 10-residue peptide stretch, which links the N terminus of CT to the irregular β-hairpin/β-strand extension of CDC2 (Supplementary Fig. 3b). The connecting region is remarkably similar in isolated CD and CthCD-CTCter structures, indicating inherent conformational stability. CD/CT contacts are only formed in direct vicinity of the covalent linkage and involve the β-hairpin extension of CDC2 as well as the loop between strands β2/β3 of the CT N-lobe, which contains a conserved RxxGxN motif. The neighbouring loop on the CT side (between CT β1/β2) is displaced by 2.5 Å compared to isolated CT structures (Supplementary Fig. 3c). On the basis of an interface area of ∼600 Å2 and its edge-to-edge connection characteristics, the interface between CT and CD might be classified as conformationally variable. Indeed, the comparison of the positioning of eight instances of the C-terminal part of CD relative to CT in crystal structures determined here, reveals flexible interdomain linking (Fig. 3a). The CDC2/CT interface acts as a true hinge with observed rotation up to 16°, which results in a translocation of the distal end of CDC2 by 8 Å. The interface between CDC2 and CDL/CDC1, which is mediated by the phosphorylated regulatory loop in the SceCD structure, is less variable than the CD–CT junction, and permits only limited rotation and tilting (Fig. 3b). Analysis of the impact of phosphorylation on the interface between CDC2 and CDL/CDC1 in CthACC variant structures is precluded by the limited crystallographic resolution. However, MS analysis of CthCD-CT and CthΔBCCP constructs revealed between 60 and 70% phosphorylation of Ser1170 (corresponding to SceACC Ser1157). The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23 Å (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of CthΔBCCP, denoted as CthCD-CT1/2 and CthΔBCCP1, respectively). In addition, CDN can rotate around hinges in the connection between CDN/CDL by 70° (Fig. 4b, observed in the second protomer of CthΔBCCP, denoted as CthΔBCCP2) and 160° (Fig. 4c, observed in SceCD) leading to displacement of the anchor site for the BCCP linker by up to 33 and 40 Å, respectively. Conformational variability in the CD thus contributes considerably to variations in the spacing between the BC and CT domains, and may extend to distance variations beyond the mobility range of the flexibly tethered BCCP. On the basis of the occurrence of related conformational changes between fungal and human ACC fragments, the observed set of conformations may well represent general states present in all eukaryotic ACCs. Large-scale conformational variability of fungal ACC To obtain a comprehensive view of fungal ACC dynamics in solution, we employed SAXS and EM. SAXS analysis of CthACC agrees with a dimeric state and an elongated shape with a maximum extent of 350 Å (Supplementary Table 1). The smooth appearance of scattering curves and derived distance distributions might indicate substantial interdomain flexibility (Supplementary Fig. 2a–c). Direct observation of individual full-length CthACC particles, according to MS results predominantly in a phosphorylated low-activity state, in negative stain EM reveals a large set of conformations from rod-like extended to U-shaped particles. Class averages, obtained by maximum-likelihood-based two-dimensional (2D) classification, are focused on the dimeric CT domain and the full BC–BCCP–CD domain of only one protomer, due to the non-coordinated motions of the lateral BC/CD regions relative to the CT dimer. They identify the connections between CDN/CDL and between CDC2/CT as major contributors to conformational heterogeneity (Supplementary Fig. 4a,b). The flexibility in the CDC2/CT hinge appears substantially larger than the variations observed in the set of crystal structures. The BC domain is not completely disordered, but laterally attached to BT/CDN in a generally conserved position, albeit with increased flexibility. Surprisingly, in both the linear and U-shaped conformations, the approximate distances between the BC and CT active sites would remain larger than 110 Å. These observed distances are considerably larger than in static structures of any other related biotin-dependent carboxylase. Furthermore, based on an average length of the BCCP–CD linker in fungal ACC of 26 amino acids, mobility of the BCCP alone would not be sufficient to bridge the active sites of BC and CT. Consequently, increased flexibility or additional modes of conformational changes may be required for productive catalysis. The most relevant candidate site for mediating such additional flexibility and permitting an extended set of conformations is the CDC1/CDC2 interface, which is rigidified by the Ser1157-phosphorylated regulatory loop, as depicted in the SceCD crystal structure. Discussion Altogether, the architecture of fungal ACC is based on the central dimeric CT domain (Fig. 4d). The CD consists of four distinct subdomains and acts as a tether from the CT to the mobile BCCP and an oriented BC domain. The CD has no direct role in substrate recognition or catalysis but contributes to the regulation of all eukaryotic ACCs. In higher eukaryotic ACCs, regulation via phosphorylation is achieved by combining the effects of phosphorylation at Ser80, Ser1201 and Ser1263. In fungal ACC, however, Ser1157 in the regulatory loop of the CD is the only phosphorylation site that has been demonstrated to be both phosphorylated in vivo and involved in the regulation of ACC activity. In its phosphorylated state, the regulatory loop containing Ser1157 wedges between CDC1/CDC2 and presumably limits the conformational freedom at this interdomain interface. However, flexibility at this hinge may be required for full ACC activity, as the distances between the BCCP anchor points and the active sites of BC and CT observed here are such large that mobility of the BCCP alone is not sufficient for substrate transfer. The current data thus suggest that regulation of fungal ACC is mediated by controlling the dynamics of the unique CD, rather than directly affecting catalytic turnover at the active sites of BC and CT. A comparison between fungal and human ACC will help to further discriminate mechanistic differences that contribute to the extended control and polymerization of human ACC. Most recently, a crystal structure of near full-length non-phosphorylated ACC from S. cerevisae (lacking only 21 N-terminal amino acids, here denoted as flACC) was published by Wei and Tong. In flACC, the ACC dimer obeys twofold symmetry and assembles in a triangular architecture with dimeric BC domains (Supplementary Fig. 5a). In their study, mutational data indicate a requirement for BC dimerization for catalytic activity. The transition from the elongated open shape, observed in our experiments, towards a compact triangular shape is based on an intricate interplay of several hinge-bending motions in the CD (Fig. 4d). Comparison of flACC with our CthΔBCCP structure reveals the CDC2/CT hinge as a major contributor to conformational flexibility (Supplementary Fig. 5b,c). In flACC, CDC2 rotates ∼120° with respect to the CT domain. A second hinge can be identified between CDC1/CDC2. On the basis of a superposition of CDC2, CDC1 of the phosphorylated SceCD is rotated by 30° relative to CDC1 of the non-phosphorylated flACC (Supplementary Fig. 5d), similar to what we have observed for the non-phosphorylated HsaBT-CD (Supplementary Fig. 1d). When inspecting all individual protomer and fragment structures in their study, Wei and Tong also identify the CDN/CDC1 connection as a highly flexible hinge, in agreement with our observations. The only bona fide regulatory phophorylation site of fungal ACC in the regulatory loop is directly participating in CDC1/CDC2 domain interactions and thus stabilizes the hinge conformation. In flACC, the regulatory loop is mostly disordered, illustrating the increased flexibility due to the absence of the phosphoryl group. Only in three out of eight observed protomers a short peptide stretch (including Ser1157) was modelled. In those instances the Ser1157 residue is located at a distance of 14–20 Å away from the location of the phosphorylated serine observed here, based on superposition of either CDC1 or CDC2. Applying the conformation of the CDC1/CDC2 hinge observed in SceCD on flACC leads to CDN sterically clashing with CDC2 and BT/CDN clashing with CT (Supplementary Fig. 6a,b). Thus, in accordance with the results presented here, phosphorylation of Ser1157 in SceACC most likely limits flexibility in the CDC1/CDC2 hinge such that activation through BC dimerization is not possible (Fig. 4d), which however does not exclude intermolecular dimerization. In addition, EM micrographs of phosphorylated and dephosphorylated SceACC display for both samples mainly elongated and U-shaped conformations and reveal no apparent differences in particle shape distributions (Supplementary Fig. 7). This implicates that the triangular shape with dimeric BC domains has a low population also in the active form, even though a biasing influence of grid preparation cannot be excluded completely. Large-scale conformational variability has also been observed in most other carrier protein-based multienzymes, including polyketide and fatty-acid synthases (with the exception of fungal-type fatty-acid synthases), non-ribosomal peptide synthetases and the pyruvate dehydrogenase complexes, although based on completely different architectures. Together, this structural information suggests that variable carrier protein tethering is not sufficient for efficient substrate transfer and catalysis in any of these systems. The determination of a set of crystal structures of SceACC in two states, unphosphorylated and phosphorylated at the major regulatory site Ser1157, provides a unique depiction of multienzyme regulation by post-translational modification (Fig. 4d). The phosphorylated regulatory loop binds to an allosteric site at the interface of two non-catalytic domains and restricts conformational freedom at several hinges in the dynamic ACC. It disfavours the adoption of a rare, compact conformation, in which intramolecular dimerization of the BC domains results in catalytic turnover. The regulation of activity thus results from restrained large-scale conformational dynamics rather than a direct or indirect influence on active site structure. To our best knowledge, ACC is the first multienzyme for which such a phosphorylation-dependent mechanical control mechanism has been visualized. However, the example of ACC now demonstrates the possibility of regulating activity by controlled dynamics of non-enzymatic linker regions also in other families of carrier-dependent multienzymes. Understanding such structural and dynamic constraints imposed by scaffolding and linking in carrier protein-based multienzyme systems is a critical prerequisite for engineering of efficient biosynthetic assembly lines. Methods Protein expression and purification All proteins were expressed in the Baculovirus Expression Vector System. The MultiBac insect cell expression plasmid pACEBACI (Geneva Biotech) was modified to host a GATEWAY (LifeTechnologies) cassette with an N-terminal 10xHis-tag, named pAB1GW-NH10 hereafter. Full-length HsaACC (Genebank accession #Q13085), SceACC (#Q00955) and CthACC (#G0S3L5) were cloned into pAB1GW-NH10 using GATEWAY according to the manufacturer's manual. Truncated variants were constructed by PCR amplification, digestion of the template DNA with DpnI, phosphorylation of the PCR product and religation of the linear fragment to a circular plasmid. The following constructs were used for this study: SceACC (1–2,233), CthACC (1–2,297), CthΔBCCP (1–2,297, Δ700–765), CthCD-CT (788–2,297), CthCD-CTCter (1,114–2,297), SceCD (768–1,494) and HsaBT-CD (622–1,584, Δ753–818). Bacmid and virus production was carried out according to MultiBac instructions. Baculovirus generation and amplification as well as protein expression were performed in Sf21 cells (Expression Systems) in Insect-Xpress medium (Lonza). The cells were harvested 68–96 h post infection by centrifugation and stored at −80 °C until being processed. Cells were lysed by sonication and the lysate was cleared by ultracentrifugation. Soluble protein was purified using Ni-NTA (Genscript) and size exclusion chromatography (Superose 6, GE Healthcare). The affinity tag was removed by tobacco etch virus (TEV) protease cleavage overnight at 4 °C. TEV protease and uncleaved protein were removed by orthogonal Ni-NTA purification before size exclusion chromatography. SceACC, CthACC and CthΔBCCP were further purified by high-resolution anion exchange chromatography before size exclusion chromatography. Purified SceCD, CthCD-CTCter, CthCD-CT, CthΔBCCP, CthACC and SceACC were concentrated to 10 mg ml−1 in 30 mM 3-(N-morpholino) propanesulfonic acid (MOPS) pH 7, 200 mM ammonium sulfate, 5% glycerol and 10 mM dithiothreitol. Purified HsaBT-CD was concentrated to 20 mg ml−1 in 20 mM bicine pH 8.0, 200 mM NaCl, 5% glycerol and 5 mM tris(2-carboxyethyl) phosphine (TCEP). Proteins were used directly or were stored at −80 °C after flash-freezing in liquid nitrogen. Protein crystallization All crystallization experiments were conducted using sitting drop vapour diffusion. SceCD crystals were grown at 19 °C by mixing protein and reservoir solution (0.1 M BisTrisPropane pH 6.5, 0.05–0.2 M di-sodium malonate, 20–30% polyethylene glycol (PEG) 3350, 10 mM trimethylamine or 2% benzamidine) in a 1:1 or 2:1 ratio. Crystals appeared after several days and continued to grow for 20–200 days. Crystals were cryoprotected by short incubation in mother liquor supplemented with 22% ethylene glycol and flash-cooled in liquid nitrogen. For heavy metal derivatization the crystals were incubated in stabilization solution supplemented with 1 mM Thimerosal or 10 mM EuCl2, and then backsoaked for 15 s in stabilization solution without heavy metal. Initial crystals of HsaBT-CD grew in 0.1 M Tris pH 8.5, 0.35 M tri-potassium citrate and 2–3.5% PEG10000 at 19 °C. After several rounds of optimization, good-quality diffraction crystals were obtained at 19 °C in 0.1 M MES pH 6, 0.25–0.35 M tri-potassium citrate, 2–5% PEG10000 and 0.01–0.04 M cadmium chloride. The protein drop contained a 1:1 ratio of protein and reservoir solution. Crystals grew immediately and stopped growing after 3 days. They were dehydrated and cryoprotected in several steps in artificial mother liquor containing incrementally increasing concentrations of tri-potassium citrate, PEG10000 and ethylene glycol and then flash-cooled in liquid nitrogen. The final solution was composed of 0.1 M MES pH 6, 0.5 M tri-potassium citrate, 6.75% PEG10000, 0.01 M cadmium chloride and 22% ethylene glycol. CthCD-CTCter crystals were grown at 19 °C by mixing protein and reservoir solution (0.1 M HEPES pH 7.5, 2–7% Tacsimate pH 7, 7.5–15% PEG monomethyl ether 5000) in a 1:1 ratio. Crystals appeared after several days and continued to grow for up to 2 weeks. Crystals were cryoprotected by short incubation in mother liquor supplemented with 22% ethylene glycol. CthCD-CT ACC crystals were grown at 19 °C by mixing protein and reservoir solution (0.1 M Bicine pH 8.5–9.5, 4–8% PEG8000) in a 1:1 or 1:2 ratio. Crystals grew 8– 10 days and were cryoprotected by short incubation in mother liquor supplemented with 22% ethylene glycol before flash-cooling in liquid nitrogen. CthΔBCCP ACC crystals were grown at 19 °C by mixing protein and reservoir solution (0.1 M Morpheus buffer 3 (Molecular Dimensions, MD2-100-102), 7–12% Morpheus ethylene glycols mix (MD2-100-74), 8–12% PEG4000, 17–23% glycerol) in a 1:1 or 1:2 ratio. Crystals grew up to 3 weeks and were cryoprotected in reservoir solution before flash-cooling in liquid nitrogen. Structure determination and analysis of phosphorylation All X-ray diffraction data were collected at beamlines X06SA (PXI) or X06DA (PXIII) at the Swiss Light Source (SLS, Paul Scherrer Institute, Villigen, Switzerland) equipped with PILATUS detectors. The wavelength of data collection was 1.000 Å for native crystals, and 1.527 and 1.907 Å for crystals derivatized with europium and cadmium, respectively. Raw data were processed using XDS. Molecular replacement was carried out using Phaser 2.5.7 and 2.6.0, density modification was performed using Parrot and resolve, multicrystal averaging was carried out using phenix. All model building procedures were conducted using Coot and figures were prepared using PyMOL (Schrödinger LLC). Diffraction of initial SceCD crystals in space group P43212 with unit cell dimensions of a=b=110.3 Å and c=131.7 Å was limited to 3.5 Å. The resolution was improved to 3 Å by addition of trimethylamine or benzamidine to the reservoir solution without significant changes in unit cell dimensions. Crystals derivatized with thimerosal and europium were used for initial SAD phase determination using the SHELXC/D package. Two mercury and four europium sites were located, and an initial model was placed in the resulting maps. Since crystals derivatized with europium were slightly non-isomorphous with a c axis length of 127 Å, multicrystal averaging was used for density modification and provided directly interpretable maps. Iterative cycles of model building and refinement in Buster (version 2.10.2; Global Phasing Ltd) converged at Rwork/Rfree of 0.20/0.24. The final model lacks the disordered N terminus (amino acids 768–789), an extended loop in the CDC1 domain (1,203–1,215), a short stretch (1,147–1,149) preceding the regulatory loop and the two very C-terminal residues (1,493–1,494). On the basis of temperature factor analysis, the start and end of the regulatory loop show higher disorder than the region around the interacting phosphoserine 1157. MS analysis of dissolved crystals detected quantitative phosphorylation of the regulatory Ser1157, as also found for full-length SceACC, and additionally albeit with much lower occurrence, phosphorylation of Ser790, Ser1137, Ser1148 and Ser1159. A modelled phosphoryl position for Ser1159 could overlap with the one of Ser1157, and might be represented in the crystal. For all other phosphorylation sites no difference density could be observed, probably because of very low occupancy. PDBeFold was used to search for structural homologues. The thresholds for lowest acceptable percentage of matched secondary structure elements were 70% for the search query and 20% for the result. Initial HsaBT-CD crystals were obtained in space group I4122 with a=b=240.1 Å and c=768.9 Å and diffracted to 7.5 Å. Optimized and dehydrated crystals also belonged to space group I4122 but with unit cell parameters a=b=267.3 Å and c=210.6 Å and diffracted to a resolution of 3.7 Å. Phase information was obtained from SAD based on bound cadmium ions from the crystallization condition. Six cadmium positions were located in a 4.0-Å resolution data set at 1.9 Å wavelength using SHELXC/D via the HKL2MAP interface. Density modification and phasing based on this anomalous data set, a 3.7-Å resolution data set at 1.0 Å wavelength and additional non-isomorphous lower-resolution data sets led to a high-quality electron density map. At the intermediate resolution obtained, the map was interpreted by a poly-alanine model, which was guided by predicted secondary structure as well as sequence and structural alignment with SceCD. The final model contains five cadmium ions and refines using phenix against experimental data with Rwork/Rfree of 0.35/0.38, as expected for a poly-alanine model. Two HsaBT-CD monomers are packed in the asymmetric unit via the CDN and BT domains. Density on top of the β-barrel of one BT most likely representing parts of the BT–CD linker guided the assignment of this BT to its linked CD partner domain. This BT-to-CD assignment was further supported by the analysis of an additional lower-resolution crystal form. Cadmium ions were found to participate in crystal packing. In HsaACC, phosphorylation at regulatory sites was detected as provided in the main text. No phosphorylation was detected for other phosphosites previously identified in large-scale phosphoproteomics studies, namely serines 5, 23, 25, 48, 53, 78, 488, 786, 1273 (refs). Two different crystal forms were obtained for CthCD-CTCter (denoted as CthCD-CTCter1 and CthCD-CTCter2), diffracting to 3.6 and 4.5 Å. Both forms packed in space group P212121 with unit cell constants of a=97.7 Å, b=165.3 Å and c=219.2 Å or a=100.2 Å, b=153.5 Å and c=249.2 Å, respectively. Phases were determined by molecular replacement using a homology model based on SceCT (pdb 1od2) as search model in Phaser; multicrystal averaging was applied in density modification. The CT domain was rebuilt and an initial homology model based on the SceCD structure was fitted into difference density for CthCD-CTCter1. Iterative cycles of rebuilding and refinement in Buster converged at Rwork/Rfree of 0.20/0.24. The refined CD fragment served as a starting model for rebuilding CthCD-CTCter2 at lower resolution. Coordinate refinement in Buster was additionally guided by reference model restraints and converged at Rwork/Rfree of 0.24/0.24. Residues 1,114–1,185, 1,213–1,252, 1,380–1,385, 1,465–1,468 and 2,188–2,195 were disordered in both crystal forms and are not included in the models. Helical regions C terminal to Glu2264 of both protomers of CthCD-CTCter1 and C terminal to Leu2259 and Arg2261 of the two protomers of CthCD-CTCter2, respectively, could not be built unambiguously and were therefore interpreted by placing poly-alanine stretches. Conservation was mapped on the CthCD-CTCter1 crystal structure using al2co based on a sequence alignment of 367 fungal ACC sequences calculated by Clustal Omega. MS analysis of purified protein detected 7% phosphorylation at Ser1170 (corresponding to Ser1157 in SceCD). CthCD-CT crystallized in space group P31212 with unit cell constants of a=b=195.0 Å and c=189.5 Å and crystals diffracted to a resolution of 7.2 Å. The structure was solved by molecular replacement using a model composed of CthCT and CDC2 as search model in Phaser. CDC1 and CDN were placed manually into the resulting maps, and the model was refined using rigid-body, domain-wise TLS and B-factor refinement and NCS- and reference model-restrained coordinate refinement in Buster to Rwork/Rfree of 0.23/0.25. Owing to the low resolution, the maximum allowed B-factor in Buster refinement was increased from the default value of 300–500 Å2, minimizing B-factor clipping to 5% of all atoms. Residues 1,033–1,035, 1,134–1,152, 1,213–1,252, 1,380–1,385, 1,465–1,468 and 2,188–2,195 were not included in the models. Helical regions C terminal to Leu2259 and Arg2261 on the two protomers, respectively, were interpreted as described for CthCD-CTCter. Loop conformations, including the regulatory loop, were modelled as observed in SceCD. MS analysis of purified protein detected 60% phosphorylation at Ser1170 (corresponding to Ser1157 in SceCD). Conservation was mapped on the CthCD-CT crystal structure as for CthCD-CTCter. CthΔBCCP ACC crystallized in space group P6422 with unit cell constants of a=b=462.2 Å and c=204.6 Å, resolution was limited to 8.4 Å. Structure determination and refinement was performed as for CthCD-CT, with a maximum allowed B-factor of 500 Å2, minimizing B-factor clipping to 3% of all atoms. Although substantial difference density is observed, no defined positions of the BT and BC domains could be derived because of disorder or partial in situ proteolysis or combinations thereof. In addition, residues 1,032–1,039, 1,134–1,152, 1,213–1,252, 1,380–1,385, 1,465–1,468 and 2,188–2,195 were not included in the model. The MissingAtom macro implemented in Buster was employed to account for missing atoms, the final Rwork/Rfree were 0.30/0.32. A region C terminal to Leu2259 on one protomer was interpreted as poly-alanine. Loop conformations, including the regulatory loop, were modelled as observed in SceCD. MS analysis of purified protein detected 70% phosphorylation at Ser1170 (corresponding to Ser1157 in SceCD). Small-angle X-ray scattering Proteins were thawed on ice and dialysed overnight against 30 mM MOPS pH 7, 200 mM ammonium sulfate, 5% glycerol and 10 mM dithiothreitol. Raw scattering data were measured at SAXS beamline B21 at Diamond Light Source. The samples were measured at concentrations of 2.5, 5 and 10 mg ml−1. Data were processed using the ATSAS package according to standard procedures. A slight increase in scattering in the very low-resolution range was observed with increasing protein concentrations, which may be because of interparticle attraction or minor aggregation. Scattering intensities were thus extrapolated to zero concentration using point-wise extrapolation implemented in Primus. Direct comparison of raw scattering curves demonstrates the similarity of CthACC and CthΔBCCP, and the derived values such as Rg and Porod Volume match within expected error margins. Molecular mass estimations based on the SAXS–MOW method derive values of 534.7 and 534.0 kDa for CthACC and CthΔBCCP, respectively. The relative discrepancies to the theoretical weights of 516.8 kDa (CthACC) and 503.0 kDa (CthΔBCCP) are 3.5% and 6.2%, respectively, which is in a typical range for this method. Electron microscopy Full-length CthACC was diluted to 0.01 mg ml−1 in 30 mM MOPS pH 7.0, 200 mM ammonium sulfate, 5% glycerol and 10 mM dithiothreitol. Protein sample was adsorbed to a 200-μm copper grid and stained with 2% uranyl acetate. Grids of CthACC were imaged on a CM-200 microscope (Philips) equipped with a TVIPS F416 4k CMOS camera (Tietz Video and Image Processing Systems). The voltage used was 200 kV, and a magnification of × 50,000 results in a pixel size of 2.14 Å. Initial image processing and particle picking was carried out using Xmipp. Overall, 22,309 particles were picked semi-automatically from 236 micrographs with a box size of 300 × 300 pixels. After extraction, particles with a z-score of more than three were discarded and 22,257 particles were aligned and classified into 48 2D class averages using the maximum-likelihood target function in Fourier space (MLF2D). After 72 iterations, 4,226 additional particles were discarded and the remaining 18,031 particles were re-aligned and classified into 36 classes using MLF2D with a high-resolution cutoff of 30 Å. After 44 iterations the alignment converged and class averages were extracted. In vitro biotinylation and activity assay To ensure full functionality, SceACC was biotinylated in vitro using the E. coli biotin ligase BirA. The reaction mixture contained 10 μM ACC, 3.7 μM BirA, 50 mM Tris-HCl, pH 8, 5.5 mM MgCl2, 0.5 mM biotin, 60 mM NaCl, 3 mM ATP and 10% glycerol, and the reaction was allowed to proceed for 7 h at 30 °C. The catalytic activity of phosphorylated and dephosphorylated SceACC was measured by following the incorporation of radioactive 14C into acid-stable non-volatile material. Dephosphorylated ACC was prepared by overnight treatment with λ protein phosphatase (New England Biolabs) of partially purified ACC before the final gel filtration step. The removal of the phosphoryl group from Ser1157 was confirmed by MS. The reaction mixture contained 0.5 μg recombinant ACC in 100 mM potassium phosphate, pH 8, 3 mM ATP, 5 mM MgCl2, 50 mM NaH14CO3 (specific activity 7.4 MBq mmol−1) and 1 mM acetyl-CoA in a total reaction volume of 100 μl. The reaction mixture was incubated for 15 min at 30 °C, stopped by addition of 200 μl 6 M HCl and subsequently evaporated to dryness at 85 °C. The non-volatile residue was redissolved in 100 μl of water, 1 ml Ultima Gold XR scintillation medium (Perkin Elmer) was added and the 14C radioactivity was measured in a Packard Tricarb 2000CA liquid scintillation analyser. Measurements were carried out in five replicates and catalytic activities were calculated using a standard curve derived from measurements of varying concentrations of NaH14CO3 in reaction buffer. Additional information Accession codes: Atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 5I6E (SceCD), 5I87 (HsaBT-CD), 5I6F/5I6G (CthCD-CTCter1/2), 5I6H (CthCD-CT) and 5I6I (CthΔBCCP). How to cite this article: Hunkeler, M. et al. The dynamic organization of fungal acetyl-CoA carboxylase. Nat. Commun. 7:11196 doi: 10.1038/ncomms11196 (2016). Supplementary Material Evidence for the participation of biotin in the enzymic synthesis of fatty acids Fatty acid synthesis and its regulation Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl-CoA carboxylase 2 Acetyl-CoA carboxylase 2 mutant mice are protected against obesity and diabetes induced by high-fat/high-carbohydrate diets Acetyl-CoA carboxylase inhibition for the treatment of metabolic syndrome Enzymes of the fatty acid synthesis pathway are highly expressed in in situ breast carcinoma Increased lipogenesis in cancer cells: new players, novel targets Structural basis of phosphopeptide recognition by the BRCT domain of BRCA1 Structural evidence for direct interactions between the BRCT domains of human BRCA1 and a phospho-peptide from human ACC1 Acetyl-coenzyme A carboxylase: crucial metabolic enzyme and attractive target for drug discovery Fatty acid metabolism: target for metabolic syndrome A mechanism for the potent inhibition of eukaryotic acetyl-coenzyme A carboxylase by soraphen A, a macrocyclic polyketide natural product Bacterial fatty acid biosynthesis: targets for antibacterial drug discovery Expression and characterization of recombinant fungal acetyl-CoA carboxylase and isolation of a soraphen-binding domain Acetyl CoA carboxylase. I. Requirement for two protein fractions Acetyl CoA carboxylase, II. Deomonstration of biotin-protein and biotin carboxylase subunits Multi-subunit acetyl-CoA carboxylases Identification of an isozymic form of acetyl-CoA carboxylase The subcellular localization of acetyl-CoA carboxylase 2 Regulation of acetyl-CoA carboxylase Regulation of mammalian acetyl-CoA carboxylase Improving production of malonyl coenzyme A-derived metabolites by abolishing Snf1-dependent regulation of Acc1 Crystal structure of the alpha(6)beta(6) holoenzyme of propionyl-coenzyme A carboxylase An unanticipated architecture of the 750-kDa alpha6beta6 holoenzyme of 3-methylcrotonyl-CoA carboxylase Three-dimensional structure of the biotin carboxylase subunit of acetyl-CoA carboxylase Structure of the biotinyl domain of acetyl-coenzyme A carboxylase determined by MAD phasing Crystal structure of the carboxyltransferase domain of acetyl-coenzyme A carboxylase The structure of the carboxyltransferase component of acetyl-coA carboxylase reveals a zinc-binding motif unique to the bacterial enzyme Structure and function of biotin-dependent carboxylases Domain architecture of pyruvate carboxylase, a biotin-dependent multifunctional enzyme Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase Induced polymerization of mammalian acetyl-CoA carboxylase by MIG12 provides a tertiary level of regulation of fatty acid synthesis ACCA phosphopeptide recognition by the BRCT repeats of BRCA1 Location and function of three sites phosphorylated on rat acetyl-CoA carboxylase by the AMP-activated protein kinase Critical phosphorylation sites for acetyl-CoA carboxylase activity Molecular mechanism for the regulation of human ACC2 through phosphorylation by AMPK Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae Yeast SNF1 is functionally related to mammalian AMP-activated protein kinase and regulates acetyl-CoA carboxylase in vivo Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution Kinetic and structural analysis of a new group of Acyl-CoA carboxylases found in Streptomyces coelicolor A3(2) Crystal structure of the 500-kDa yeast acetyl-CoA carboxylase holoenzyme dimer Effect of interdomain dynamics on the structure determination of modular proteins by small-angle scattering Architecture of the polyketide synthase module: surprises from electron cryo-microscopy The crystal structure of a mammalian fatty acid synthase Asturias FJ. Conformational flexibility of metazoan fatty acid synthase enables catalysis Nonribosomal peptide synthetases: structures and dynamics The pyruvate dehydrogenase complexes: structure-based function and regulation Protein complex expression by using multigene baculoviral vectors XDS Combining constraints for electron-density modification Overview of the CCP4 suite and current developments PHENIX: a comprehensive Python-based system for macromolecular structure solution Features and development of Coot A short history of SHELX Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions HKL2MAP: a graphical user interface for macromolecular phasing with SHELX programs Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis An enzyme assisted RP-RPLC approach for in-depth analysis of human liver phosphoproteome Combining protein-based IMAC, peptide-based IMAC, and MudPIT for efficient phosphoproteomic analysis Phaser crystallographic software SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information AL2CO: calculation of positional conservation in a protein sequence alignment Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega New developments in the program package for small-angle scattering data analysis Synchrotron-based small-angle X-ray scattering of proteins in solution Small-angle scattering for structural biology-Expanding the frontier while avoiding the pitfalls PRIMUS: a Windows PC-based system for small-angle scattering data analysis Determination of the molecular weight of proteins in solution from a single small-angle X-ray scattering measurement on a relative scale A Bayesian view on cryo-EM structure determination Xmipp: an image processing package for electron microscopy Author contributions M.H. cloned, expressed, purified and crystallized fungal ACC constructs, determined their structure and carried out SAXS analysis. E.S. cloned, expressed and crystallized human ACC CD and determined its structure. EM analysis was carried out by E.S., M.H. and A.H. S.I. contributed to structural analysis and figure preparation. T.M. designed and supervised work and analysed crystallographic data; all authors contributed to manuscript preparation. The phosphorylated central domain of yeast ACC. (a) Schematic overview of the domain organization of eukaryotic ACCs. Crystallized constructs are indicated. (b) Cartoon representation of the SceCD crystal structure. CDN is linked by a four-helix bundle (CDL) to two α–β-fold domains (CDC1 and CDC2). The regulatory loop is shown as bold cartoon, and the phosphorylated Ser1157 is marked by a red triangle. The N- and C termini are indicated by spheres. (c) Superposition of CDC1 and CDC2 reveals highly conserved folds. (d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group. (e) Structural overview of HsaBT-CD. The attachment points to the N-terminal BCCP domain and the C-terminal CT domain are indicated with spheres. All colourings are according to scheme a. Architecture of the CD–CT core of fungal ACC. Cartoon representation of crystal structures of multidomain constructs of CthACC. One protomer is shown in colour and one in grey. Individual domains are labelled; the active site of CT and the position of the conserved regulatory phosphoserine site based on SceCD are indicated by an asterisk and a triangle, respectively. Variability of the connections of CDC2 to CT and CDC1 in fungal ACC. (a) Hinge properties of the CDC2–CT connection analysed by a CT-based superposition of eight instances of the CDC2-CT segment. For clarity, only one protomer of CthCD-CTCter1 is shown in full colour as reference. For other instances, CDC2 domains are shown in transparent tube representation with only one helix each highlighted. The range of hinge bending is indicated and the connection points between CDC2 and CT (blue) as well as between CDC1 and CDC2 (green and grey) are marked as spheres. (b) The interdomain interface of CDC1 and CDC2 exhibits only limited plasticity. Representation as in a, but the CDC1 and CDC2 are superposed based on CDC2. One protomer of CthΔBCCP is shown in colour, the CDL domains are omitted for clarity and the position of the phosphorylated serine based on SceCD is indicated with a red triangle. The connection points from CDC1 to CDC2 and to CDL are represented by green spheres. The conformational dynamics of fungal ACC. (a–c) Large-scale conformational variability of the CDN domain relative to the CDL/CDC1 domain. CthCD-CT1 (in colour) serves as reference, the compared structures (as indicated, numbers after construct name differentiate between individual protomers) are shown in grey. Domains other than CDN and CDL/CDC1 are omitted for clarity. The domains are labelled and the distances between the N termini of CDN (spheres) in the compared structures are indicated. (d) Schematic model of fungal ACC showing the intrinsic, regulated flexibility of CD in the phosphorylated inhibited or the non-phosphorylated activated state. Flexibility of the CDC2/CT and CDN/CDL hinges is illustrated by arrows. The Ser1157 phosphorylation site and the regulatory loop are schematically indicated in magenta. Crystallographic data collection and refinement statistics.  \tSceCD\tSceCD Thimerosal\tSceCD Eu\tHsaBT-CD\tHsaBT-CD Cd2+\tCthCD-CTCter1\tCthCD-CTCter2\tCthCD-CT\tCthΔBCCP\t \tData collection\t \t Space group\tP43212\tP43212\tP43212\tI4122\tI4122\tP212121\tP212121\tP31212\tP6422\t \t Cell dimensions\t \t \t \t \t \t \t \t \t \t \t  a, b, c (Å)\t110.86, 110.86, 131.12\t111.22, 111.22, 131.49\t108.65, 108.65, 127.36\t267.27, 267.27, 210.61\t267.67, 267.67, 210.46\t97.66, 165.34, 219.23\t100.17, 153.45, 249,24\t295.02, 295.02, 189.52\t462.20, 462.20, 204.64\t \t  α, β, γ (°)\t90, 90, 90\t90, 90, 90\t90, 90, 90\t90, 90, 90\t90, 90, 90\t90, 90, 90\t90, 90, 90\t90, 90, 120\t90, 90, 120\t \t Resolution* (Å)\t3.0\t3.4\t4.0\t3.7\t4.1\t3.6\t4.5\t7.2\t8.4\t \t RMerge†\t18.2 (389.6)\t20.5 (306.1)\t40.6 (327.0)\t7.5 (400.9)\t15 (730.5)\t14.5 (384.5)\t27.4 (225.6)\t5.6 (302.6)\t29.4 (381.7)\t \t CC ½*†\t100 (58.3)\t99.9 (42.6)\t99.9 (48.5)\t100 (59.4)\t99.8 (73.2)\t99.9 (50.9)\t99.5 (46.7)\t100 (33.3)\t99.7 (35)\t \t I/σI†\t24.68 (1.46)\t7.99 (0.89)\t17.92 (1.85)\t21.24 (1.07)\t16.53 (1.41)\t10.61 (0.97)\t6.35 (1.00)\t18.95 (0.92)\t9.05 (0.9)\t \t Completeness†\t99.9 (99.9)\t99.6 (100)\t99.7 (96.8)\t99.8 (99.1)\t99.8 (99.7)\t99.7 (99.9)\t99.4 (98.6)\t99.6 (100)\t99.1 (99.9)\t \t Redundancy†\t39.1 (39.8)\t12.1 (14.3)\t81.6 (65.2)\t13.7 (13.7)\t20.9 (19.1)\t12.7 (13.5)\t6.1 (6.5)\t9.9 (10.4)\t18.5 (18.2)\t \t \t \t \t \t \t \t \t \t \t \t \tRefinement\t \t Resolution* (Å)\t46.4–3.0\t \t \t84.5–3.7\t \t49.2–3.6\t49.1–4.5\t49.9–7.2\t50.0–8.4\t \t Reflections\t16,928\t—\t—\t40,647\t—\t41,799\t23,340\t14,046\t12,111\t \t Rwork/Rfree\t0.20/0.24\t—\t—\t0.35/0.38‡\t—\t0.20/0.24\t0.24/0.24\t0.23/0.25\t0.30/0.32\t \t Number of atoms\t \t \t \t \t \t \t \t \t \t \t  Protein\t5,465\t \t \t6,925\t \t16,592\t16,405\t22,543\t22,445\t \t  Waters\t43\t—\t—\t—\t—\t—\t—\t—\t—\t \t  Ligand/ion\t7\t—\t—\t5\t—\t—\t—\t—\t—\t \t B-factors\t \t \t \t \t \t \t \t \t \t \t  Protein\t130\t \t \t158\t \t226\t275\t272\t250\t \t  Waters\t84\t—\t—\t—\t—\t—\t—\t—\t—\t \t  Ligand/ion\t90\t—\t—\t189\t—\t—\t—\t—\t—\t \t R.m.s.d.\t \t \t \t \t \t \t \t \t \t \t  RMS (angles, °)\t0.97\t—\t—\t0.83\t—\t1.07\t1.11\t1.15\t1.01\t \t  RMS (bonds, Å)\t0.01\t—\t—\t0.01\t—\t0.01\t0.01\t0.01\t0.01\t \t *Resolution cutoffs determined based on internal correlation significant at the 0.1% level as calculated by XDS. †Highest-resolution shell is shown in parentheses. ‡Modelled only as 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diff --git a/annotated_BioC_JSON/PMC4841544_ann.json b/annotated_BioC_JSON/PMC4841544_ann.json
new file mode 100644
index 0000000000000000000000000000000000000000..6758a7c8e04cba6d746db792b3f54427b179e22f
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+[{"sourceid":"4841544","sourcedb":"","project":"","target":"","text":"Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA  Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. Overall, this study deepens our molecular understanding of the sophisticated regulatory mechanisms of the expression of nadA and other genes governed by NadR, dependent on interactions with niche-specific signal molecules that may play important roles during meningococcal pathogenesis. Author Summary Serogroup B meningococcus (MenB) causes fatal sepsis and invasive meningococcal disease, particularly in young children and adolescents, as highlighted by recent MenB outbreaks in universities of the United States and Canada. The Bexsero vaccine protects against MenB and has recently been approved in \u003e 35 countries worldwide. Neisseria adhesin A (NadA) present on the meningococcal surface can mediate binding to human cells and is one of the three MenB vaccine protein antigens. The amount of NadA exposed on the meningococcal surface also influences the antibody-mediated serum bactericidal response measured in vitro. A deep understanding of nadA expression is therefore important, otherwise the contribution of NadA to vaccine-induced protection against meningococcal meningitis may be underestimated. The abundance of surface-exposed NadA is regulated by the ligand-responsive transcriptional repressor NadR. Here, we present functional, biochemical and high-resolution structural data on NadR. Our studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by ‘conformational selection’ of inactive forms. These findings shed light on the regulation of NadR, a key MarR-family virulence factor of this important human pathogen. Introduction The ‘Reverse Vaccinology’ approach was pioneered to identify antigens for a protein-based vaccine against serogroup B Neisseria meningitidis (MenB), a human pathogen causing potentially-fatal sepsis and invasive meningococcal disease. Indeed, Reverse Vaccinology identified Neisseria adhesin A (NadA), a surface-exposed protein involved in epithelial cell invasion and found in ~30% of clinical isolates. Recently, we reported the crystal structure of NadA, providing insights into its biological and immunological functions. Recombinant NadA elicits a strong bactericidal immune response and is therefore included in the Bexsero vaccine that protects against MenB and which was recently approved in over 35 countries worldwide. Previous studies revealed that nadA expression levels are mainly regulated by the Neisseria adhesin A Regulator (NadR). Although additional factors influence nadA expression, we focused on its regulation by NadR, the major mediator of NadA phase variable expression. Studies of NadR also have broader implications, since a genome-wide analysis of MenB wild-type and nadR knock-out strains revealed that NadR influences the regulation of \u003e 30 genes, including maf genes, from the multiple adhesin family. These genes encode a wide variety of proteins connected to many biological processes contributing to bacterial survival, adaptation in the host niche, colonization and invasion. NadR belongs to the MarR (Multiple Antibiotic Resistance Regulator) family, a group of ligand-responsive transcriptional regulators ubiquitous in bacteria and archaea. MarR family proteins can promote bacterial survival in the presence of antibiotics, toxic chemicals, organic solvents or reactive oxygen species and can regulate virulence factor expression. MarR homologues can act either as transcriptional repressors or as activators. Although \u003e 50 MarR family structures are known, a molecular understanding of their ligand-dependent regulatory mechanisms is still limited, often hampered by lack of identification of their ligands and/or DNA targets. A potentially interesting exception comes from the ligand-free and salicylate-bound forms of the Methanobacterium thermoautotrophicum protein MTH313 which revealed that two salicylate molecules bind to one MTH313 dimer and induce large conformational changes, apparently sufficient to prevent DNA binding. However, the homologous archeal Sulfolobus tokodaii protein ST1710 presented essentially the same structure in ligand-free and salicylate-bound forms, apparently contrasting the mechanism proposed for MTH313. Despite these apparent differences, MTH313 and ST1710 bind salicylate in approximately the same site, between their dimerization and DNA-binding domains. However, it is unknown whether salicylate is a relevant in vivo ligand of either of these two proteins, which share ~20% sequence identity with NadR, rendering unclear the interpretation of these findings in relation to the regulatory mechanisms of NadR or other MarR family proteins. NadR binds the nadA promoter and represses gene transcription. NadR binds nadA on three different operators (OpI, OpII and OpIII). The DNA-binding activity of NadR is attenuated in vitro upon addition of various hydroxyphenylacetate (HPA) derivatives, including 4-HPA. 4-HPA is a small molecule derived from mammalian aromatic amino acid catabolism and is released in human saliva, where it has been detected at micromolar concentration. In the presence of 4-HPA, NadR is unable to bind the nadA promoter and nadA gene expression is induced. In vivo, the presence of 4-HPA in the host niche of N. meningitidis serves as an inducer of NadA production, thereby promoting bacterial adhesion to host cells. Further, we recently reported that 3Cl,4-HPA, produced during inflammation, is another inducer of nadA expression. Extending our previous studies based on hydrogen-deuterium exchange mass spectrometry (HDX-MS), here we sought to reveal the molecular mechanisms and effects of NadR/HPA interactions via X-ray crystallography, NMR spectroscopy and complementary biochemical and in vivo mutagenesis studies. We obtained detailed new insights into ligand specificity, how the ligand allosterically influences the DNA-binding ability of NadR, and the regulation of nadA expression, thus also providing a deeper structural understanding of the ligand-responsive MarR super-family. Moreover, these findings are important because the activity of NadR impacts the potential coverage provided by anti-NadA antibodies elicited by the Bexsero vaccine and influences host-bacteria interactions that contribute to meningococcal pathogenesis. Results NadR is dimeric and is stabilized by specific hydroxyphenylacetate ligands Recombinant NadR was produced in E. coli using an expression construct prepared from N. meningitidis serogroup B strain MC58. Standard chromatographic techniques were used to obtain a highly purified sample of NadR (see Materials and Methods). In analytical size-exclusion high-performance liquid chromatography (SE-HPLC) experiments coupled with multi-angle laser light scattering (MALLS), NadR presented a single species with an absolute molecular mass of 35 kDa (S1 Fig). These data showed that NadR was dimeric in solution, since the theoretical molecular mass of the NadR dimer is 33.73 kDa; and, there was no change in oligomeric state on addition of 4-HPA. The thermal stability of NadR was examined using differential scanning calorimetry (DSC). Since ligand-binding often increases protein stability, we also investigated the effect of various HPAs (Fig 1A) on the melting temperature (Tm) of NadR. As a control of specificity, we also tested salicylate, a known ligand of some MarR proteins previously reported to increase the Tm of ST1710 and MTH313. The Tm of NadR was 67.4 ± 0.1°C in the absence of ligand, and was unaffected by salicylate. However, an increased thermal stability was induced by 4-HPA and, to a lesser extent, by 3-HPA. Interestingly, NadR displayed the greatest Tm increase upon addition of 3Cl,4-HPA (Table 1 and Fig 1B). Stability of NadR is increased by small molecule ligands. \n(A) Molecular structures of 3-HPA (MW 152.2), 4-HPA (MW 152.2), 3Cl,4-HPA (MW 186.6) and salicylic acid (MW 160.1). (B) DSC profiles, colored as follows: apo-NadR (violet), NadR+salicylate (red), NadR+3-HPA (green), NadR+4-HPA (blue), NadR+3Cl,4-HPA (pink). All DSC profiles are representative of triplicate experiments. Melting-point (Tm) and its ligand-induced increase (ΔTm) derived from DSC thermostability experiments. Dissociation constants (KD) of the NadR/ligand interactions from SPR steady-state binding experiments. Ligand\tTm (°C)\tΔTm (°C)\tKD (mM)\t \tNo ligand\t67.4 ± 0.1\tn.a.\tn.a.\t \tSalicylate\t67.5 ± 0.1\t0\tn.d.\t \t3-HPA\t70.0 ± 0.1\t2.7\t2.7 ± 0.1\t \t4-HPA\t70.7 ± 0.1\t3.3\t1.5 ± 0.1\t \t3Cl,4-HPA\t71.3 ± 0.2\t3.9\t1.1 ± 0.1\t \t n.a.: not applicable; n.d.: not determinable NadR displays distinct binding affinities for hydroxyphenylacetate ligands To further investigate the binding of HPAs to NadR, we used surface plasmon resonance (SPR). The SPR sensorgrams revealed very fast association and dissociation events, typical of small molecule ligands, thus prohibiting a detailed study of binding kinetics. However, steady-state SPR analyses of the NadR-HPA interactions allowed determination of the equilibrium dissociation constants (KD) (Table 1 and S2 Fig). The interactions of 4-HPA and 3Cl,4-HPA with NadR exhibited KD values of 1.5 mM and 1.1 mM, respectively. 3-HPA showed a weaker interaction, with a KD of 2.7 mM, while salicylate showed only a very weak response that did not reach saturation, indicating a non-specific interaction with NadR. A ranking of these KD values showed that 3Cl,4-HPA was the tightest binder, and thus matched the ranking of ligand-induced Tm increases observed in the DSC experiments. Although these KD values indicate rather weak interactions, they are similar to the values reported previously for the MarR/salicylate interaction (KD ~1 mM) and the MTH313/salicylate interaction (KD 2–3 mM), and approximately 20-fold tighter than the ST1710/salicylate interaction (KD ~20 mM). Crystal structures of holo-NadR and apo-NadR To fully characterize the NadR/HPA interactions, we sought to determine crystal structures of NadR in ligand-bound (holo) and ligand-free (apo) forms. First, we crystallized NadR (a selenomethionine-labelled derivative) in the presence of a 200-fold molar excess of 4-HPA. The structure of the NadR/4-HPA complex was determined at 2.3 Å resolution using a combination of the single-wavelength anomalous dispersion (SAD) and molecular replacement (MR) methods, and was refined to R work/R free values of 20.9/26.0% (Table 2). Despite numerous attempts, we were unable to obtain high-quality crystals of NadR complexed with 3Cl,4-HPA, 3,4-HPA, 3-HPA or DNA targets. However, it was eventually possible to crystallize apo-NadR, and the structure was determined at 2.7 Å resolution by MR methods using the NadR/4-HPA complex as the search model. The apo-NadR structure was refined to R work/R free values of 19.1/26.8% (Table 2). Data collection and refinement statistics for NadR structures. \tNadR SeMet + 4-HPA (SAD peak) (PDB code 5aip)\tNadR apo-form (PDB code 5aiq)\t \tData collection\t\t\t \tWavelength (Å)\t0.9792\t1.0\t \tBeamline\tSLS (PXII-X10SA)\tSLS (PXII-X10SA)\t \tResolution range (Å)\t39.2–2.3\t48.2–2.7\t \tSpace group\tP 43 21 2\tP 43 21 2\t \tUnit cell dimensions (Å)\t75.3, 75.3, 91.8\t69.4, 69.4, 253.8\t \tTotal reflections\t291132 (41090)\t225521 (35809)\t \tUnique reflections\t12320 (1773)\t17700 (2780)\t \tMultiplicity\t23.6 (23.2)\t12.7 (12.8)\t \tCompleteness (%)\t100.0 (100.00)\t99.9 (99.7)\t \tMean I/sigma(I)\t25.5 (9.0)\t22.6 (3.8)\t \tWilson B-factor\t23.9\t49.1\t \tRsym*\t10.9 (39.4)\t11.4 (77.6)\t \tRmeas**\t11.3\t11.8\t \tRefinement\t\t\t \tRwork♯\t20.9\t21.7\t \tRfree♯♯\t26.0\t27.2\t \tNumber of atoms\t\t\t \t  Non-hydrogen atoms\t2263\t4163\t \t  Macromolecules\t2207\t4144\t \t  Ligands\t11\t0\t \t  Water\t45\t19\t \tProtein residues\t275\t521\t \tRMS(bonds)\t0.008\t0.003\t \tRMS(angles)\t1.09\t0.823\t \tRamachandran (%)§\t\t\t \t  Favored\t100\t98.4\t \t  Outliers\t0\t0\t \tClash score\t5.0\t3.9\t \tAverage B-factor\t\t\t \t  Macromolecules\t34.8\t53.3\t \t  Ligands\t32.9\t-\t \t  Solvent\t37.3 (H2O)\t29.0 (H2O)\t \t Statistics for the highest-resolution shell are shown in parentheses. *R\nsym = Σhkl Σi |Ii(hkl)—\u003cI(hkl)\u003e| / Σhkl Σi Ii(hkl) ** R\nmeas = redundancy-independent (multiplicity-weighted) R\nmerge as reported from AIMLESS. \n♯\nR\nwork = Σ||F(obs)|- |F(calc)||/Σ|F(obs)| \n♯♯\nR\nfree = as for R\nwork, calculated for 5.0% of the total reflections, chosen at random, and omitted from refinement. \n§ Values obtained using Molprobity. The asymmetric unit of the NadR/4-HPA crystals (holo-NadR) contained one NadR homodimer, while the apo-NadR crystals contained two homodimers. In the apo-NadR crystals, the two homodimers were related by a rotation of ~90°; the observed association of the two dimers was presumably merely an effect of crystal packing, since the interface between the two homodimers is small (\u003c 550 Å2 of buried surface area), and is not predicted to be physiologically relevant by the PISA software. Moreover, our SE-HPLC/MALLS analyses (see above) revealed that in solution NadR is dimeric, and previous studies using native mass spectrometry (MS) revealed dimers, not tetramers. The NadR homodimer bound to 4-HPA has a dimerization interface mostly involving the top of its ‘triangular’ form, while the two DNA-binding domains are located at the base (Fig 2A). High-quality electron density maps allowed clear identification of the bound ligand, 4-HPA (Fig 2B). The overall structure of NadR shows dimensions of ~50 × 65 × 50 Å and a large homodimer interface that buries a total surface area of ~ 4800 Å2. Each NadR monomer consists of six α-helices and two short β-strands, with helices α1, α5, and α6 forming the dimer interface. Helices α3 and α4 form a helix-turn-helix motif, followed by the “wing motif” comprised of two short antiparallel β-strands (β1-β2) linked by a relatively long and flexible loop. Interestingly, in the α4-β2 region, the stretch of residues from R64-R91 presents seven positively-charged side chains, all available for potential interactions with DNA. Together, these structural elements constitute the winged helix-turn-helix (wHTH) DNA-binding domain and, together with the dimeric organization, are the hallmarks of MarR family structures. The crystal structure of NadR in complex with 4-HPA. \n(A) The holo-NadR homodimer is depicted in green and blue for chains A and B respectively, while yellow sticks depict the 4-HPA ligand (labelled). For simplicity, secondary structure elements are labelled for chain B only. Red dashes show hypothetical positions of chain B residues 88–90 that were not modeled due to lack of electron density. (B) A zoom into the pocket occupied by 4-HPA shows that the ligand contacts both chains A and B; blue mesh shows electron density around 4-HPA calculated from a composite omit map (omitting 4-HPA), using phenix. The map is contoured at 1σ and the figure was prepared with a density mesh carve factor of 1.7, using Pymol (www.pymol.org). A single conserved leucine residue (L130) is crucial for dimerization The NadR dimer interface is formed by at least 32 residues, which establish numerous inter-chain salt bridges or hydrogen bonds, and many hydrophobic packing interactions (Fig 3A and 3B). To determine which residues were most important for dimerization, we studied the interface in silico and identified several residues as potential mediators of key stabilizing interactions. Using site-directed mutagenesis, a panel of eight mutant NadR proteins was prepared (including mutations H7A, S9A, N11A, D112A, R114A, Y115A, K126A, L130K and L133K), sufficient to explore the entire dimer interface. Each mutant NadR protein was purified, and then its oligomeric state was examined by analytical SE-HPLC. Almost all the mutants showed the same elution profile as the wild-type (WT) NadR protein. Only the L130K mutation induced a notable change in the oligomeric state of NadR (Fig 3C). Further, in SE-MALLS analyses, the L130K mutant displayed two distinct species in solution, approximately 80% being monomeric (a 19 kDa species), and only 20% retaining the typical native dimeric state (a 35 kDa species) (Fig 3D), demonstrating that Leu130 is crucial for stable dimerization. It is notable that L130 is usually present as Leu, or an alternative bulky hydrophobic amino acid (e.g. Phe, Val), in many MarR family proteins, suggesting a conserved role in stabilizing the dimer interface. In contrast, most of the other residues identified in the NadR dimer interface were poorly conserved in the MarR family. Analysis of the NadR dimer interface. \n(A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). (C) SE-HPLC analyses of all mutant forms of NadR are compared with the wild-type (WT) protein. The WT and most of the mutants show a single elution peak with an absorbance maximum at 17.5 min. Only the mutation L130K has a noteworthy effect on the oligomeric state, inducing a second peak with a longer retention time and a second peak maximum at 18.6 min. To a much lesser extent, the L133K mutation also appears to induce a ‘shoulder’ to the main peak, suggesting very weak ability to disrupt the dimer. (D) SE-HPLC/MALLS analyses of the L130K mutant, shows 20% dimer and 80% monomer. The curves plotted correspond to Absorbance Units (mAU) at 280nm wavelength (green), light scattering (red), and refractive index (blue). The holo-NadR structure presents only one occupied ligand-binding pocket The NadR/4-HPA structure revealed the ligand-binding site nestled between the dimerization and DNA-binding domains (Fig 2). The ligand showed a different position and orientation compared to salicylate complexed with MTH313 and ST1710 (see Discussion). The binding pocket was almost entirely filled by 4-HPA and one water molecule, although there also remained a small tunnel 2-4Å in diameter and 5-6Å long leading from the pocket (proximal to the 4-hydroxyl position) to the protein surface. The tunnel was lined with rather hydrophobic amino acids, and did not contain water molecules. Unexpectedly, only one monomer of the holo-NadR homodimer contained 4-HPA in the binding pocket, whereas the corresponding pocket of the other monomer was unoccupied by ligand, despite the large excess of 4-HPA used in the crystallization conditions. Inspection of the protein-ligand interaction network revealed no bonds from NadR backbone groups to the ligand, but several key side chain mediated hydrogen (H)-bonds and ionic interactions, most notably between the carboxylate group of 4-HPA and Ser9 of chain A (SerA9), and chain B residues TrpB39, ArgB43 and TyrB115 (Fig 4A). At the other ‘end’ of the ligand, the 4-hydroxyl group was proximal to AspB36, with which it may establish an H-bond (see bond distances in Table 3). The water molecule observed in the pocket was bound by the carboxylate group and the side chains of SerA9 and AsnA11. Atomic details of NadR/HPA interactions. \nA) A stereo-view zoom into the binding pocket showing side chain sticks for all interactions between NadR and 4-HPA. Green and blue ribbons depict NadR chains A and B, respectively. 4-HPA is shown in yellow sticks, with oxygen atoms in red. A water molecule is shown by the red sphere. H-bonds up to 3.6Å are shown as dashed lines. The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually). Residues AsnA11 and ArgB18 likely make indirect yet local contributions to ligand binding, mainly by stabilizing the position of AspB36. Bond distances for interacting polar atoms are provided in Table 3. Side chains mediating hydrophobic interactions are shown in orange. (B) A model was prepared to visualize putative interactions of 3Cl,4-HPA (pink) with NadR, revealing the potential for additional contacts (dashed lines) of the chloro moiety (green stick) with LeuB29 and AspB36. List of 4-HPA atoms bound to NadR via ionic interactions and/or H-bonds. 4-HPA atom\tNadR residue/atom\tDistance (Å)\t \tO2\tTrpB39/NE1\t2.83\t \tO2\tArgB43/NH1\t2.76\t \tO1\tArgB43/NH1\t3.84\t \tO1\tSerA9/OG\t2.75\t \tO1\tTyrB115/OH\t2.50\t \tO2\tWater (*Ser9/Asn11)\t2.88\t \tOH\tAspB36/OD1/OD2\t3.6/3.7\t \t * Bond distance between the ligand carboxylate group and the water molecule, which in turn makes H-bond to the SerA9 and AsnA11 side chains. In addition to the H-bonds involving the carboxylate and hydroxyl groups of 4-HPA, binding of the phenyl moiety appeared to be stabilized by several van der Waals’ contacts, particularly those involving the hydrophobic side chain atoms of LeuB21, MetB22, PheB25, LeuB29 and ValB111 (Fig 4A). Notably, the phenyl ring of PheB25 was positioned parallel to the phenyl ring of 4-HPA, potentially forming π-π parallel-displaced stacking interactions. Consequently, residues in the 4-HPA binding pocket are mostly contributed by NadR chain B, and effectively created a polar ‘floor’ and a hydrophobic ‘ceiling’, which house the ligand. Collectively, this mixed network of polar and hydrophobic interactions endows NadR with a strong recognition pattern for HPAs, with additional medium-range interactions potentially established with the hydroxyl group at the 4-position. Structure-activity relationships: molecular basis of enhanced stabilization by 3Cl,4-HPA We modelled the binding of other HPAs by in silico superposition onto 4-HPA in the holo-NadR structure, and thereby obtained molecular explanations for the binding specificities of diverse ligands. For example, similar to 4-HPA, the binding of 3Cl,4-HPA could involve multiple bonds towards the carboxylate group of the ligand and some to the 4-hydroxyl group. Additionally, the side chains of LeuB29 and AspB36 would be only 2.6–3.5 Å from the chlorine atom, thus providing van der Waals’ interactions or H-bonds to generate the additional binding affinity observed for 3Cl,4-HPA (Fig 4B). The presence of a single hydroxyl group at position 2, as in 2-HPA, rather than at position 4, would eliminate the possibility of favorable interactions with AspB36, resulting in the lack of NadR regulation by 2-HPA described previously. Finally, salicylate is presumably unable to specifically bind NadR due to the 2-hydroxyl substitution and the shorter aliphatic chain connecting its carboxylate group (Fig 1A): the compound simply seems too small to simultaneously establish the network of beneficial bonds observed in the NadR/HPA interactions. Analysis of the pockets reveals the molecular basis for asymmetric binding and stoichiometry We attempted to investigate further the binding stoichiometry using solution-based techniques. However, studies based on tryptophan fluorescence were confounded by the fluorescence of the HPA ligands, and isothermal titration calorimetry (ITC) was unfeasible due to the need for very high concentrations of NadR in the ITC chamber (due to the relatively low affinity), which exceeded the solubility limits of the protein. However, it was possible to calculate the binding stoichiometry of the NadR-HPA interactions using an SPR-based approach. In SPR, the signal measured is proportional to the total molecular mass proximal to the sensor surface; consequently, if the molecular weights of the interactors are known, then the stoichiometry of the resulting complex can be determined. This approach relies on the assumption that the captured protein (‘the ligand’, according to SPR conventions) is 100% active and freely-accessible to potential interactors (‘the analytes’). This assumption is likely valid for this pair of interactors, for two main reasons. Firstly, NadR is expected to be covalently immobilized on the sensor chip as a dimer in random orientations, since it is a stable dimer in solution and has sixteen lysines well-distributed around its surface, all able to act as potential sites for amine coupling to the chip, and none of which are close to the ligand-binding pocket. Secondly, the HPA analytes are all very small (MW 150–170, Fig 1A) and therefore are expected to be able to diffuse readily into all potential binding sites, irrespective of the random orientations of the immobilized NadR dimers on the chip.The stoichiometry of the NadR-HPA interactions was determined using Eq 1 (see Materials and Methods), and revealed stoichiometries of 1.13 for 4-HPA, 1.02 for 3-HPA, and 1.21 for 3Cl,4-HPA, strongly suggesting that one NadR dimer bound to 1 HPA analyte molecule. The crystallographic data, supported by the SPR studies of binding stoichiometry, revealed the lack of a second 4-HPA molecule in the homodimer, suggesting negative co-operativity, a phenomenon previously described for the MTH313/salicylate interaction and for other MarR family proteins. To explore the molecular basis of asymmetry in holo-NadR, we superposed its ligand-free monomer (chain A) onto the ligand-occupied monomer (chain B). Overall, the superposition revealed a high degree of structural similarity (Cα root mean square deviation (rmsd) of 1.5Å), though on closer inspection a rotational difference of ~9 degrees along the long axis of helix α6 was observed, suggesting that 4-HPA induced a slight conformational change (Fig 5A). However, since residues of helix α6 were not directly involved in ligand binding, an explanation for the lack of 4-HPA in monomer A did not emerge by analyzing only these backbone atom positions, suggesting that a more complex series of allosteric events may occur. Indeed, we noted interesting differences in the side chains of Met22, Phe25 and Arg43, which in monomer B are used to contact the ligand while in monomer A they partially occupied the pocket and collectively reduced its volume significantly. Specifically, upon analysis with the CASTp software, the pocket in chain B containing the 4-HPA exhibited a total volume of approximately 370 Å3, while the pocket in chain A was occupied by these three side chains that adopted ‘inward’ positions and thereby divided the space into a few much smaller pockets, each with volume \u003c 50 Å3, evidently rendering chain A unfavorable for ligand binding. Most notably, atomic clashes between the ligand and the side chains of MetA22, PheA25 and ArgA43 would occur if 4-HPA were present in the monomer A pocket (Fig 5B). Subsequently, analyses of the pockets in apo-NadR revealed that in the absence of ligand the long Arg43 side chain was always in the open ‘outward’ position compatible with binding to the 4-HPA carboxylate group. In contrast, the apo-form Met22 and Phe25 residues were still encroaching the spaces of the 4-hydroxyl group and the phenyl ring of the ligand, respectively (Fig 5C). The ‘outward’ position of Arg43 generated an open apo-form pocket with volume approximately 380Å3. Taken together, these observations suggest that Arg43 is a major determinant of ligand binding, and that its ‘inward’ position inhibits the binding of 4-HPA to the empty pocket of holo-NadR. Structural differences of NadR in ligand-bound or free forms. \n(A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. In these crystals, the ArgA43 side chain showed two alternate conformations, modelled with 50% occupancy in each state, as indicated by the two ‘mirrored’ arrows. The inner conformer is the one that would display major clashes if 4-HPA were present. (C) Comparison of the empty pocket from holo-NadR (green residues) with the four empty pockets of apo-NadR (grey residues), shows that in the absence of 4-HPA the Arg43 side chain is always observed in the ‘outward’ conformation. Finally, we applied 15N heteronuclear solution NMR spectroscopy to examine the interaction of 4-HPA with apo NadR. We collected NMR spectra on NadR in the presence and absence of 4-HPA (see Materials and Methods). The 1H-15N TROSY-HSQC spectrum of apo-NadR, acquired at 25°C, displayed approximately 140 distinct peaks (Fig 6A), most of which correspond to backbone amide N-H groups. The broad spectral dispersion and the number of peaks observed, which is close to the number of expected backbone amide N-H groups for this polypeptide, confirmed that apo-NadR is well-folded under these conditions and exhibits one conformation appreciable on the NMR timescale, i.e. in the NMR experiments at 25°C, two or more distinct conformations of apo-NadR monomers were not readily apparent. Upon the addition of 4-HPA, over 45 peaks showed chemical shift perturbations, i.e. changed position in the spectrum or disappeared, while the remaining peaks remained unchanged. This observation showed that 4-HPA was able to bind NadR and induce notable changes in specific regions of the protein. NMR spectra of NadR in the presence and absence of 4-HPA. \n(A) Superposition of two 1H-15N TROSY-HSQC spectra recorded at 25°C on apo-NadR (cyan) and on NadR in the presence of 4-HPA (red). (B,C) Overlay of selected regions of the 1H-15N TROSY-HSQC spectra acquired at 25°C of apo-NadR (cyan) and NadR/4-HPA (red) superimposed with the spectra acquired at 10°C of apo-NadR (blue) and NadR/4-HPA (green). The spectra acquired at 10°C are excluded from panel A for simplicity. However, in the presence of 4-HPA, the 1H-15N TROSY-HSQC spectrum of NadR displayed approximately 140 peaks, as for apo-NadR, i.e. two distinct stable conformations (that might have potentially revealed the molecular asymmetry observed crystallographically) were not notable. Considering the small size, fast diffusion and relatively low binding affinity of 4-HPA, it would not be surprising if the ligand associates and dissociates rapidly on the NMR time scale, resulting in only one set of peaks whose chemical shifts represent the average environment of the bound and unbound states. Interestingly, by cooling the samples to 10°C, we observed that a number of those peaks strongly affected by 4-HPA (and therefore likely to be in the ligand-binding site) demonstrated evidence of peak splitting, i.e. a tendency to become two distinct peaks rather than one single peak (Fig 6B and 6C). These doubled peaks may therefore reveal that the cooler temperature partially trapped the existence in solution of two distinct states, in presence or absence of 4-HPA, with minor conformational differences occurring at least in proximity to the binding pocket. Although more comprehensive NMR experiments and full chemical shift assignment of the spectra would be required to precisely define this multi-state behavior, the NMR data clearly demonstrate that NadR exhibits conformational flexibility which is modulated by 4-HPA in solution. Apo-NadR structures reveal intrinsic conformational flexibility The apo-NadR crystal structure contained two homodimers in the asymmetric unit (chains A+B and chains C+D). Upon overall structural superposition, these dimers revealed a few minor differences in the α6 helix (a major component of the dimer interface) and the helices α4-α5 (the DNA binding region), and an rmsd of 1.55Å (Fig 7A). Similarly, the entire holo-homodimer could be closely superposed onto each of the apo-homodimers, showing rmsd values of 1.29Å and 1.31Å, and with more notable differences in the α6 helix positions (Fig 7B). The slightly larger rmsd between the two apo-homodimers, rather than between apo- and holo-homodimers, further indicate that apo-NadR possesses a notable degree of intrinsic conformational flexibility. Overall apo- and holo-NadR structures are similar. \n(A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. Here, larger differences are observed in the α6 helices (top). 4-HPA stabilizes concerted conformational changes in NadR that prevent DNA-binding To further investigate the conformational rearrangements of NadR, we performed local structural alignments using only a subset of residues in the DNA-binding helix (α4). By selecting and aligning residues Arg64-Ala77 of one α4 helix per dimer, superposition of the holo-homodimer onto the two apo-homodimers revealed differences in the monomer conformations of each structure. While one monomer from each structure was closely superimposable (Fig 8A, left side), the second monomer displayed quite large differences (Fig 8A, right side). Most notably, the position of the DNA-binding helix α4 shifted by as much as 6 Å (Fig 8B). Accordingly, helix α4 was also found to be one of the most dynamic regions in previous HDX-MS analyses of apo-NadR in solution. Structural comparisons of NadR and modelling of interactions with DNA. \n(A) The holo-homodimer structure is shown as green and blue cartoons, for chain A and B, respectively, while the two homodimers of apo-NadR are both cyan and pale blue for chains A/C and B/D, respectively. The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). The α4 helices aligned closely, Cα rmsd 0.2Å for 14 residues. (B) The relative positions of the α4 helices of the 4-HPA-bound holo homodimer chain B (blue), and of apo homodimers AB and CD (showing chains B and D) in pale blue. Dashes indicate the Ala77 Cα atoms, in the most highly shifted region of the ‘non-fixed’ α4 helix. (C) The double-stranded DNA molecule (grey cartoon) from the OhrR-ohrA complex is shown after superposition with NadR, to highlight the expected positions of the NadR α4 helices in the DNA major grooves. The proteins share ~30% amino acid sequence identity. For clarity, only the α4 helices are shown in panels (B) and (C). (D) Upon comparison with the experimentally-determined OhrR:ohrA structure (grey), the α4 helix of holo-NadR (blue) is shifted ~8Å out of the major groove. However, structural comparisons revealed that the shift of holo-NadR helix α4 induced by the presence of 4-HPA was also accompanied by several changes at the holo dimer interface, while such extensive structural differences were not observed in the apo dimer interfaces, particularly notable when comparing the α6 helices (S3 Fig). In summary, compared to ligand-stabilized holo-NadR, apo-NadR displayed an intrinsic flexibility focused in the DNA-binding region. This was also evident in the greater disorder (i.e. less well-defined electron density) in the β1-β2 loops of the apo dimers (density for 16 residues per dimer was missing) compared to the holo dimer (density for only 3 residues was missing). In holo-NadR, the distance separating the two DNA-binding α4 helices was 32 Å, while in apo-NadR it was 29 Å for homodimer AB, and 34 Å for homodimer CD (Fig 8C). Thus, the apo-homodimer AB presented the DNA-binding helices in a conformation similar to that observed in the protein:DNA complex of OhrR:ohrA from Bacillus subtilis (Fig 8C). Interestingly, OhrR contacts ohrA across 22 base pairs (bp), and similarly the main NadR target sites identified in the nadA promoter (the operators Op I and Op II) both span 22 bp. Pairwise superpositions showed that the NadR apo-homodimer AB was the most similar to OhrR (rmsd 2.6 Å), while the holo-homodimer was the most divergent (rmsd 3.3 Å) (Fig 8C). Assuming the same DNA-binding mechanism is used by OhrR and NadR, the apo-homodimer AB seems ideally pre-configured for DNA binding, while 4-HPA appeared to stabilize holo-NadR in a conformation poorly suited for DNA binding. Specifically, in addition to the different inter-helical translational distances, the α4 helices in the holo-NadR homodimer were also reoriented, resulting in movement of α4 out of the major groove, by up to 8Å, and presumably preventing efficient DNA binding in the presence of 4-HPA (Fig 8D). When aligned with OhrR, the apo-homodimer CD presented yet another different intermediate conformation (rmsd 2.9Å), apparently not ideally pre-configured for DNA binding, but which in solution can presumably readily adopt the AB conformation due to the intrinsic flexibility described above. NadR residues His7, Ser9, Asn11 and Phe25 are essential for regulation of NadA expression in vivo  While previous studies had correctly suggested the involvement of several NadR residues in ligand binding, the crystal structures presented here revealed additional residues with previously unknown roles in dimerization and/or binding to 4-HPA. To explore the functional involvement of these residues, we characterized the behavior of four new NadR mutants (H7A, S9A, N11A and F25A) in an in vivo assay using the previously described MC58-Δ1843 nadR-null mutant strain, which was complemented either by wild-type nadR or by the nadR mutants. NadA protein abundance levels were assessed by Western blotting to evaluate the ability of the NadR mutants to repress the nadA promoter, in the presence or absence of 4-HPA. The nadR H7A, S9A and F25A complemented strains showed hyper-repression of nadA expression in vivo, i.e. these mutants repressed nadA more efficiently than the NadR WT protein, either in the presence or absence of 4-HPA, while complementation with wild-type nadR resulted in high production of NadA only in the presence of 4-HPA (Fig 9). Interestingly, and on the contrary, the nadR N11A complemented strain showed hypo-repression (i.e. exhibited high expression of nadA both in absence and presence of 4-HPA). This mutagenesis data revealed that NadR residues His7, Ser9, Asn11 and Phe25 play key roles in the ligand-mediated regulation of NadR; they are each involved in the controlled de-repression of the nadA promoter and synthesis of NadA in response to 4-HPA in vivo. Structure-based point mutations shed light on ligand-induced regulation of NadR. Western blot analyses of wild-type (WT) strain (lanes 1–2) or isogenic nadR knockout strains (ΔNadR) complemented to express the indicated NadR WT or mutant proteins (lanes 3–12) or not complemented (lanes 13–14), grown in the presence (even lanes) or absence (odd lanes) of 5mM 4-HPA, showing NadA and NadR expression. Complementation of ΔNadR with WT NadR enables induction of nadA expression by 4-HPA. The H7A, S9A and F25A mutants efficiently repress nadA expression but are less ligand-responsive than WT NadR. The N11A mutant does not efficiently repress nadA expression either in presence or absence of 4-HPA. (The protein abundance levels of the meningococcal factor H binding protein (fHbp) were used as a gel loading control). Discussion NadA is a surface-exposed meningococcal protein contributing to pathogenesis, and is one of three main antigens present in the vaccine Bexsero. A detailed understanding of the in vitro repression of nadA expression by the transcriptional regulator NadR is important, both because it is a relevant disease-related model of how small-molecule ligands can regulate MarR family proteins and thereby impact bacterial virulence, and because nadA expression levels are linked to the prediction of vaccine coverage. The repressive activity of NadR can be relieved by hydroxyphenylacetate (HPA) ligands, and HDX-MS studies previously indicated that 4-HPA stabilizes dimeric NadR in a configuration incompatible with DNA binding. Despite these and other studies, the molecular mechanisms by which ligands regulate MarR family proteins are relatively poorly understood and likely differ depending on the specific ligand. Given the importance of NadR-mediated regulation of NadA levels in the contexts of meningococcal pathogenesis, we sought to characterize NadR, and its interaction with ligands, at atomic resolution. Firstly, we confirmed that NadR is dimeric in solution and demonstrated that it retains its dimeric state in the presence of 4-HPA, indicating that induction of a monomeric status is not the manner by which 4-HPA regulates NadR. These observations were in agreement with (i) a previous study of NadR performed using SEC and mass spectrometry, and (ii) crystallographic studies showing that several MarR homologues are dimeric. We also used structure-guided site-directed mutagenesis to identify an important conserved residue, Leu130, which stabilizes the NadR dimer interface, knowledge of which may also inform future studies to explore the regulatory mechanisms of other MarR family proteins. Secondly, we assessed the thermal stability and unfolding of NadR in the presence or absence of ligands. All DSC profiles showed a single peak, suggesting that a single unfolding event simultaneously disrupted the dimer and the monomer. HPA ligands specifically increased the stability of NadR. The largest effects were induced by the naturally-occurring compounds 4-HPA and 3Cl,4-HPA, which, in SPR assays, were found to bind NadR with KD values of 1.5 mM and 1.1 mM, respectively. Although these NadR/HPA interactions appeared rather weak, their distinct affinities and specificities matched their in vitro effects and their biological relevance appears similar to previous proposals that certain small molecules, including some antibiotics, in the millimolar concentration range may be broad inhibitors of MarR family proteins. Indeed, 4-HPA is found in human saliva and 3Cl,4-HPA is produced during inflammatory processes, suggesting that these natural ligands are encountered by N. meningitidis in the mucosa of the oropharynx during infections. It is also possible that NadR responds to currently unidentified HPA analogues. Indeed, in the NadR/4-HPA complex there was a water molecule close to the carboxylate group and also a small unfilled tunnel ~5Å long, both factors suggesting that alternative larger ligands could occupy the pocket. It is conceivable that such putative ligands may establish different bonding networks, potentially binding in a 2:2 ratio, rather than the 1:2 ratio observed herein. The ability to respond to various ligands might enable NadR in vivo to orchestrate multiple response mechanisms and modulate expression of genes other than nadA. Ultimately, confirmation of the relevance of each ligand will require a deeper understanding of the available concentration in vivo in the host niche during bacterial colonization and inflammation. Here, we determined the first crystal structures of apo-NadR and holo-NadR. These experimentally-determined structures enabled a new detailed characterization of the ligand-binding pocket. In holo-NadR, 4-HPA interacted directly with at least 11 polar and hydrophobic residues. Several, but not all, of these interactions were predicted previously by homology modelling combined with ligand docking in silico. Subsequently, we established the functional importance of His7, Ser9, Asn11 and Phe25 in the in vitro response of meningococcus to 4-HPA, via site-directed mutagenesis. More unexpectedly, the crystal structure revealed that only one molecule of 4-HPA was bound per NadR dimer. We confirmed this stoichiometry in solution using SPR methods. We also used heteronuclear NMR spectroscopy to detect substantial conformational changes of NadR occurring in solution upon addition of 4-HPA. Moreover, NMR spectra at 10°C suggested the existence of two distinct conformations of NadR in the vicinity of the ligand-binding pocket. More powerfully, our unique crystallographic observation of this ‘occupied vs unoccupied site’ asymmetry in the NadR/4-HPA interaction is, to our knowledge, the first example reported for a MarR family protein. Structural analyses suggested that ‘inward’ side chain positions of Met22, Phe25 and especially Arg43 precluded binding of a second ligand molecule. Such a mechanism indicates negative cooperativity, which may enhance the ligand-responsiveness of NadR. Comparisons of the NadR/4-HPA complex with available MarR family/salicylate complexes revealed that 4-HPA has a previously unobserved binding mode. Briefly, in the M. thermoautotrophicum MTH313 dimer, one molecule of salicylate binds in the pocket of each monomer, though with two rather different positions and orientations, only one of which (site-1) is thought to be biologically relevant (Fig 10A). In the S. tokodaii protein ST1710, salicylate binds to the same position in each monomer of the dimer, in a site equivalent to the putative biologically relevant site of MTH313 (Fig 10B). Unlike other MarR family proteins which revealed multiple ligand binding interactions, we observed only 1 molecule of 4-HPA bound to NadR, suggesting a more specific and less promiscuous interaction. In NadR, the single molecule of 4-HPA binds in a position distinctly different from the salicylate binding site: translated by \u003e 10 Å and with a 180° inverted orientation (Fig 10C). NadR shows a ligand binding site distinct from other MarR homologues. \n(A) A structural alignment of MTH313 chains A and B shows that salicylate is bound in distinct locations in each monomer; site-1 (thought to be the biologically relevant site) and site-2 differ by ~7Å (indicated by black dotted line) and also by ligand orientation. (B) A structural alignment of MTH313 chain A and ST1710 (pink) (Cα rmsd 2.3Å), shows that they bind salicylate in equivalent sites (differing by only ~3Å) and with the same orientation. (C) Addition of holo-NadR (chain B, blue) to the alignment reveals that bound 4-HPA differs in position by \u003e 10 Å compared to salicylate, and adopts a novel orientation. Interestingly, a crystal structure was previously reported for a functionally-uncharacterized meningococcal homologue of NadR, termed NMB1585, which shares 16% sequence identity with NadR. The two structures can be closely aligned (rmsd 2.3 Å), but NMB1585 appears unsuited for binding HPAs, since its corresponding ‘pocket’ region is occupied by several bulky hydrophobic side chains. It can be speculated that MarR family members have evolved separately to engage distinct signaling molecules, thus enabling bacteria to use the overall conserved MarR scaffold to adapt and respond to diverse changing environmental stimuli experienced in their natural niches. Alternatively, it is possible that other MarR homologues (e.g. NMB1585) may have no extant functional binding pocket and thus may have lost the ability to respond to a ligand, acting instead as constitutive DNA-binding regulatory proteins. The apo-NadR crystal structures revealed two dimers with slightly different conformations, most divergent in the DNA-binding domain. It is not unusual for a crystal structure to reveal multiple copies of the same protein in very slightly different conformations, which are likely representative of the lowest-energy conformations sampled by the dynamic ensemble of molecular states occurring in solution, and which likely have only small energetic differences, as described previously for MexR (a MarR protein) or more recently for the solute-binding protein FhuD2. Further, the holo-NadR structure was overall more different from the two apo-NadR structures (rmsd values ~1.3Å), suggesting that the ligand selected and stabilized yet another conformation of NadR. These observations suggest that 4-HPA, and potentially other similar ligands, can shift the molecular equilibrium, changing the energy barriers that separate active and inactive states, and stabilizing the specific conformation of NadR poorly suited to bind DNA. Comparisons of the apo- and holo-NadR structures revealed that the largest differences occurred in the DNA-binding helix α4. The shift of helix α4 in holo-NadR was also accompanied by rearrangements at the dimer interface, involving helices α1, α5, and α6, and this holo-form appeared poorly suited for DNA-binding when compared with the known OhrR:ohrA complex. While some flexibility of helix α4 was also observed in the two apo-structures, concomitant changes in the dimer interfaces were not observed, possibly due to the absence of ligand. One of the two conformations of apo-NadR appeared ideally suited for DNA-binding. Overall, these analyses suggest that the apo-NadR dimer has a pre-existing equilibrium that samples a variety of conformations, some of which are compatible with DNA binding. This intrinsically dynamic nature underlies the possibility for different conformations to inter-convert or to be preferentially selected by a regulatory ligand, as generally described in the ‘conformational selection’ model for protein-ligand interactions (the Monod-Wyman-Changeux model), rather than an ‘induced fit’ model (Koshland-Nemethy-Filmer). The noted flexibility may also explain how NadR can adapt to bind various DNA target sequences with slightly different structural features. Subsequently, upon ligand binding, holo-NadR adopts a structure less suited for DNA-binding and this conformation is selected and stabilized by a network of protein-ligand interactions and concomitant rearrangements at the NadR holo dimer interface. In an alternative and less extensive manner, the binding of two salicylate molecules to the M. thermoautotrophicum protein MTH313 appeared to induce large changes in the wHTH domain, which was associated with reduced DNA-binding activity. Here we have presented two new crystal structures of the transcription factor, NadR, which regulates expression of the meningococcal surface protein, virulence factor and vaccine antigen NadA. Detailed structural analyses provided a molecular explanation for the ligand-responsive regulation by NadR on the majority of the promoters of meningococcal genes regulated by NadR, including nadA. Intriguingly, NadR exhibits a reversed regulatory mechanism on a second class of promoters, including mafA of the multiple adhesin family–i.e. NadR represses these genes in the presence but not absence of 4-HPA. The latter may influence the surface abundance or secretion of maf proteins, an emerging class of highly conserved meningococcal putative adhesins and toxins with many important roles. Further work is required to investigate how the two different promoter types influence the ligand-responsiveness of NadR during bacterial infection and may provide insights into the regulatory mechanisms occurring during these host-pathogen interactions. Ultimately, knowledge of the ligand-dependent activity of NadR will continue to deepen our understanding of nadA expression levels, which influence meningococcal pathogenesis. Materials and Methods Bacterial strains, culture conditions and mutant generation In this study we used N. meningitidis MC58 wild type strain and related mutant derivatives. The MC58 isolate was kindly provided to us by Professor E. Richard Moxon, University of Oxford, UK, and was previously submitted to the Meningococcal Reference Laboratory, Manchester, UK. Strains were routinely cultured, stocked, and transformed as described previously. To generate N. meningitidis MC58 mutant strains expressing only the amino acid substituted forms of NadR, plasmids containing the sequence of nadR mutated to insert alanine codons to replace His7, Ser9, Asn11 or Phe25 were constructed using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene). The nadR gene (also termed NMB1843) was mutated in the pComEry-1843 plasmid using couples of mutagenic primers (forward and reverse). The resulting plasmids were named pComEry-1843H7A, -1843S9A, -1843N11A or -1843F25A, and contained a site-directed mutant allele of the nadR gene, in which the selected codons were respectively substituted by a GCG alanine codon, and were used for transformation of the MC-Δ1843 strain. Total lysates from single colonies of all transformants were used as a template for PCR analysis to confirm the correct insertion by double homologous recombinant event. When indicated, bacterial strains were grown in presence of 5 mM 4-HPA (MW 152, Sigma-Aldrich). Molecular cloning The preparation of the expression construct enabling production of soluble NadR with an N-terminal His-tag followed by a thrombin cleavage site (MGSSHHHHHHSSGLVPR↓GSH-) (where the arrow indicates the cleavage site) and then NadR residues M1-S146 (Uniprot code Q7DD70), and methods to generate site-directed mutants, were described previously. Briefly, site-directed mutagenesis was performed using two overlapping primers containing the desired mutation to amplify pET15b containing several NadR variants. (Full oligonucleotide sequences of primers are available upon request). 1–10 ng of plasmid DNA template were amplified using Kapa HiFi DNA polymerase (Kapa Biosystems) and with the following cycling conditions: 98°C for 5 min, 15 cycles of (98°C for 30 s, 60°C for 30 s, 72°C for 6 min) followed by a final extension of 10 min at 72°C. Residual template DNA was digested by 30 min incubation with FastDigest DpnI (Thermo Scientific) at 37°C and 1 μl of this reaction was used to transform E. coli DH5α competent cells. The full recombinant tagged NadR protein generated contained 166 residues, with a theoretical MW of 18746, while after thrombin-cleavage the untagged protein contained 149 residues, with a theoretical MW of 16864. Protein production and purification The NadR expression constructs (wild-type or mutant clones) were transformed into E. coli BL21 (DE3) cells and were grown at 37°C in Luria-Bertani (LB) medium supplemented with 100 μg/mL ampicillin, until an OD600 of 0.5 was reached. Target protein production was induced by the addition of 1 mM IPTG followed by incubation with shaking overnight at 21°C. For production of the selenomethionine (SeMet) derivative form of NadR for crystallization studies, essentially the same procedure was followed, but using the E. coli B834 strain grown in a modified M9 minimal medium supplemented with 40 mg/L L-SeMet. For production of 15N-labeled NadR for NMR analyses, the EnPresso B Defined Nitrogen-free medium (Sigma-Aldrich) was used; in brief, BL21 (DE3) cells were grown in BioSilta medium at 30°C for 30 h, and production of the 15N-labeled NadR was enabled by the addition of 2.5 g/L 15NH4Cl and further incubation for 2 days. Cells were harvested by centrifugation (6400 g, 30 min, 4°C), resuspended in 20 mM HEPES pH 8.0, 300 mM NaCl, 20 mM imidazole, and were lysed by sonication (Qsonica Q700). Cell lysates were clarified by centrifugation at 2800 g for 30 min, and the supernatant was filtered using a 0.22 μm membrane (Corning filter system) prior to protein purification. NadR was purified by affinity chromatography using an AKTA purifier (GE Healthcare). All steps were performed at room temperature (18–26°C), unless stated otherwise. The filtered supernatant was loaded onto an Ni-NTA resin (5 mL column, GE Healthcare), and NadR was eluted using 4 steps of imidazole at 20, 30, 50 and 250 mM concentration, at a flow rate of 5 mL/min. Eluted fractions were examined by reducing and denaturing SDS-PAGE analysis. Fractions containing NadR were identified by a band migrating at ~17 kDa, and were pooled. The N-terminal 6-His tag was removed enzymatically using the Thrombin CleanCleave Kit (Sigma-Aldrich). Subsequently, the sample was reloaded on the Ni-NTA resin to capture the free His tag (or unprocessed tagged protein), thus allowing elution in the column flow-through of tagless NadR protein, which was used in all subsequent studies. The NadR sample was concentrated and loaded onto a HiLoad Superdex 75 (16/60) preparative size-exclusion chromatography (SEC) column equilibrated in buffer containing 20 mM HEPES pH 8.0, 150 mM NaCl, at a flow-rate of 1 mL/min. NadR protein was collected and the final yield of purified protein obtained from 0.5 L LB growth medium was approximately 8 mg (~2 mg protein per g wet biomass). Samples were used immediately for crystallization or analytical experiments, or were frozen for storage at -20°C. SE-HPLC/MALLS analyses MALLS analyses were performed online with SE-HPLC using a Dawn TREOS MALLS detector (Wyatt Corp., Santa Barbara, CA, USA) and an incident laser wavelength of 658 nm. The intensity of the scattered light was measured at 3 angles simultaneously. Data analysis was performed using the Astra V software (Wyatt) to determine the weighted-average absolute molecular mass (MW), the polydispersity index (MW/Mn) and homogeneity (Mz/Mn) for each oligomer present in solution. Normalization of the MALLS detectors was performed in each analytical session by use of bovine serum albumin. Differential scanning calorimetry The thermal stability of NadR proteins was assessed by differential scanning calorimetry (DSC) using a MicroCal VP-Capillary DSC instrument (GE Healthcare). NadR samples were prepared at a protein concentration of 0.5 mg/mL (~30 μM) in buffer containing 20 mM HEPES, 300 mM NaCl, pH 7.4, with or without 6 mM HPA or salicylate. The DSC temperature scan ranged from 10°C to 110°C, with a thermal ramping rate of 200°C per hour and a 4 second filter period. Data were analyzed by subtraction of the reference data for a sample containing buffer only, using the Origin 7 software. All experiments were performed in triplicate, and mean values of the melting temperature (Tm) were determined. Surface plasmon resonance (SPR) Determination of equilibrium dissociation constant, KD  Surface plasmon resonance binding analyses were performed using a Biacore T200 instrument (GE Healthcare) equilibrated at 25°C. The ligand (NadR) was covalently immobilized by amine-coupling on a CM-5 sensor chip (GE Healthcare), using 20 μg/mL purified protein in 10 mM sodium acetate buffer pH 5, injected at 10 μl/min for 120 s until ~9000 response units (RU) were captured. A high level of ligand immobilization was required due to the small size of the analytes. An unmodified surface was used as the reference channel. Titrations with analytes (HPAs or salicylate) were performed with a flow-rate of 30 μl/min, injecting the compounds in a concentration range of 10 μM to 20 mM, using filtered running buffer containing Phosphate Buffered Saline (PBS) with 0.05% Tween-20, pH 7.4. Following each injection, sensor chip surfaces were regenerated with a 30-second injection of 10 mM Glycine pH 2.5. Each titration series contained 20 analyte injections and was performed in triplicate. Titration experiments with long injection phases (\u003e 15 mins) were used to enable steady-state analyses. Data were analyzed using the BIAcore T200 evaluation software and the steady-state affinity model. A buffer injection was subtracted from each curve, and reference sensorgrams were subtracted from experimental sensorgrams to yield curves representing specific binding. The equilibrium dissociation constant, KD, was determined from the plot of RUeq against analyte concentration (S2 Fig), as described previously. Determination of binding stoichiometry: From each plot of RUeq against analyte concentration, obtained from triplicate experiments, the Rmax value (maximum analyte binding capacity of the surface) was extrapolated from the experimental data (S2 Fig). Stoichiometry was calculated using the molecular weight of dimeric NadR as ligand molecule (MWligand) and the molecular weights of the HPA analyte molecules (MWanalyte), and the following equation:  where Rligand is recorded directly from the sensorgram during ligand immobilization prior to the titration series, as described previously. The stoichiometry derived therefore represented the number of HPA molecules bound to one dimeric NadR protein. Crystallization of NadR in the presence or absence of 4-HPA Purified NadR was concentrated to 2.7 mg/mL (~160 μM) using a centrifugal concentration device (Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-10 membrane with cut-off size 10 kDa; Millipore) running at 600 g in a bench top centrifuge (Thermo Scientific IEC CL40R) refrigerated at 2–8°C. To prepare holo-NadR samples, HPA ligands were added at a 200-fold molar excess prior to the centrifugal concentration step. The concentrated holo- or apo-NadR was subjected to crystallization trials performed in 96-well low-profile Intelli-Plates (Art Robbins) or 96-well low-profile Greiner crystallization plates, using a nanodroplet sitting-drop vapour-diffusion format and mixing equal volumes (200 nL) of protein samples and crystallization buffers using a Gryphon robot (Art Robbins). Crystallization trays were incubated at 20°C. Crystals of apo-NadR were obtained in 50% PEG 3350 and 0.13 M di-Ammonium hydrogen citrate, whereas crystals of SeMet–NadR in complex with 4-HPA grew in condition H4 of the Morpheus screen (Molecular Dimensions), which contains 37.5% of the pre-mixed precipitant stock MPD_P1K_PEG 3350, buffer system 1 and 0.1 M amino acids, at pH 6.5. All crystals were mounted in cryo-loops using 10% ethylene glycol or 10% glycerol as cryo-protectant before cooling to 100 K for data collection. X-ray diffraction data collection and structure determination X-ray diffraction data from crystals of apo-NadR and SeMet–NadR/4-HPA were collected on beamline PXII-X10SA of the Swiss Light Source (SLS) at the Paul Scherrer Institut (PSI), Villigen, Switzerland. All diffraction data were processed with XDS and programs from the CCP4 suite. Crystals of apo-NadR and 4-HPA-bound SeMet-NadR belonged to space group P43 21 2 (see Table 2). Apo-NadR crystals contained four molecules (two dimers) in the asymmetric unit (Matthews coefficient 2.25 Å3 Da−1, for a solvent content of 45%), while crystals of SeMet–NadR/4-HPA contained two molecules (one dimer) in the asymmetric unit (Matthews coefficient 1.98 Å3 Da−1, for a solvent content of 38%). In solving the holo-NadR structure, an initial and marginal molecular replacement (MR) solution was obtained using as template search model the crystal structure of the transcriptional regulator PA4135 (PBD entry 2FBI), with which NadR shares ~54% sequence identity. This solution was combined with SAD data to aid identification of two selenium sites in NadR, using autosol in phenix and this allowed generation of high-quality electron density maps that were used to build and refine the structure of the complex. Electron densities were clearly observed for almost the entire dimeric holo-NadR protein, except for a new N-terminal residues and residues 88–90 of chain B. The crystal structure of apo-NadR was subsequently solved by MR in Phaser at 2.7 Å, using the final refined model of SeMet-NadR/4-HPA as the search model. For apo-NadR, electron densities were clearly observed for almost the entire protein, although residues 84–91 of chains A, C, and D, and residues 84–90 of chain B lacked densities suggesting local disorder. Both structures were refined and rebuilt using phenix and Coot, and structural validation was performed using Molprobity. Data collection and refinement statistics are reported in Table 2. Atomic coordinates of the two NadR structures have been deposited in the Protein Data Bank, with entry codes 5aip (NadR bound to 4-HPA) and 5aiq (apo-NadR). All crystallographic software was compiled, installed and maintained by SBGrid. NMR spectroscopy For heteronuclear NMR experiments, the NadR protein concentration used was 85 μM (~ 1.4 mg/mL) in a solution containing 100 mM sodium phosphate buffer (90% H2O and 10% D2O) and 200 mM NaCl, prepared in the apo-form or in the presence of a 200-fold molar excess of 4-HPA, at pH 6.5. The stability, integrity and dimeric state of the protein in the NMR buffer was confirmed by analytical SEC (Superdex 75, 10/300 column) prior to NMR studies. 1H-15N transverse relaxation-optimized spectroscopy (TROSY)-heteronuclear single quantum coherence (HSQC) experiments on apo-NadR and NadR in the presence of 4-HPA were acquired using an Avance 950 Bruker spectrometer, operating at a proton frequency of 949.2 MHz and equipped with triple resonance cryogenically-cooled probe at two different temperatures (298 K and 283 K). 1H-15N TROSY-HSQC experiments were recorded for 12 h, with a data size of 1024 x 232 points. Spectra were processed using the Bruker TopSpin 2.1 and 3.1 software packages. Western blot Western blot analysis was performed as described previously. Supporting Information References Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing  Neisseria adhesin A variation and revised nomenclature scheme  Neisseria meningitidis NadA is a new invasin which promotes bacterial adhesion to and penetration into human epithelial cells NadA, a novel vaccine candidate of Neisseria meningitidis  Structure of the meningococcal vaccine antigen NadA and epitope mapping of a bactericidal antibody A multi-component meningococcal serogroup B vaccine (4CMenB): the clinical development program Expression of the meningococcal adhesin NadA is controlled by a transcriptional regulator of the MarR family . MtrR control of a transcriptional regulatory pathway in Neisseria meningitidis that influences expression of a gene (nadA) encoding a vaccine candidate A novel phase variation mechanism in the meningococcus driven by a ligand-responsive repressor and differential spacing of distal promoter elements In the NadR Regulon, Adhesins and Diverse Meningococcal Functions Are Regulated in Response to Signals in Human Saliva A new family of secreted toxins in pathogenic Neisseria species Emergence of a new epidemic Neisseria meningitidis serogroup A Clone in the African meningitis belt: high-resolution picture of genomic changes that mediate immune evasion The mar regulon: multiple resistance to antibiotics and other toxic chemicals MarR family transcription factors Regulation of virulence by members of the MarR/SlyA family Molecular mechanisms of ligand-mediated attenuation of DNA binding by MarR family transcriptional regulators Structural insight on the mechanism of regulation of the MarR family of proteins: high-resolution crystal structure of a transcriptional repressor from Methanobacterium thermoautotrophicum  ST1710-DNA complex crystal structure reveals the DNA binding mechanism of the MarR family of regulators The presence of 4-hydroxyphenylacetic acid in human saliva and the possibility of its nitration by salivary nitrite in the stomach Transcriptional regulation of the nadA gene in Neisseria meningitidis impacts the prediction of coverage of a multicomponent meningococcal serogroup B vaccine Structural Insight into the Mechanism of DNA-Binding Attenuation of the Neisserial Adhesin Repressor NadR by the Small Natural Ligand 4-Hydroxyphenylacetic Acid Binding of purified multiple antibiotic-resistance repressor protein (MarR) to mar operator sequences Inference of macromolecular assemblies from crystalline state BiaCore analysis of leptin-leptin receptor interaction: evidence for 1:1 stoichiometry Analytical shape computation of macromolecules: I. Molecular area and volume through alpha shape Structure of an OhrR-ohrA operator complex reveals the DNA binding mechanism of the MarR family Qualitative and quantitative assessment of meningococcal antigens to evaluate the potential strain coverage of protein-based vaccines The metabolism and dechlorination of chlorotyrosine in vivo The structure of NMB1585, a MarR-family regulator from Neisseria meningitidis  Crystal structure of the MexR repressor of the mexRAB-oprM multidrug efflux operon of Pseudomonas aeruginosa  Structural and functional characterization of the Staphylococcus aureus virulence factor and vaccine candidate FhuD2 Crystal and solution structure analysis of FhuD2 from Staphylococcus aureus in multiple unliganded conformations and bound to ferrioxamine-B Conformational selection or induced fit? 50 years of debate resolved Point mutation in meningococcal por A gene associated with increased endemic disease Mechanistic implications for LDL receptor degradation from the PCSK9/LDLR structure at neutral pH Xds The CCP4 suite: programs for protein crystallography PHENIX: a comprehensive Python-based system for macromolecular structure solution Phaser crystallographic software Features and development of Coot MolProbity: all-atom structure validation for macromolecular crystallography Collaboration gets the most out of software Scaling and assessment of data quality PDBsum 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+[{"sourceid":"4848090","sourcedb":"","project":"","target":"","text":"Mechanistic insight into a peptide hormone signaling complex mediating floral organ abscission Plants constantly renew during their life cycle and thus require to shed senescent and damaged organs. Floral abscission is controlled by the leucine-rich repeat receptor kinase (LRR-RK) HAESA and the peptide hormone IDA. It is unknown how expression of IDA in the abscission zone leads to HAESA activation. Here we show that IDA is sensed directly by the HAESA ectodomain. Crystal structures of HAESA in complex with IDA reveal a hormone binding pocket that accommodates an active dodecamer peptide. A central hydroxyproline residue anchors IDA to the receptor. The HAESA co-receptor SERK1, a positive regulator of the floral abscission pathway, allows for high-affinity sensing of the peptide hormone by binding to an Arg-His-Asn motif in IDA. This sequence pattern is conserved among diverse plant peptides, suggesting that plant peptide hormone receptors may share a common ligand binding mode and activation mechanism. DOI: http://dx.doi.org/10.7554/eLife.15075.001 eLife digest Plants can shed their leaves, flowers or other organs when they no longer need them. But how does a leaf or a flower know when to let go? A receptor protein called HAESA is found on the surface of the cells that surround a future break point on the plant. When its time to shed an organ, a hormone called IDA instructs HAESA to trigger the shedding process. However, the molecular details of how IDA triggers organ shedding are not clear. The shedding of floral organs (or leaves) can be easily studied in a model plant called Arabidopsis. Santiago et al. used protein biochemistry, structural biology and genetics to uncover how the IDA hormone activates HAESA. The experiments show that IDA binds directly to a canyon shaped pocket in HAESA that extends out from the surface of the cell. IDA binding to HAESA allows another receptor protein called SERK1 to bind to HAESA, which results in the release of signals inside the cell that trigger the shedding of organs. The next step following on from this work is to understand what signals are produced when IDA activates HAESA. Another challenge will be to find out where IDA is produced in the plant and what causes it to accumulate in specific places in preparation for organ shedding. DOI: http://dx.doi.org/10.7554/eLife.15075.002 Introduction The HAESA ectodomain folds into a superhelical assembly of 21 leucine-rich repeats. (A) SDS PAGE analysis of the purified Arabidopsis thaliana HAESA ectodomain (residues 20–620) obtained by secreted expression in insect cells. The calculated molecular mass is 65.7 kDa, the actual molecular mass obtained by mass spectrometry is 74,896 Da, accounting for the N-glycans. (B) Ribbon diagrams showing front (left panel) and side views (right panel) of the isolated HAESA LRR domain. The N- (residues 20–88) and C-terminal (residues 593–615) capping domains are shown in yellow, the central 21 LRR motifs are in blue and disulphide bonds are highlighted in green (in bonds representation). (C) Structure based sequence alignment of the 21 leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. Conserved hydrophobic residues are shaded in gray, N-glycosylation sites visible in our structures are highlighted in blue, cysteine residues involved in disulphide bridge formation in green. (D) Asn-linked glycans mask the N-terminal portion of the HAESA ectodomain. Oligomannose core structures (containing two N-actylglucosamines and three terminal mannose units) as found in Trichoplusia ni cells and in plants were modeled onto the seven glycosylation sites observed in our HAESA structures, to visualize the surface areas potentially not masked by carbohydrate. The HAESA ectodomain is shown in blue (in surface representation), the glycan structures are shown in yellow. Molecular surfaces were calculated with the program MSMS, with a probe radius of 1.5 Å. DOI:\nhttp://dx.doi.org/10.7554/eLife.15075.004 Hydrophobic contacts and a hydrogen-bond network mediate the interaction between HAESA and the peptide hormone IDA. (A) Details of the IDA binding pocket. HAESA is shown in blue (ribbon diagram), the C-terminal Arg-His-Asn motif (left panel), the central Hyp anchor (center) and the N-terminal Pro-rich motif in IDA (right panel) are shown in yellow (in bonds representation). HAESA interface residues are shown as sticks, selected hydrogen bond interactions are denoted as dotted lines (in magenta). (B) View of the complete IDA (in bonds representation, in yellow) binding pocket in HAESA (surface view, in blue). Orientation as in (A). (C) Structure based sequence alignment of leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. Residues mediating hydrophobic interactions with the IDA peptide are highlighted in blue, residues contributing to hydrogen bond interactions and/or salt bridges are shown in red. The IDA binding pocket covers LRRs 2–14 and all residues originate from the inner surface of the HAESA superhelix. DOI:\nhttp://dx.doi.org/10.7554/eLife.15075.005 The IDA-HAESA and SERK1-HAESA complex interfaces are conserved among HAESA and HAESA-like proteins from different plant species. Structure-based sequence alignment of the HAESA family members: Arabidopsis thaliana HAESA (Uniprot (http://www.uniprot.org) ID P47735), Arabidopsis thaliana HSL2 (Uniprot ID C0LGX3), Capsella rubella HAESA (Uniprot ID R0F2U6), Citrus clementina HSL2 (Uniprot ID V4U227), Vitis vinifera HAESA (Uniprot ID F6HM39). The alignment includes a secondary structure assignment calculated with the program DSSP and colored according to Figure 1, with the N- and C-terminal caps and the 21 LRR motifs indicated in orange and blue, respectively. Cysteine residues engaged in disulphide bonds are depicted in green. HAESA residues interacting with the IDA peptide and/or the SERK1 co-receptor kinase ectodomain are highlighted in blue and orange, respectively. DOI:\nhttp://dx.doi.org/10.7554/eLife.15075.006 The peptide hormone IDA binds to the HAESA LRR ectodomain. (A) Multiple sequence alignment of selected IDA family members. The conserved PIP motif is highlighted in yellow, the central Hyp in blue. The PKGV motif present in our N-terminally extended IDA peptide is highlighted in red. (B) Isothermal titration calorimetry of the HAESA ectodomain vs. IDA and including the synthetic peptide sequence. (C) Structure of the HAESA – IDA complex with HAESA shown in blue (ribbon diagram). IDA (in bonds representation, surface view included) is depicted in yellow. The peptide binding pocket covers HAESA LRRs 2–14. (D) Close-up view of the entire IDA (in yellow) peptide binding site in HAESA (in blue). Details of the interactions between the central Hyp anchor in IDA and the C-terminal Arg-His-Asn motif with HAESA are highlighted in (E) and (F), respectively. Hydrogren bonds are depicted as dotted lines (in magenta), a water molecule is shown as a red sphere. DOI:\nhttp://dx.doi.org/10.7554/eLife.15075.003 During their growth, development and reproduction plants use cell separation processes to detach no-longer required, damaged or senescent organs. Abscission of floral organs in Arabidopsis is a model system to study these cell separation processes in molecular detail. The LRR-RKs HAESA (greek: to adhere to) and HAESA-LIKE 2 (HSL2) redundantly control floral abscission. Loss-of-function of the secreted small protein INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) causes floral organs to remain attached while its over-expression leads to premature shedding. Full-length IDA is proteolytically processed and a conserved stretch of 20 amino-acids (termed EPIP) can rescue the IDA loss-of-function phenotype (Figure 1A). It has been demonstrated that a dodecamer peptide within EPIP is able to activate HAESA and HSL2 in transient assays in tobacco cells. This sequence motif is highly conserved among IDA family members (IDA-LIKE PROTEINS, IDLs) and contains a central Pro residue, presumed to be post-translationally modified to hydroxyproline (Hyp; Figure 1A). The available genetic and biochemical evidence suggests that IDA and HAESA together control floral abscission, but it is poorly understood if IDA is directly sensed by the receptor kinase HAESA and how IDA binding at the cell surface would activate the receptor. Results IDA directly binds to the LRR domain of HAESA Active IDA-family peptide hormones are hydroxyprolinated dodecamers. Close-up views of (A) IDA, (B) the N-terminally extended PKGV-IDA and (C) IDL1 bound to the HAESA hormone binding pocket (in bonds representation, in yellow) and including simulated annealing 2Fo–Fc omit electron density maps contoured at 1.0 σ. Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. no detectable binding). (E) Structural superposition of the active IDA (in bonds representation, in gray) and IDL1 peptide (in yellow) hormones bound to the HAESA ectodomain. Root mean square deviation (r.m.s.d.) is 1.0 Å comparing 100 corresponding atoms. DOI:\nhttp://dx.doi.org/10.7554/eLife.15075.007 The receptor kinase SERK1 acts as a HAESA co-receptor and promotes high-affinity IDA sensing. (A) Petal break-strength assays measure the force (expressed in gram equivalents) required to remove the petals from the flower of serk mutant plants compared to haesa/hsl2 mutant and Col-0 wild-type flowers. Petal break-strength is measured from positions 1 to 8 along the primary inflorescence where positions 1 is defined as the flower at anthesis (n=15, bars=SD). This treatment-by-position balanced two-way layout was analyzed separately per position, because of the serious interaction, by means of a Dunnett-type comparison against the Col-0 control, allowing for heterogeneous variances. Petal break-strength was found significantly increased in almost all positions (indicated with a *) for haesa/hsl2 and serk1-1 mutant plants with respect to the Col-0 control. Calculations were performed in R (version 3.2.3). (B) Analytical size-exclusion chromatography. The HAESA LRR domain elutes as a monomer (black dotted line), as does the isolated SERK1 ectodomain (blue dotted line). A HAESA – IDA – SERK1 complex elutes as an apparent heterodimer (red line), while a mixture of HAESA and SERK1 yields two isolated peaks that correspond to monomeric HAESA and SERK1, respectively (black line). Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). A SDS PAGE of the peak fractions is shown alongside. Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. (C) Isothermal titration calorimetry of wild-type and Hyp64→Pro IDA versus the HAESA and SERK1 ectodomains. The titration of IDA wild-type versus the isolated HAESA ectodomain from Figure 1B is shown for comparison (red line; n.d. no detectable binding) (D) Analytical size-exclusion chromatography in the presence of the IDA Hyp64→Pro mutant peptide reveals no complex formation between HAESA and SERK1 ectodomains. A SDS PAGE of the peak fractions is shown alongside. (E) In vitro kinase assays of the HAESA and SERK1 kinase domains. Wild-type HAESA and SERK1 kinase domains (KDs) exhibit auto-phosphorylation activities (lanes 1 + 3). Mutant (m) versions, which carry point mutations in their active sites (Asp837HAESA→Asn, Asp447SERK1→Asn) possess no autophosphorylation activity (lanes 2+4). Transphosphorylation activity from the active kinase to the mutated form can be observed in both directions (lanes 5+6). A coomassie-stained gel loading control is shown below. DOI:\nhttp://dx.doi.org/10.7554/eLife.15075.008 Crystallographic data collection, phasing and refinement statistics for the isolated A. thaliana HAESA ectodomain. DOI:\nhttp://dx.doi.org/10.7554/eLife.15075.009 \tHAESA NaI shortsoak\tHAESA apo\t \tPDB-ID\t\t5IXO\t \tData collection\t\t\t \tSpace group\tP31 21\tP31 21\t \tCell dimensions\t\t\t \ta, b, c (Å)\t148.55, 148.55, 58.30\t149.87, 149.87, 58.48\t \tα, β, γ (°)\t90, 90, 120\t90, 90, 120\t \tResolution (Å)\t48.63–2.39 (2.45–2.39)\t45.75–1.74 (1.85–1.74)\t \tRmeas#\t0.096 (0.866)\t0.038 (1.02)\t \tCC(1/2)#\t100/86.6\t100/75.6\t \tI/σ I#\t27.9 (4.9)\t18.7 (1.8)\t \tCompleteness (%)#\t99.9 (98.6)\t99.6 (97.4)\t \tRedundancy#\t53.1 (29.9)\t14.4 (14.0)\t \tWilson B-factor (Å2)#\t84.45\t81.10\t \tRefinement\t\t\t \tResolution (Å)\t\t45.75 – 1.74\t \tNo. reflections\t\t71,213\t \tRwork/Rfree$\t\t0.188/0.218\t \tNo. atoms\t\t\t \tProtein/glycan\t\t4,533/126\t \tWater\t\t71\t \tRes. B-factors (Å2)$\t\t\t \tProtein\t\t77.54\t \tGlycan\t\t95.98\t \tWater\t\t73.20\t \tR.m.s deviations$\t\t\t \tBond lengths (Å)\t\t0.0095\t \tBond angles (°)\t\t1.51\t \t Highest resolution shell is shown in parenthesis. #As defined in XDS. $As defined in Refmac5. Crystallographic data collection and refinement statistics for the HAESA – IDA, – PKGV-IDA, – IDL1 and – IDA – SERK1 complexes. DOI:\nhttp://dx.doi.org/10.7554/eLife.15075.010 \tHAESA – IDA\tHAESA – PKGV-IDA\tHAESA – IDL1\tHAESA – IDA – SERK1\t \tPDB-ID\t5IXQ\t5IXT\t5IYN\t5IYX\t \tData collection\t\t\t\t\t \tSpace group\tP31 21\tP31 21\tP31 21\tP212121\t \tCell dimensions\t\t\t\t\t \ta, b, c (Å)\t148.55, 148.55, 58.30\t148.92, 148.92, 58.02\t150.18, 150.18, 60.07\t74.51, 100.46, 142.76\t \tα, β, γ (°)\t90, 90, 120\t90, 90, 120\t90, 90, 120\t90, 90, 90\t \tResolution (Å)\t48.54–1.86 (1.97–1.86)\t48.75–1.94 (2,06–1.94)\t49.16–2.56 (2.72–2.56)\t47.59–2.43 (2.57–2.43)\t \tRmeas#\t0.057 (1.35)\t0.037 (0.97)\t0.056 (1.27)\t0.113 (1.37)\t \tCC(1/2)#\t100/77.9\t100/80.3\t100/89.5\t100/77.6\t \tI/σI#\t16.7 (2.0)\t20.9 (2.4)\t26.0 (1.9)\t16.12 (2.0)\t \tCompleteness# (%)\t99.8 (98.6)\t99.4 (97.9)\t99.5 (98.8)\t99.4 (96.4s\t \tRedundancy#\t20.3 (19.1)\t11.2 (11.1)\t14.7 (14.7)\t9.7 (9.3)\t \tWilson B-factor (Å2)#\t80.0\t81.7\t89.5\t59.3\t \tRefinement\t\t\t\t\t \tResolution (Å)\t48.54–1.86\t48.75–1.94\t49.16–2.56\t47.59–2.43\t \tNo. reflections\t58,551\t51,557\t23,835\t38,969\t \tRwork/Rfree$\t0.190/0.209\t0.183/0.208\t0.199/0.236\t0.199/0.235\t \tNo. atoms\t\t\t\t\t \tProtein/Glycan\t4,541/176\t4,545/176\t4,499/176\t5,965/168\t \tPeptide\t93\t93\t90\t112\t \tWater\t39\t40\t9\t136\t \tRes. B-factors (Å2)$\t\t\t\t\t \tProtein/Glycan\t79.48/109.02\t79.63/113.24\t102.12/132.49\t60.05/73.48\t \tPeptide\t87.19\t89.50\t125.74\t51.06\t \tWater\t75.32\t71.92\t74.65\t51.47\t \tR.m.s deviations$\t\t\t\t\t \tBond lengths (Å)\t0.0087\t0.0091\t0.0081\t0.0074\t \tBond angles (°)\t1.48\t1.47\t1.36\t1.34\t \t Highest resolution shell is shown in parenthesis. #As defined in XDS. $As defined in Refmac5. We purified the HAESA ectodomain (residues 20–620) from baculovirus-infected insect cells (Figure 1—figure supplement 1A, see Materials and methods) and quantified the interaction of the ~75 kDa glycoprotein with synthetic IDA peptides using isothermal titration calorimetry (ITC). A Hyp-modified dodecamer comprising the highly conserved PIP motif in IDA (Figure 1A) interacts with HAESA with 1:1 stoichiometry (N) and with a dissociation constant (Kd) of ~20 μM (Figure 1B). We next determined crystal structures of the apo HAESA ectodomain and of a HAESA-IDA complex, at 1.74 and 1.86 Å resolution, respectively (Figure 1C; Figure 1—figure supplement 1B–D; Tables 1,2). IDA binds in a completely extended conformation along the inner surface of the HAESA ectodomain, covering LRRs 2–14 (Figure 1C,D, Figure 1—figure supplement 2). The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8–10, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1—figure supplement 3). The restricted size of the Hyp pocket suggests that IDA does not require arabinosylation of Hyp64IDA for activity in vivo, a modification that has been reported for Hyp residues in plant CLE peptide hormones. The C-terminal Arg-His-Asn motif in IDA maps to a cavity formed by HAESA LRRs 11–14 (Figure 1D,F). The COO- group of Asn69IDA is in direct contact with Arg407HAESA and Arg409HAESA and HAESA cannot bind a C-terminally extended IDA-SFVN peptide (Figures 1D,F, 2D). This suggests that the conserved Asn69IDA may constitute the very C-terminus of the mature IDA peptide in planta and that active IDA is generated by proteolytic processing from a longer pre-protein. Mutation of Arg417HSL2 (which corresponds to Arg409HAESA) causes a loss-of-function phenotype in HSL2, which indicates that the peptide binding pockets in different HAESA receptors have common structural and sequence features. Indeed, we find many of the residues contributing to the formation of the IDA binding surface in HAESA to be conserved in HSL2 and in other HAESA-type receptors in different plant species (Figure 1—figure supplement 3). A N-terminal Pro-rich motif in IDA makes contacts with LRRs 2–6 of the receptor (Figure 1D, Figure 1—figure supplement 2A–C). Other hydrophobic and polar interactions are mediated by Ser62IDA, Ser65IDA and by backbone atoms along the IDA peptide (Figure 1D, Figure 1—figure supplement 2A–C). HAESA specifically senses IDA-family dodecamer peptides We next investigated whether HAESA binds N-terminally extended versions of IDA. We obtained a structure of HAESA in complex with a PKGV-IDA peptide at 1.94 Å resolution (Table 2). In this structure, no additional electron density accounts for the PKGV motif at the IDA N-terminus (Figure 2A,B). Consistently, PKGV-IDA and IDA have similar binding affinities in our ITC assays, further indicating that HAESA senses a dodecamer peptide comprising residues 58-69IDA (Figure 2D). We next tested if HAESA binds other IDA peptide family members. IDL1, which can rescue IDA loss-of-function mutants when introduced in abscission zone cells, can also be sensed by HAESA, albeit with lower affinity (Figure 2D). A 2.56 Å co-crystal structure with IDL1 reveals that different IDA family members use a common binding mode to interact with HAESA-type receptors (Figure 2A–C,E, Table 2). We do not detect interaction between HAESA and a synthetic peptide missing the C-terminal Asn69IDA (ΔN69), highlighting the importance of the polar interactions between the IDA carboxy-terminus and Arg407HAESA/Arg409HAESA (Figures 1F, 2D). Replacing Hyp64IDA, which is common to all IDLs, with proline impairs the interaction with the receptor, as does the Lys66IDA/Arg67IDA → Ala double-mutant discussed below (Figure 1A, 2D). Notably, HAESA can discriminate between IDLs and functionally unrelated dodecamer peptides with Hyp modifications, such as CLV3 (Figures 2D, 7). The co-receptor kinase SERK1 allows for high-affinity IDA sensing Our binding assays reveal that IDA family peptides are sensed by the isolated HAESA ectodomain with relatively weak binding affinities (Figures 1B, 2A–D). It has been recently reported that SOMATIC EMBRYOGENESIS RECEPTOR KINASES (SERKs) are positive regulators of floral abscission and can interact with HAESA and HSL2 in an IDA-dependent manner. As all five SERK family members appear to be expressed in the Arabidopsis abscission zone, we quantified their relative contribution to floral abscission in Arabidopsis using a petal break-strength assay. Our experiments suggest that among the SERK family members, SERK1 is a positive regulator of floral abscission. We found that the force required to remove the petals of serk1-1 mutants is significantly higher than that needed for wild-type plants, as previously observed for haesa/hsl2 mutants, and that floral abscission is delayed in serk1-1 (Figure 3A). The serk2-2, serk3-1, serk4-1 and serk5-1 mutant lines showed a petal break-strength profile not significantly different from wild-type plants. Possibly because SERKs have additional roles in plant development such as in pollen formation and brassinosteroid signaling, we found that higher-order SERK mutants exhibit pleiotropic phenotypes in the flower, rendering their analysis and comparison by quantitative petal break-strength assays difficult. We thus focused on analyzing the contribution of SERK1 to HAESA ligand sensing and receptor activation. In vitro, the LRR ectodomain of SERK1 (residues 24–213) forms stable, IDA-dependent heterodimeric complexes with HAESA in size exclusion chromatography experiments (Figure 3B). We next quantified the contribution of SERK1 to IDA recognition by HAESA. We found that HAESA senses IDA with a ~60 fold higher binding affinity in the presence of SERK1, suggesting that SERK1 is involved in the specific recognition of the peptide hormone (Figure 3C). We next titrated SERK1 into a solution containing only the HAESA ectodomain. In this case, there was no detectable interaction between receptor and co-receptor, while in the presence of IDA, SERK1 strongly binds HAESA with a dissociation constant in the mid-nanomolar range (Figure 3C). This suggests that IDA itself promotes receptor – co-receptor association, as previously described for the steroid hormone brassinolide and for other LRR-RK complexes. Importantly, hydroxyprolination of IDA is critical for HAESA-IDA-SERK1 complex formation (Figure 3C,D). Our calorimetry experiments now reveal that SERKs may render HAESA, and potentially other receptor kinases, competent for high-affinity sensing of their cognate ligands. Upon IDA binding at the cell surface, the kinase domains of HAESA and SERK1, which have been shown to be active protein kinases, may interact in the cytoplasm to activate each other. Consistently, the HAESA kinase domain can transphosphorylate SERK1 and vice versa in in vitro transphosphorylation assays (Figure 3E). Together, our genetic and biochemical experiments implicate SERK1 as a HAESA co-receptor in the Arabidopsis abscission zone. SERK1 senses a conserved motif in IDA family peptides Crystal structure of a HAESA – IDA – SERK1 signaling complex. (A) Overview of the ternary complex with HAESA in blue (surface representation), IDA in yellow (bonds representation) and SERK1 in orange (surface view). (B) The HAESA ectodomain undergoes a conformational change upon SERK1 co-receptor binding. Shown are Cα traces of a structural superposition of the unbound (yellow) and SERK1-bound (blue) HAESA ectodomains (r.m.s.d. is 1.5 Å between 572 corresponding Cα atoms). SERK1 (in orange) and IDA (in red) are shown alongside. The conformational change in the C-terminal LRRs and capping domain is indicated by an arrow. (C) SERK1 forms an integral part of the receptor's peptide binding pocket. The N-terminal capping domain of SERK1 (in orange) directly contacts the C-terminal part of IDA (in yellow, in bonds representation) and the receptor HAESA (in blue). Polar contacts of SERK1 with IDA are shown in magenta, with the HAESA LRR domain in gray. (D) Details of the zipper-like SERK1-HAESA interface. Ribbon diagrams of HAESA (in blue) and SERK1 (in orange) are shown with selected interface residues (in bonds representation). Polar interactions are highlighted as dotted lines (in magenta). DOI:\nhttp://dx.doi.org/10.7554/eLife.15075.011 To understand in molecular terms how SERK1 contributes to high-affinity IDA recognition, we solved a 2.43 Å crystal structure of the ternary HAESA – IDA – SERK1 complex (Figure 4A, Table 2). HAESA LRRs 16–21 and its C-terminal capping domain undergo a conformational change upon SERK1 binding (Figure 4B). The SERK1 ectodomain interacts with the IDA peptide binding site using a loop region (residues 51-59SERK1) from its N-terminal cap (Figure 4A,C). SERK1 loop residues establish multiple hydrophobic and polar contacts with Lys66IDA and the C-terminal Arg-His-Asn motif in IDA (Figure 4C). SERK1 LRRs 1–5 and its C-terminal capping domain form an additional zipper-like interface with residues originating from HAESA LRRs 15–21 and from the HAESA C-terminal cap (Figure 4D). SERK1 binds HAESA using these two distinct interaction surfaces (Figure 1—figure supplement 3), with the N-cap of the SERK1 LRR domain partially covering the IDA peptide binding cleft. The IDA C-terminal motif is required for HAESA-SERK1 complex formation and for IDA bioactivity. (A) Size exclusion chromatography experiments similar to Figure 3B,D reveal that IDA mutant peptides targeting the C-terminal motif do not form biochemically stable HAESA-IDA-SERK1 complexes. Deletion of the C-terminal Asn69IDA completely inhibits complex formation. Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. Left panel: IDA K66A/R67A; center: IDA ΔN69, right panel: SDS-PAGE of peak fractions. Note that the HAESA and SERK1 input lanes have already been shown in Figure 3D. (B) Isothermal titration thermographs of wild-type and mutant IDA peptides titrated into a HAESA - SERK1 mixture in the cell. Table summaries for calorimetric binding constants and stoichoimetries for different IDA peptides binding to the HAESA – SERK1 ectodomain mixture ( ± fitting errors; n.d. no detectable binding) are shown alongside. (C) Quantitative petal break-strength assay for Col-0 wild-type flowers and 35S::IDA wild-type and 35S::IDA K66A/R67A mutant flowers. Petal break is measured from positions 1 to 8 along the primary inflorescence where positions 1 is defined as the flower at anthesis (n=15, bars=SD). The three treatment groups in this unbalanced one-way layout were compared by Tukey’s all-pairs comparison procedure using the package multcomp in R (version 3.2.3). 35S::IDA plants showed significantly increased abscission compared to Col-0 controls in inflorescence positions 2 and 3 (a). Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. 15 out of 15 35S::IDA plants, 0 out of 15 Col-0 plants and 0 out of 15 35S::IDA K66A/R67A double-mutant plants, showed an enlarged abscission zone, respectively (3 independent lines were analyzed). DOI:\nhttp://dx.doi.org/10.7554/eLife.15075.012 The four C-terminal residues in IDA (Lys66IDA-Asn69IDA) are conserved among IDA family members and are in direct contact with SERK1 (Figures 1A, 4C). We thus assessed their contribution to HAESA – SERK1 complex formation. Deletion of the buried Asn69IDA completely inhibits receptor – co-receptor complex formation and HSL2 activation (Figure 5A,B). A synthetic Lys66IDA/Arg67IDA → Ala mutant peptide (IDA K66A/R66A) showed a 10 fold reduced binding affinity when titrated in a HAESA/SERK1 protein solution (Figures 5A,B, 2D). We over-expressed full-length wild-type IDA or this Lys66IDA/Arg67IDA → Ala double-mutant to similar levels in Col-0 Arabidopsis plants (Figure 5D). We found that over-expression of wild-type IDA leads to early floral abscission and an enlargement of the abscission zone (Figure 5C–E). In contrast, over-expression of the IDA Lys66IDA/Arg67IDA → Ala double mutant significantly delays floral abscission when compared to wild-type control plants, suggesting that the mutant IDA peptide has reduced activity in planta (Figure 5C–E). Comparison of 35S::IDA wild-type and mutant plants further indicates that mutation of Lys66IDA/Arg67IDA → Ala may cause a weak dominant negative effect (Figure 5C–E). In agreement with our structures and biochemical assays, this experiment suggests a role of the conserved IDA C-terminus in the control of floral abscission. Discussion In contrast to animal LRR receptors, plant LRR-RKs harbor spiral-shaped ectodomains and thus they require shape-complementary co-receptor proteins for receptor activation. For a rapidly growing number of plant signaling pathways, SERK proteins act as these essential co-receptors (; ). SERK1 has been previously reported as a positive regulator in plant embryogenesis, male sporogenesis, brassinosteroid signaling and in phytosulfokine perception. Recent findings by and our mechanistic studies now also support a positive role for SERK1 in floral abscission. As serk1-1 mutant plants show intermediate abscission phenotypes when compared to haesa/hsl2 mutants, SERK1 likely acts redundantly with other SERKs in the abscission zone (Figure 3A). It has been previously suggested that SERK1 can inhibit cell separation. However our results show that SERK1 also can activate this process upon IDA sensing, indicating that SERKs may fulfill several different functions in the course of the abscission process. While the sequence of the mature IDA peptide has not been experimentally determined in planta, our HAESA-IDA complex structures and calorimetry assays suggest that active IDLs are hydroxyprolinated dodecamers. It will be thus interesting to see if proteolytic processing of full-length IDA in vivo is regulated in a cell-type or tissue-specific manner. The central Hyp residue in IDA is found buried in the HAESA peptide binding surface and thus this post-translational modification may regulate IDA bioactivity. Our comparative structural and biochemical analysis further suggests that IDLs share a common receptor binding mode, but may preferably bind to HAESA, HSL1 or HSL2 in different plant tissues and organs. In our quantitative biochemical assays, the presence of SERK1 dramatically increases the HAESA binding specificity and affinity for IDA. This observation is consistent with our complex structure in which receptor and co-receptor together form the IDA binding pocket. The fact that SERK1 specifically interacts with the very C-terminus of IDLs may allow for the rational design of peptide hormone antagonists, as previously demonstrated for the brassinosteroid pathway. Importantly, our calorimetry assays reveal that the SERK1 ectodomain binds HAESA with nanomolar affinity, but only in the presence of IDA (Figure 3C). This ligand-induced formation of a receptor – co-receptor complex may allow the HAESA and SERK1 kinase domains to efficiently trans-phosphorylate and activate each other in the cytoplasm. It is of note that our reported binding affinities for IDA and SERK1 have been measured using synthetic peptides and the isolated HAESA and SERK1 ectodomains, and thus might differ in the context of the full-length, membrane-embedded signaling complex. SERK1 uses partially overlapping surface areas to activate different plant signaling receptors. (A) Structural comparison of plant steroid and peptide hormone membrane signaling complexes. Left panel: Ribbon diagram of HAESA (in blue), SERK1 (in orange) and IDA (in bonds and surface represention). Right panel: Ribbon diagram of the plant steroid receptor BRI1 (in blue) bound to brassinolide (in gray, in bonds representation) and to SERK1, shown in the same orientation (PDB-ID. 4lsx). (B) View of the inner surface of the SERK1 LRR domain (PDB-ID 4lsc, surface representation, in gray). A ribbon diagram of SERK1 in the same orientation is shown alongside. Residues interacting with the HAESA or BRI1 LRR domains are shown in orange or magenta, respectively. DOI:\nhttp://dx.doi.org/10.7554/eLife.15075.013 Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A). Several residues in the SERK1 N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B). In addition, residues 53-55SERK1 from the SERK1 N-terminal cap mediate specific interactions with the IDA peptide (Figures 4C, 6B). These residues are not involved in the sensing of the steroid hormone brassinolide. In both cases however, the co-receptor completes the hormone binding pocket. This fact together with the largely overlapping SERK1 binding surfaces in HAESA and BRI1 allows us to speculate that SERK1 may promote high-affinity peptide hormone and brassinosteroid sensing by simply slowing down dissociation of the ligand from its cognate receptor. Different plant peptide hormone families contain a C-terminal (Arg)-His-Asn motif, which in IDA represents the co-receptor recognition site. Structure-guided multiple sequence alignment of IDA and IDA-like peptides with other plant peptide hormone families, including CLAVATA3 – EMBRYO SURROUNDING REGION-RELATED (CLV3/CLE), ROOT GROWTH FACTOR – GOLVEN (RGF/GLV), PRECURSOR GENE PROPEP1 (PEP1) from Arabidopsis thaliana. The conserved (Arg)-His-Asn motif is highlighted in red, the central Hyp residue in IDLs and CLEs is marked in blue. DOI:\nhttp://dx.doi.org/10.7554/eLife.15075.014 Our experiments reveal that SERK1 recognizes a C-terminal Arg-His-Asn motif in IDA. Importantly, this motif can also be found in other peptide hormone families (Figure 7). Among these are the CLE peptides regulating stem cell maintenance in the shoot and the root. It is interesting to note, that CLEs in their mature form are also hydroxyprolinated dodecamers, which bind to a surface area in the BARELY ANY MERISTEM 1 receptor that would correspond to part of the IDA binding cleft in HAESA. Diverse plant peptide hormones may thus also bind their LRR-RK receptors in an extended conformation along the inner surface of the LRR domain and may also use small, shape-complementary co-receptors for high-affinity ligand binding and receptor activation. Materials and methods Protein expression and purification Synthetic genes coding for the Arabidopsis thaliana HAESA (residues 20–620) and SERK1 ectodomains (residues 24–213, carrying Asn115→Asp and Asn163→Gln mutations), codon optimized for expression in Trichoplusia ni (Geneart, Germany), were cloned into a modified pBAC-6 transfer vector (Novagen, Billerica, MA), providing an azurocidin signal peptide and a C-terminal TEV (tobacco etch virus protease) cleavable Strep-9xHis tandem affinity tag. Recombinant baculoviruses were generated by co-transfecting transfer vectors with linearised baculovirus DNA (ProFold-ER1, AB vector, San Diego, CA) followed by viral amplification in Spodoptera frugiperda Sf9 cells. The HAESA and SERK1 ectodomains were individually expressed in Trichoplusia ni Tnao38 cells using a multiplicity of infection of 3, and harvested from the medium 2 days post infection by tangential flow filtration using 30 kDa MWCO and 10 kDa MWCO (molecular weight cut-off) filter membranes (GE Healthcare Life Sciences, Pittsburgh, PA), respectively. Proteins were purified separately by sequential Ni2+ (HisTrap HP, GE Healthcare) and Strep (Strep-Tactin Superflow high-capacity, IBA, Germany) affinity chromatography. Next, affinity tags were removed by incubating the purified proteins with recombinant Strep-tagged TEV protease in 1:100 molar ratio. The cleaved tag and the protease were separated from HAESA and SERK1 by a second Strep affinity step. The purified HAESA ectodomain was incubated with a synthetic IDA peptide (YVPIPPSA-Hyp-SKRHN, the N-terminal Tyr residue was added to allow for peptide quantification by UV absorbance) and the SERK1 ectodomain in 1:1:1.5 molar ratio. The HAESA-IDA-SERK1 complex was purified by size exclusion chromatography on a Superdex 200 HR10/30 column (GE Healthcare) equilibrated in 20 mM citric acid pH 5.0, 100 mM NaCl). Peak fractions containing the complex were concentrated to ~10 mg/mL and immediately used for crystallization. About 0.2 mg of purified HAESA and 0.1 mg of purified SERK1 protein were obtained from 1 L of insect cell culture, respectively. Crystallization and data collection Hexagonal crystals of the isolated HAESA ectodomain developed at room-temperature in hanging drops composed of 1.0 μL of protein solution (5.5 mg/mL) and 1.0 μL of crystallization buffer (21% PEG 3,350, 0.2 M MgCl2 · 6 H2O, 0.1 M citric acid pH 4.0), suspended above 1.0 mL of crystallization buffer. For structure solution crystals were derivatized and cryo-protected by serial transfer into crystallization buffer supplemented with 0.5 M NaI and 15% ethylene glycol and cryo-cooled in liquid nitrogen. Redundant single-wavelength anomalous diffraction (SAD) data to 2.39 Å resolution were collected at beam-line PXII at the Swiss Light Source (SLS), Villigen, CH with λ=1.7 Å. A native data set to 1.74 Å resolution was collected on a crystal from the same drop cryo-protected by serial transfer into crystallization buffer supplemented with 15% (v/v) ethylene glycol only (λ=1.0 Å; Table 1). HAESA complexes with IDA (PIPPSA-Hyp-SKRHN), PKGV-IDA (YPKGVPIPPSA-Hyp-SKRHN) and IDL1 (LVPPSG-Hyp-SMRHN) peptide hormones were obtained by soaking apo crystals in crystallization buffer containing the respective synthetic peptide at a final concentration of 15 mM. Soaked crystals diffracted to 1.86 Å (HAESA – IDA), 1.94 Å (HAESA-PKGV-IDA) and 2.56 Å resolution (HAESA – IDL1), respectively (Table 2). Orthorhombic crystals of the HAESA-IDA-SERK1 complex developed in 18% PEG 8000, MgCl2 · 6 H2O, 0.1 M citric acid and diffracted to 2.43 Å resolution (Table 2). Data processing and scaling was done in XDS (version: Nov 2014). Structure solution and refinement The SAD method was used to determine the structure of the isolated HAESA ectodomain. SHELXD located 32 iodine sites (CC All/Weak 37.7/14.9). 20 consistent sites were input into the program SHARP for phasing and identification of 8 additional sites at 2.39 Å resolution. Refined heavy atom sites and phases were provided to PHENIX.AUTOBUILD for density modification and automated model building. The structure was completed in alternating cycles of model building in COOT and restrained TLS refinement in REFMAC5 (version 5.8.0107) against an isomorphous high resolution native data set. Crystals contain one HAESA monomer per asymmetric unit with a solvent content of ~55%, the final model comprises residues 20 – 615. The refined structure has excellent stereochemistry, with 93.8% of all residues in the favored region of the Ramachandran plot, no outliers and a PHENIX.MOLPROBITY score of 1.34 (Table 1). The HAESA – IDA – SERK1 complex structure was determined by molecular replacement with the program PHASER, using the isolated HAESA and SERK1 (PDB-ID: 4LSC) LRR domain structures as search models. The solution comprises one HASEA-IDA-SERK1 complex in the asymmetric unit. The structure was completed in iterative cycles of manual model-building in COOT and restrained TLS refinement in REFMAC5. Amino acids whose side-chain position could not be modeled with confidence were truncated to alanine (0.6 – 1% of total residues), the stereochemistry of N-linked glycan structures was assessed with the CCP4 program PRIVATEER-VALIDATE. The refined model has 94.44% of all residues in the favored region of the Ramachandran plot, no outliers and a PHENIX.MOLPROBITY score of 1.17 (Table 2). Structural visualization was done with POVScript+ and POV-Ray (http://www.povray.org). Size-exclusion chromatography Gel filtration experiments were performed using a Superdex 200 HR 10/30 column (GE Healthcare) pre-equilibrated in 20 mM citric acid (pH 5) and 100 mM NaCl. 100 μL of the isolated HAESA ectodomain (5.5 mg/mL), of the purified SERK1 LRR domain (3 mg/mL) or of mixtures of HAESA and SERK1 (either in the presence or absence of synthetic wild-type IDA, wild-type IDL1 or mutant IDA peptides at a concentration of 25 μM; 10 mg/mL; samples contained HAESA and SERK1 in 1:1 molar ratio) were loaded sequentially onto the column and elution at 0.5 mL/min was monitored by ultraviolet absorbance at 280 nm. Isothermal titration calorimetry ITC experiments were performed using a Nano ITC (TA Instruments, New Castle, DE) with a 1.0 mL standard cell and a 250 μL titration syringe. Proteins were dialyzed extensively against ITC buffer (20 mM citric acid pH 5.0, 100 mM NaCl) and synthetic wild-type or point-mutant peptides (with wild-type IDA sequence YVPIPPSA-Hyp-SKRHN, PKGV-IDA YPKGVPIPPSA-Hyp-SKRHN, IDA-SFVN YPIPPSA-Hyp-SKRHNSFVN, IDL1 YLVPPSG-Hyp-SMRHN and CLV3 sequence YRTV-Hyp-SG-Hyp-DPLHH) were dissolved in ITC buffer prior to all titrations. Molar protein concentrations for SERK1 and HAESA were calculated using their molar extinction coefficient and a molecular weight of 27,551 and 74,896 Da, respectively (determined by MALDI-TOF mass spectrometry). Experiments were performed at 25°C. A typical experiment consisted of injecting 10 μL aliquots of peptide solution (250 μM) into 20 μM HAESA. The concentrations for the complex titrations were 150 μM of ligand (either wild-type or point-mutant IDA peptides) in the syringe and 10 μM of a 1:1 HAESA – SERK1 protein mixture in the cell at time intervals of 150 s to ensure that the titration peak returned to the baseline. Binding of SERK1 to HAESA was assessed by titrating SERK1 (100 μM) into a solution containing HAESA (10 μM) in the pre- or absence of 150 μM wild-type IDA peptide. ITC data were corrected for the heat of dilution by subtracting the mixing enthalpies for titrant solution injections into protein free ITC buffer. Data were analyzed using the NanoAnalyze program (version 2.3.6) as provided by the manufacturer. In vitro kinase trans-phosphorylation assay Coding sequences of SERK1 kinase domain (SERK1-KD) (residues 264–625) and HAESA-KD (residues 671–969) were cloned into a modified pET (Novagen) vector providing an TEV-cleavable N-terminal 8xHis-StrepII-Thioredoxin tag. Point mutations were introduced into the SERK1 (Asp447→Asn; mSERK1) and HAESA (Asp837→Asn; mHAESA) coding sequences by site directed mutagenesis, thereby rendering the kinases inactive. The plasmids were transformed into E.coli Rosetta 2 (DE3) (Novagen). Protein expression was induced by adding IPTG to final concentration of 0.5 mM to cell cultures grown to an OD600 = 0.6. Cells were then incubated at 16°C for 18 hr, pelleted by centrifugation at 5000 x g and 4°C for 15 min, and resuspended in buffer A (20 mM Tris-HCl pH 8, 500 mM NaCl, 4 mM MgCl2 and 2 mM β-Mercaptoethanol) supplemented with 15 mM Imidazole and 0.1% (v/v) Igepal. After cell lysis by sonication, cell debris was removed by centrifugation at 35,000 x g and 4°C for 30 min. The recombinant proteins were isolated by Co2+ metal affinity purification using a combination of batch and gravity flow approaches (HIS-Select Cobalt Affinity Gel, Sigma, St. Louis, MO). After washing the resin with the wash buffer (buffer A + 15 mM Imidazole) proteins were eluted in buffer A supplemented with 250 mM Imidazole. All elutions were then dialyzed against 20 mM Tris-HCl pH 8, 250 mM NaCl, 4 mM MgCl2 and 0.5 mM TCEP. For SERK1-KD and mSERK1-KD the 8xHis-StrepII-Thioredoxin tag was removed with 6xHis tagged TEV protease. TEV and the cleaved tag were removed by a second metal affinity purification step. Subsequently, all proteins were purified by gel filtration on a Superdex 200 10/300 GL column equilibrated in 20 mM Tris pH 8, 250 mM NaCl, 4 mM MgCl2 and 0.5 mM TCEP. Peak fractions were collected and concentrated using Amicon Ultra centrifugation devices (10,000 MWCO). For in vitro kinase assays, 1 μg of HAESA-KD, 0.25 μg of SERK1-KD and 2 μg of mSERK1 and mHAESA were used in a final reaction volume of 20 μl. The reaction buffer consisted of 20 mM Tris pH 8, 250 mM NaCl, 4 mM MgCl2 and 0.5 mM TCEP. The reactions were started by the addition of 4 μCi [γ-32P]-ATP (Perkin-Elmer, Waltham, MA), incubated at room temperature for 45 min and stopped by the addition of 6x SDS-loading dye immediately followed by incubating the samples at 95°C. Proteins of the whole reaction were subsequently separated via SDS-PAGE in 4–15% gradient gels (TGX, Biorad, Hercules, CA) and stained with Instant Blue (Expedeon, San Diego, CA). After pictures were taken of the stained gel, 32P-derived signals were visualized by exposing the gel to an X-ray film (Fuji, SuperRX, Valhalla, NY). Plant material and generation of transgenic lines 35S::IDA wild-type and 35S::IDA (R66 → Ala/K67 → Ala) over-expressing transgenic lines in Col-0 background were generated as follows: The constructs were introduced in the destination vector pB7m34GW2 and transferred to A. tumefaciens strain pGV2260. Plants were transformed using the floral dip method. Transformants were selected in medium supplemented with BASTA up to the T3 generation. For phenotyping, plants were grown at 21°C with 50% humidity and a 16h light: 8 hr dark cycle. RNA analyses Plants were grown on ½ Murashige and Skoog (MS) plates supplemented with 1% sucrose. After 7 d, ∼30 to 40 seedlings were collected and frozen in liquid nitrogen. Total RNA was extracted using a RNeasy plant mini kit (Qiagen, Valencia, CA), and 1 μg of the RNA solution obtained was reverse-transcribed using the SuperScritpVILO cDNA synthesis kit (Invitrogen, Grand Island, NY). RT-qPCR amplifications and measurements were performed using a 7900HT Fast Real Time PCR-System by Applied Biosystems (Carlsbad, CA). RT-qPCR amplifications were monitored using SYBR-Green fluorescent stain (Applied Biosystems). Relative quantification of gene expression data was performed using the 2−ΔΔCT (or comparative CT) method. Expression levels were normalized using the CT values obtained for the actin2 gene (forward: TGCCAATCTACGAGGGTTTC; reverse: TTCTCGATGGAAGAGCTGGT). For detection and amplification of IDA sequence we used specific primers (forward: TCGTACGATGATGGTTCTGC; reverse: GAATGGGAACGCCTTTAGGT). The presence of a single PCR product was further verified by dissociation analysis in all amplifications. All quantifications were made in quadruplicates on RNA samples obtained from three independent experiments. Petal break measurements serk1-1, serk2-2, serk3-1, serk4-1 and serk5-1 and Col-0 wild-type plants were grown in growth chambers at 22°C under long days (16 hr day/8 hr dark) at a light intensity of 100 µE·m-2·sec-1. Petal break-strength was quantified as the force in gram equivalents required for removal of a petal from a flower when the plants had a minimum of twenty flowers and siliques. Measurements were performed using a load transducer as described in. Break-strength was measured for 15 plants and a minimum of 15 measurements at each position. Funding Information This paper was supported by the following grants:   to Michael Hothorn.   to Michael Hothorn.   to Michael Hothorn.   to Melinka A Butenko.   to Benjamin Brandt.   to Julia Santiago. Additional information Competing interests The authors declare that no competing interests exist. Author contributions JS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. BB, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. MW, Acquisition of data, Analysis and interpretation of data. UH, Acquisition of data, Analysis and interpretation of data. LAH, Analysis and interpretation of data, Drafting or revising the article. MAB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. MH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. References Ida: A peptide ligand regulating cell separation processes in Arabidopsis The Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASES1 and 2 control male sporogenesis Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE proteins serve brassinosteroid-dependent and -independent signaling pathways Crystal structures of the phosphorylated BRI1 kinase domain and implications for brassinosteroid signal initiation SERK co-receptor kinases Generation, representation and flow of phase information in structure determination: Recent developments in and around SHARP 2.0 Inflorescence deficient in abscission controls floral organ abscission in Arabidopsis and identifies a novel family of putative ligands in plants Plant peptides in signalling: Looking for new partners Tools and strategies to match peptide-ligand receptor pairs Regulation of floral organ abscission in Arabidopsis thaliana CLAVATA3 is a specific regulator of shoot and floral meristem development affecting the same processes as CLAVATA1 Floral dip: A simplified method for agrobacterium-mediated transformation of Arabidopsis thaliana Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASES1 and 2 are essential for tapetum development and microspore maturation Molprobity: All-atom contacts and structure validation for proteins and nucleic acids Coot : Model-building tools for molecular graphics Povscript+ : A program for model and data visualization using persistence of vision ray-tracing Genetic evidence for an indispensable role of somatic embryogenesis receptor kinases in brassinosteroid signaling Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae), is permissive for AcMNPV infection and produces high levels of recombinant proteins Multiple contrast tests in the presence of heteroscedasticity The Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 gene is expressed in developing ovules and embryos and enhances embryogenic competence in culture Simultaneous inference in general parametric models Structural basis of steroid hormone perception by the receptor kinase BRI1 HAESA, an Arabidopsis leucine-rich repeat receptor kinase, controls floral organ abscission Dictionary of protein secondary structure: Pattern recognition of hydrogen-bonded and geometrical features Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants A plant peptide encoded by CLV3 identified by in situ MALDI-TOF MS analysis A petal breakstrength meter for Arabidopsis abscission studies The SERK1 receptor-like kinase regulates organ separation in Arabidopsis flowers Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method Secreted peptide signals required for maintenance of root stem cell niche in Arabidopsis Phaser crystallographic software Differential function of Arabidopsis SERK family receptor-like kinases in stomatal patterning Ligand-induced receptor-like kinase complex regulates floral organ abscission in Arabidopsis Refinement of macromolecular structures by the maximum-likelihood method Brassinolide-2,3-acetonide: A brassinolide-induced rice lamina joint inclination antagonist Letting go is never easy: Abscission and receptor-like protein kinases Transcriptional profiling of the Arabidopsis abscission mutant hae hsl2 by RNA-Seq Arabidopsis CLV3 peptide directly binds CLV1 ectodomain A glycopeptide regulating stem cell fate in Arabidopsis thaliana  AtSERK1 expression precedes and coincides with early somatic embryogenesis in Arabidopsis thaliana Reduced surface: An efficient way to compute molecular surfaces Molecular mechanism for plant steroid receptor activation by somatic embryogenesis co-receptor kinases Role of threonines in the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 activation loop in phosphorylation A short history of SHELX Biochemical mapping of a ligand-binding domain within Arabidopsis BAM1 reveals diversified ligand recognition mechanisms of plant LRR-RKs Overexpression of INFLORESCENCE DEFICIENT IN ABSCISSION activates cell separation in vestigial abscission zones in Arabidopsis The EPIP peptide of INFLORESCENCE DEFICIENT IN ABSCISSION is sufficient to induce abscission in Arabidopsis through the receptor-like kinases HAESA and HAESA-LIKE2 Structure reveals that BAK1 as a co-receptor recognizes the BRI1-bound brassinolide Structural basis for recognition of an endogenous peptide by the plant receptor kinase PEPR1 Analysis of phosphorylation of the receptor-like protein kinase HAESA during arabidopsis floral abscission Iterative model building, structure refinement and density modification with the PHENIX autobuild wizard Allosteric receptor activation by the plant peptide hormone phytosulfokine 10.7554/eLife.15075.017 Decision letter Zhang Mingjie In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included. Thank you for submitting your article \"Mechanistic insight into a peptide hormone signaling complex mediating floral organ abscission\" to eLife for consideration by eLife. Your article has been favorably evaluated by John Kuyiyan (Senior editor) and three reviewers, one of whom, (Mingjie Zhang) is a member of our Board of Reviewing Editors. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. As you will see that the required revisions are essentially clarifications and some additional analysis of existing data in nature. Summary: In this work, Hothorn and colleagues investigated the structural basis governing the recognition of peptide hormone IDA during plant floral abscission process. Through an array of complex structures, supplemented with biochemical and genetic experiments, the authors uncovered the IDA recognition mechanism by a co-receptor (HAESA and SERK1) detection mechanism. The structures also reveal the specific recognition mechanism of the 12-residue IDA core peptide sequence by the co-receptors, and suggest that this 12-residue IDA sequence is likely to be the mature peptide hormone functioning in plants. The comparison of the structures of the HAESA/SERK1/IDA complex and the previously determined BRA1/SERK1/brassinolide complex by the same group also suggests a co-receptor pairing mechanism for various plant hormones. The story gives detailed and novel mechanistic insights in the perception of IDA during floral abscission, and is convincing and worthy to be considered for publication in eLife with the following revisions. Key issues which need to be addressed: 1) As the results reported here rest largely on the interpretation of the structural data, the following points need to be addressed by the authors. i) Temperature factors (Wilson B and residual) are unusually high for the reported resolution. Are there portions of the structure that exhibit more disorder or are these high temperature factors throughout the structure? Are there potential problems with radiation damage due to the high multiplicity? ii) A simulated annealing omit map figure should be provided as the peptides all exhibit very high temperature factors. iii) How many side chains were trimmed due to poor electron density? If this is a significant percentage, it should be noted. 2) A further discussion comparing the mechanisms of BRI1-BL-SERK1 and HAESA-IDA-SERK1 would be helpful as a structural comparison is presented. As BR1 binds its ligand with high affinity, the low affinity of HAESA for IDA could be further discussed. 3) Are the results here applicable to the related receptor HSL2? Are the residues that interact with SERK1 and IDA conserved in HSL2? 4) The authors show that N-terminal extension of the peptide does not impact on binding efficiency, but what would happen if the peptide was extended at the C-terminal end, at the suggested cleavage site? Would cleavage be required for recognition? A brief discussion on this point may help. 5) Figure 3C: Could the authors comment on the difference between the blue (SERK1 vs. HAESA-IDA) and the black (IDA vs. HAESA-SERK1) line? SERK1 vs. HAESA-IDA gives a Kd of 75 nM, yet IDA vs. SERK1-HAESA gives a Kd of 350 nM. In the text the authors keep referring to the 75 nM Kd, but not the 350 nM Kd. Is it really fair to say the Kd is 75 nM? 6) In the Discussion, can the authors comment further on the discrepancy between their study and the study of Lewis et al. (Plant J, 2010) concerning the role of SERK1 in floral abscission? Similarly, could the authors comment on the fact that the LRR-RLP EVD/SOBIR seems to be a negative regulator of the HAESA/HSL2 pathway (Leslie et al., Development, 2010), which seems puzzling given that EVD/SOBIR function is normally restricted to LRR-RLPs (Gust \u0026 Felix, Curr Op Plant Biol, 2014). 7) Given that the central hydroxyproline in IDA is of such crucial importance for binding, isn't it surprising that IDAΔ69N does not bind to HAESA at all? Wouldn't it be expected that the remaining part of the peptide still binds to HAESA? 8) Figure 1—figure supplement 2: How is it possible that charged amino acids are involved in hydrophobic interactions? 9) Are the distances shown in the graphical representation of the structures proportional? It would seem that some of the aromatic rings could cause steric hindrance. 10) Why did the authors decide to express HAESA and SERK1 without signal peptide? Would it make a difference for binding of IDA, if they leave the SP on? 11) Figure 7: Are the homologous regions also the active parts of these peptides? And could the authors display amino acids numbers on either side of the fragments? 12) Have the authors ever measured dissociation of the peptide from the complex? And in this regard, to what does \"highly stable receptor – co-receptor complex\" refer/compare to? 13) Figures 3A and 5C require statistical analyses. 10.7554/eLife.15075.018 Author response  1) As the results reported here rest largely on the interpretation of the structural data, the following points need to be addressed by the authors. i) Temperature factors (Wilson B and residual) are unusually high for the reported resolution.  We thank the reviewer(s) for pointing out this issue: Indeed our mean B-values deviate substantially from the expected mean B-values (resolution 1.75 – 2.00 A, B(Wilson) ~ 27.0 over 5,510 structures in the PDB; Pavel Afonine, personal communication). We would like to note that due to the many N-glycosylation sites dispersed over the HAESA LRR domain (shown in Figure 1—figure supplement 1D), we find relatively few crystal contacts in our P3121 crystal form, which may rationalize our high B-values. We have reanalyzed our space group assignment (using the CCP4 program ZANUDA) and checked for any signs of problems during data collection (ice rings, multiple crystal lattices, splitting, using the programs XDS and XDSSTAT), as well as for twinning and pseudosymmetry (using phenix.xtriage). No such problems appear to exist, our structures refine very well and our refined B-values are in good agreement with our Wilson B-factors (see Table 2). Thus, the high B-values appear to represent an intrinsic property of our crystals and are not the result of a poor data collection strategy or inappropriate crystallographic analysis. Are there portions of the structure that exhibit more disorder or are these high temperature factors throughout the structure?  Cα trace of the HAESA LRR domain and IDA peptide colored according to B-factor from low (60.9, in blue) to high (134.7, in red). Mean B-value is 79.5. DOI:\nhttp://dx.doi.org/10.7554/eLife.15075.015 Yes. As shown in revised Figure 4B, the Cterminal LRRs of HAESA in contact with SERK1 in our complex structure appear to be somewhat flexible. Author response image 1. illustrates that the B-values are significantly higher in the C-terminal part of the HAESA LRR domain (with the Cterminal capping domain being the most flexible), while both the N-terminal LRRs of HAESA (with exception of the LRR N-terminal capping domain) and the IDA peptide appear better ordered in our P3121 crystals form. Are there potential problems with radiation damage due to the high multiplicity?  No. Data were collected at SLS beamline PXIII equipped with a Dectris Pilatus 2M-F detector. We perform all our data collections at very low dose and high multiplicity of measurement, which at this beam-line produces similar results compared to exposing the crystal at higher dose for a smaller angular range. We collected 360 deg with 0.1 deg slices and obtained a Wilson B-value of 80, with no sign of radiation damage in our data processing (subroutine COLSPOT in XDS over all frames). To test the reviewer's hypothesis we cut the data after 90 deg (when completeness approaches 100%) and we obtained a Wilson B-value of 78 and a refined mean B-value of around 75. These value do not significantly differ from our presented 360 deg data set and thus it is unlikely that radiation damage produces these high B-values. Again, they rather appear to be an intrinsic property of our crystals. ii) A simulated annealing omit map figure should be provided as the peptides all exhibit very high temperature factors. Thank you for this suggestion. In our first submission, we presented 2Fo-Fc omit electron density maps for our HAESA-IDA/IDL complex structures. As suggested, we now present simulated annealing omit maps in revised Figure 2A, B, C. The maps were generated like this: phenix.composite_omit_map *.pdb *.mtz *.cif nproc=8 anneal=True We would like to note that our peptide are well ordered in our structures, and their B-values match the B-values of their interacting LRR surface (compare Author response image 1). iii) How many side chains were trimmed due to poor electron density? If this is a significant percentage, it should be noted.  Here are the requested numbers (trimmed residues out of total residues in asymmetric unit, percentage): HAESA apo: 7 out of 595 (1%) HAESA IDA: 6 out of 597 + 12 (1%) HAESA IDL1: 6 out of 597 + 12 (1%) HAESA – IDA – SERK1: 5 out of 594 + 12 + 185 (0.6%) We have included a statement in the Methods section that reads: “Amino acids whose side-chain position could not be modeled with confidence were truncated to alanine (0.6 – 1% of total residues)[…]”  2) A further discussion comparing the mechanisms of BRI1-BL-SERK1 and HAESA-IDA-SERK1 would be helpful as a structural comparison is presented.  We have expanded our discussion of the HAESA – SERK1 and BRI1 – SERK1 interfaces. We now specify the SERK1 residues in contact with both receptors and the SERK1 residues unique to HAESA/IDA sensing. We also comment on the very different ligand binding modes in HAESA and BRI1 and specify that different LRR segments contribute to the formation of the respective steroid and peptide hormone binding pockets. We feel however that an in-depth comparison of the interacting surfaces is beyond the scope of this report and partially redundant with our earlier work (Santiago et al., Science, 2013). In our opinion, such an analysis seems more appropriate for a review on the subject, which we are currently preparing. Our revised Discussion now reads: “Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor (Santiago, Henzler, and Hothorn 2013), reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A).[…] These residues are not involved in the sensing of the steroid hormone brassinolide (Santiago, Henzler, and Hothorn 2013). In both cases however, the co-receptor completes the hormone binding pocket.” As BR1 binds its ligand with high affinity, the low affinity of HAESA for IDA could be further discussed.  High affinity brassinosteroid binding to BRI1 was previously shown using BRI1-enriched plant extracts and radiolabeled brassinolide (Wang et al., Nature410:380-383, 2001). We now know that co-immunoprecipitations of BRI1 from Arabidopsis contain SERK proteins (compare for example Jaillais et al., PNAS, 2011) and thus the reported binding constants likely correspond to steroid binding to BRI1-SERK complexes, not to BRI1 alone. We would thus prefer not to compare the binding affinities for brassinosteroid and peptide hormone ligands at this point. 3) Are the results here applicable to the related receptor HSL2? Are the residues that interact with SERK1 and IDA conserved in HSL2?  Yes. We present a structure-based sequence alignment of AtHAESA and AtHSL2, as well as other HAESA-type receptors from different plant species in Figure 1—figure supplement 3. In the peptide binding surface, 17 out of 26 contributing amino-acids are conserved among AtHAESA and AtHSL2. 13 out of 19 interacting residues in the HAESA – SERK1 complex are also present in AtHSL2. We feel that this is strong conservation given that the AtHAESA and AtHSL2 ectodomains share 45% overall sequence identity. We have included a statement in our manuscript that reads: “Indeed, we find many of the residues contributing to the formation of the IDA binding surface in HAESA to be conserved in HSL2 and other HAESA-type receptors in different plant species (Figure 1—figure supplement 3).” 4) The authors show that N-terminal extension of the peptide does not impact on binding efficiency, but what would happen if the peptide was extended at the C-terminal end, at the suggested cleavage site?  Isothermal titration calorimetry thermograph of the C-terminally extended IDA-SFVN peptide (200 μM) titrated into a solution containing 20 μM of the purified HAESA ectodomain. No detectable binding is observed. DOI:\nhttp://dx.doi.org/10.7554/eLife.15075.016 Thank you for suggesting this experiment. We synthesized a C-terminally extended version of the IDA peptide (IDA-SFVN with sequence YPIPPSA-Hyp- SKRHN SFVN) and performed quantitative binding assays by ITC. As shown in Author response image 2, we cannot observe any detectable binding of this C-terminally extended peptide to the HAESA ectodomain, consistent with our crystallographic models that suggest that HAESA specifically senses an active IDA 12mer. We have incorporated this new result in Figure 2D. We have included a new statement in the manuscript that reads: “The COO- group of Asn69IDA is in direct contact with Arg407HAESA and Arg409HAESA and HAESA cannot bind a C-terminally extended IDA-SFVN peptide (Figures 1D, F, 2D).” Would cleavage be required for recognition? A brief discussion on this point may help.  Yes. We have modified our manuscript accordingly: “This suggests that the conserved Asn69 may constitute the very C-terminus of the mature IDA peptide in planta and that active IDA is generated by proteolytic processing from a longer pre-protein (Stenvik et al. 2008).“ 5) Figure 3C: Could the authors comment on the difference between the blue (SERK1 vs. HAESA-IDA) and the black (IDA vs. HAESA-SERK1) line? SERK1 vs. HAESA-IDA gives a Kd of 75 nM, yet IDA vs. SERK1-HAESA gives a Kd of 350 nM. In the text the authors keep referring to the 75 nM Kd, but not the 350 nM Kd. Is it really fair to say the Kd is 75 nM? Yes and No. These are two different experiments. We first measured (by ITC) the binding affinity of IDA (in the syringe) binding to a protein solution containing equimolar ratios of HAESA and SERK1 (Kdis 350 nM in this case). Next, we titrated a concentrated SERK1 solution (in the syringe) into a solution of HAESA containing IDA in 10fold molar excess (Kdin this case is 75 nM). Given that the experimental conditions (protein and peptide concentrations and molar ratios between the components) are very different, we feel that the Kd 's obtained by these experiments are in good agreement (4.5 fold difference vs. a 60-260 fold difference when compared to the isolated HAESA ectodomain). Nevertheless, we addressed the reviewer's concern by modifying our manuscript which now states: “In this case, there was no detectable interaction between receptor and co-receptor, while in the presence of IDA, SERK1 strongly binds HAESA with a dissociation constant in the mid-nanomolar range. (Figure 3C).” 6) In the Discussion, can the authors comment further on the discrepancy between their study and the study of Lewis et al. (Plant J, 2010) concerning the role of SERK1 in floral abscission?  The process of floral organ abscission in Arabidopsis is divided into distinct steps where a gradual loosening of the cell wall between abscising cells can be measured as a reduction in petal breakstrength (Bleecker and Patterson, 1997). During floral abscission in wild-type plants a significant drop in breakstrength occurs shortly before the petals drop (shown in Figure 3A in our manuscript). Previously reported negative regulators of abscission, such as the transcription factor KNAT1, have an earlier reduction in breakstrength, indicative of early cell wall remodeling (Shi et al. 2011). Our results show that serk1 mutant plants, contrary to knat1 mutants and wild type, have a delay in cell wall loosening and organ separation (Figure 3A) thus positively regulating organ separation during abscission. The weaker phenotype when compared to haesa/hsl2 mutants is likely due to the redundant nature of other SERKs inthe abscission zone (recent work of Meng et al. 2016, cited in the Results and Discussion sections of our manuscript). It has previously been reported that mutations in SERK1 can rescue the block in abscission in plants without the functional ADP-ribosylation factor GTPase-activating protein NEVERSHED (NEV) (Lewis et al. 2010). However, as a mutation in SERK1 is not capable of rescuing the ida mutant phenotype (Lewis et al. 2010) and revertant mutants capable of rescuing the abscission defect of ida do not complement nev, it has been suggested that NEV and IDA function in parallel pathways to promote cell separation (Liu 2013). Our work does not rule out a function for SERK1 in such a parallel pathway, we merely report SERK1 can ALSO act as a positive regulator of abscission by interacting with HAESA in an IDA-dependent manner. We do not observe negative regulation of floral abscission using our SERK1 mutant alleles. Based on the available evidence there is thus little to discuss and speculate about the different functions of SERK1 in abscission, as no molecular mechanism for the negative role of SERK1 in this pathway has been reported thus far. We feel that it is beyond the scope of our manuscript to clarify the different roles of SERK1 in the Arabidopsis abscission zone. Similarly, could the authors comment on the fact that the LRR-RLP EVD/SOBIR seems to be a negative regulator of the HAESA/HSL2 pathway (Leslie et al., Development, 2010), which seems puzzling given that EVD/SOBIR function is normally restricted to LRR-RLPs (Gust \u0026 Felix, Curr Op Plant Biol, 2014). We did attempt to express and purify the EVR/SOBIR extracellular domain, but in our hands the protein is not properly secreted and hence unfolded. We thus could not further investigate the potential mechanism of EVR/SOBIR in the HAESA pathway. 7) Given that the central hydroxyproline in IDA is of such crucial importance for binding, isn't it surprising that IDAΔ69N does not bind to HAESA at all? Wouldn't it be expected that the remaining part of the peptide still binds to HAESA?  We thank the reviewers for pointing this out to us. Indeed, we find several structural and sequence features in IDA peptide to be important determinants for HAESA binding, namely the correct size of the peptide, the presence of a central Hyp residue and an intact C-terminal Arg-His-Asn motif that is buried in the structure. In the revised manuscript we now provide new experiments (binding of a C-terminal extended IDA peptide to HAESA) that clarifies this point (summarized in revised Figure 2D). We have revised our statement in the Discussion accordingly: “The central Hyp residue in IDA is found buried in the HAESA peptide binding surfaceand thus this post-translational modification may regulateIDA bioactivity.” 8) Figure 1—figure supplement 2: How is it possible that charged amino acids are involved in hydrophobic interactions?  We apologize for this confusing statement. It now reads: “A N-terminal Pro-rich motif in IDA makes contacts LRRs 2-6 of the receptor(Figure 1D, Figure 1—figure supplement 2A-C).” 9) Are the distances shown in the graphical representation of the structures proportional? It would seem that some of the aromatic rings could cause steric hindrance.  No. The graphical representation are proportional and e.g. Trp218 in the back of the binding pocket is not producing steric clashes with the peptide with the closest distance being 4.5 A. 10) Why did the authors decide to express HAESA and SERK1 without signal peptide? Would it make a difference for binding of IDA, if they leave the SP on?  No. We did express both the HAESA and SERK1 ectodomains fused to the signal peptide of human azurocidin, which provides very efficient secretion of LRR proteins in insect cells (Olczak \u0026 Olczak, Anal. Biochem., 2006) (see Methods, subsection “Protein Expression and Purification“). Both the native signal peptides for SERK1 and HAESA as well as the azurocidin signal peptide are being recognized and cleaved by the Trichoplusia ni signal peptidase. This results, just like in planta, in a mature receptor/coreceptor ectodomain starting with the first α-helix of the N-terminal capping domain (residues 20 and 24, respectively). Thus, there is no reason to believe that the signal peptide would play a role in IDA sensing. Using our system, we cannot produce HAESA and/or SERK1 ectodomains with an intact signal peptide, as this would impair folding and proper secretion of the recombinant proteins. 11) Figure 7: Are the homologous regions also the active parts of these peptides?  Yes. We have included three additional references in the Discussion section of our manuscript, which report the bioactive regions of CLV3/CLE, RGF and PEP peptides shown in Figure 7. The revised section now reads: “Importantly, this motif can also be found in other peptide hormone families (Kondo et al. 2006; Matsuzaki et al. 2010; Tang et al. 2015)(Figure 7). Among these are the CLE peptides regulating stem cell maintenance in the shoot and the root (Clark, Running, and Meyerowitz 1995). It is interesting to note, that CLEs in their mature form are also hydroxyprolinated dodecamers, which bind to a surface area in the BARELY ANY MERISTEM 1 receptor that would correspond to part of the IDA binding cleft in HAESA (Kondo et al. 2006;Ogawa et al. 2008; Shinohara et al. 2012).” And could the authors display amino acids numbers on either side of the fragments?  Yes. We have now included the residues number of each peptide in Figure 7. 12) Have the authors ever measured dissociation of the peptide from the complex?  No. We have not performed any biochemical experiment that would allow us to quantify the dissociation of the peptide from the ternary complex. In qualitative terms it is however of note that HAESA-IDA-SERK1 complexes do not dissociate in size exclusion chromatography experiments, even when the peptide is not provided in excess or supplied in the running buffer. And in this regard, to what does \"highly stable receptor – co-receptor complex\" refer/compare to?  The reviewers are correct, we should not claim that the complex is 'highly stable' if we have not quantified the dissociation rate. The revised sentence reads: “This ligand-induced formation of a receptor – co-receptor complex may allow the HAESA and SERK1 kinase domains to efficiently trans-phosphorylate and activate each other in the cytoplasm.” 13) Figures 3A and 5C require statistical analyses. Thank you for pointing this out to us. The statistical analysis of the petal break-strength assays shown in Figures 3A and 5C has been carried out by Prof. Ludwig A. Hothorn, Institute for Biostatistics, University of Hannover, Germany, whom we have added as an author on our manuscript: Statistical analysis for Figure 3A: The statistical analysis is described in the figure legend of Figure 3A; statistical significant changes are indicated by a * in the Figure itself. The revised figure legend reads: “Petal break-strength assays measure the force (expressed in gram equivalents) required to remove the petals from the flower of serk mutant plants compared to haesa/hsl2 mutant and Col-0 wild-type flowers. […] Petal break was found significantly increased in almost all positions (indicated with a *) for haesa/hsl2 and serk1-1 mutant plants with respect to the Col-0 control. Calculations were performed in R (R Core Team 2014) (version 3.2.3).” The two new references have been added to the Reference section of the manuscript. We have changed our Results section accordingly: “Our experiments suggest that among the SERK family members, SERK1 is a positive regulator offloral abscission. We found that the force required to remove the petals of serk1-1 mutants is significantlyhigher than that needed for wild-type plants, as previously observed for haesa/hsl2 mutants (Stenvik et al. 2008), and that floral abscission is delayed in serk1-1 (Figure 3A). The serk2-2, serk3-1, serk4-1 and serk5-1 mutant lines (Albrecht et al. 2008) showed a petal break-strength profile not significantly differentfrom wild-type plants.” Statistical analysis for Figure 5C: The statistical analysis is described in the figure legend of Figure 5C; statistical significant changes are indicated by * and # symbols in the Figure itself. The revised figure legend reads:”Quantitative petal break assay for Col-0 wild-type flowers and 35S::IDA wild-type and 35S::IDA K66A/R67A mutant flowers. […] Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c).” We have changed our Results section accordingly: “We overexpressed full-length wild-type IDA or this Lys66IDA/Arg67IDA → Ala double-mutant to similar levels in Col-0 Arabidopsis plants (Figure 5D). […] Comparison of 35S::IDA wild-type and mutant plants further indicates that mutation of Lys66IDA/Arg67IDA→ Ala may cause a weak dominant negative effect (Figure 5C-E).” Following the suggestions from for example Nuzzo (Nature506:150-152, 2014) and Trafirmow and Marks (Basic and Applied Social Psychology37:1-2, 2015), we decided not to report 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+[{"sourceid":"4848761","sourcedb":"","project":"","target":"","text":"Predictive features of ligand‐specific signaling through the estrogen receptor Abstract Some estrogen receptor‐α (ERα)‐targeted breast cancer therapies such as tamoxifen have tissue‐selective or cell‐specific activities, while others have similar activities in different cell types. To identify biophysical determinants of cell‐specific signaling and breast cancer cell proliferation, we synthesized 241 ERα ligands based on 19 chemical scaffolds, and compared ligand response using quantitative bioassays for canonical ERα activities and X‐ray crystallography. Ligands that regulate the dynamics and stability of the coactivator‐binding site in the C‐terminal ligand‐binding domain, called activation function‐2 (AF‐2), showed similar activity profiles in different cell types. Such ligands induced breast cancer cell proliferation in a manner that was predicted by the canonical recruitment of the coactivators NCOA1/2/3 and induction of the GREB1 proliferative gene. For some ligand series, a single inter‐atomic distance in the ligand‐binding domain predicted their proliferative effects. In contrast, the N‐terminal coactivator‐binding site, activation function‐1 (AF‐1), determined cell‐specific signaling induced by ligands that used alternate mechanisms to control cell proliferation. Thus, incorporating systems structural analyses with quantitative chemical biology reveals how ligands can achieve distinct allosteric signaling outcomes through ERα. Introduction Many drugs are small‐molecule ligands of allosteric signaling proteins, including G protein‐coupled receptors (GPCRs) and nuclear receptors such as ERα. These receptors regulate distinct phenotypic outcomes (i.e., observable characteristics of cells and tissues, such as cell proliferation or the inflammatory response) in a ligand‐dependent manner. Small‐molecule ligands control receptor activity by modulating recruitment of effector enzymes to distal regions of the receptor, relative to the ligand‐binding site. Some of these ligands achieve selectivity for a subset of tissue‐ or pathway‐specific signaling outcomes, which is called selective modulation, functional selectivity, or biased signaling, through structural mechanisms that are poorly understood (Frolik et al, 1996; Nettles \u0026 Greene, 2005; Overington et al, 2006; Katritch et al, 2012; Wisler et al, 2014). For example, selective estrogen receptor modulators (SERMs) such as tamoxifen (Nolvadex®; AstraZeneca) or raloxifene (Evista®; Eli Lilly) (Fig 1A) block the ERα‐mediated proliferative effects of the native estrogen, 17β‐estradiol (E2), on breast cancer cells, but promote beneficial estrogenic effects on bone mineral density and adverse estrogenic effects such as uterine proliferation, fatty liver, or stroke (Frolik et al, 1996; Fisher et al, 1998; McDonnell et al, 2002; Jordan, 2003). Allosteric control of ERα activity Chemical structures of some common ERα ligands. BSC, basic side chain. E2‐rings are numbered A‐D. The E‐ring is the common site of attachment for BSC found in many SERMS. ERα domain organization lettered, A‐F. DBD, DNA‐binding domain; LBD, ligand‐binding domain; AF, activation function Schematic illustration of the canonical ERα signaling pathway. Linear causality model for ERα‐mediated cell proliferation. Branched causality model for ERα‐mediated cell proliferation. ERα contains structurally conserved globular domains of the nuclear receptor superfamily, including a DNA‐binding domain (DBD) that is connected by a flexible hinge region to the ligand‐binding domain (LBD), as well as unstructured AB and F domains at its amino and carboxyl termini, respectively (Fig 1B). The LBD contains a ligand‐dependent coactivator‐binding site called activation function‐2 (AF‐2). However, the agonist activity of SERMs derives from activation function‐1 (AF‐1)—a coactivator recruitment site located in the AB domain (Berry et al, 1990; Shang \u0026 Brown, 2002; Abot et al, 2013). AF‐1 and AF‐2 bind distinct but overlapping sets of coregulators (Webb et al, 1998; Endoh et al, 1999; Delage‐Mourroux et al, 2000; Yi et al, 2015). AF‐2 binds the signature LxxLL motif peptides of coactivators such as NCOA1/2/3 (also known as SRC‐1/2/3). AF‐1 binds a separate surface on these coactivators (Webb et al, 1998; Yi et al, 2015). Yet, it is unknown how different ERα ligands control AF‐1 through the LBD, and whether this inter‐domain communication is required for cell‐specific signaling or anti‐proliferative responses. In the canonical model of the ERα signaling pathway (Fig 1C), E2‐bound ERα forms a homodimer that binds DNA at estrogen‐response elements (EREs), recruits NCOA1/2/3 (Metivier et al, 2003; Johnson \u0026 O'Malley, 2012), and activates the GREB1 gene, which is required for proliferation of ERα‐positive breast cancer cells (Ghosh et al, 2000; Rae et al, 2005; Deschenes et al, 2007; Liu et al, 2012; Srinivasan et al, 2013). However, ERα‐mediated proliferative responses vary in a ligand‐dependent manner (Srinivasan et al, 2013); thus, it is not known whether this canonical model is widely applicable across diverse ERα ligands. Our long‐term goal is to be able to predict proliferative or anti‐proliferative activity of a ligand in different tissues from its crystal structure by identifying different structural perturbations that lead to specific signaling outcomes. The simplest response model for ligand‐specific proliferative effects is a linear causality model, where the degree of NCOA1/2/3 recruitment determines GREB1 expression, which in turn drives ligand‐specific cell proliferation (Fig 1D). Alternatively, a more complicated branched causality model could explain ligand‐specific proliferative responses (Fig 1E). In this signaling model, multiple coregulator binding events and target genes (Won Jeong et al, 2012; Nwachukwu et al, 2014), LBD conformation, nucleocytoplasmic shuttling, the occupancy and dynamics of DNA binding, and other biophysical features could contribute independently to cell proliferation (Lickwar et al, 2012). To test these signaling models, we profiled a diverse library of ERα ligands using systems biology approaches to X‐ray crystallography and chemical biology (Srinivasan et al, 2013), including a series of quantitative bioassays for ERα function that were statistically robust and reproducible, based on the Z’‐statistic (Fig EV1A and B; see Materials and Methods). We also determined the structures of 76 distinct ERα LBD complexes bound to different ligand types, which allowed us to understand how diverse ligand scaffolds distort the active conformation of the ERα LBD. Our findings here indicate that specific structural perturbations can be tied to ligand‐selective domain usage and signaling patterns, thus providing a framework for structure‐based design of improved breast cancer therapeutics, and understanding the different phenotypic effects of environmental estrogens. High‐throughput screens for ERα ligand profiling Summary of ligand screening assays used to measure ER‐mediated activities. ERE, estrogen‐response element; Luc, luciferase reporter gene; M2H, mammalian 2‐hybrid; UAS, upstream‐activating sequence. Controls for screening assays described in panel (A), above. Error bars indicate mean ± SEM, n = 3. Results Strength of AF‐1 signaling does not determine cell‐specific signaling To compare ERα signaling induced by diverse ligand types, we synthesized and assayed a library of 241 ERα ligands containing 19 distinct molecular scaffolds. These include 15 indirect modulator series, which lack a SERM‐like side chain and modulate coactivator binding indirectly from the ligand‐binding pocket (Fig 2A–E; Dataset EV1) (Zheng et al, 2012) (Zhu et al, 2012) (Muthyala et al, 2003; Seo et al, 2006) (Srinivasan et al, 2013) (Wang et al, 2012) (Liao et al, 2014) (Min et al, 2013). We also generated four direct modulator series with side chains designed to directly dislocate h12 and thereby completely occlude the AF‐2 surface (Fig 2C and E; Dataset EV1) (Kieser et al, 2010). Ligand profiling using our quantitative bioassays revealed a wide range of ligand‐induced GREB1 expression, reporter gene activities, ERα‐coactivator interactions, and proliferative effects on MCF‐7 breast cancer cells (Figs EV1 and EV2A–J). This wide variance enabled us to probe specific features of ERα signaling using ligand class analyses, and identify signaling patterns shared by specific ligand series or scaffolds. Classes of compounds in the ERα ligand library Structure of the E2‐bound ERα LBD in complex with an NCOA2 peptide of (PDB 1GWR). Structural details of the ERα LBD bound to the indicated ligands. Unlike E2 (PDB 1GWR), TAM is a direct modulator with a BSC that dislocates h12 to block the NCOA2‐binding site (PDB 3ERT). OBHS is an indirect modulator that dislocates the h11 C‐terminus to destabilize the h11–h12 interface (PDB 4ZN9). The ERα ligand library contains 241 ligands representing 15 indirect modulator scaffolds, plus 4 direct modulator scaffolds. The number of compounds per scaffold is shown in parentheses (see Dataset EV1 for individual compound information and Appendix Supplementary Methods for synthetic protocols). ERα ligands induced a range of agonist activity profiles Screening data from individual ligands are shown, grouped by scaffold. Each data point represents the activity of a distinct compound. Error bars indicate the class average (mean) ± range. *Direct modulator. \nSource data are available online for this figure.\n We first asked whether direct modulation of the receptor with an extended side chain is required for cell‐specific signaling. To this end, we compared the average ligand‐induced GREB1 mRNA levels in MCF‐7 cells and 3×ERE‐Luc reporter gene activity in Ishikawa endometrial cancer cells (E‐Luc) or in HepG2 cells transfected with wild‐type ERα (L‐Luc ERα‐WT) (Figs 3A and EV2A–C). Direct modulators showed significant differences in average activity between cell types except OBHS‐ASC analogs, which had similar low agonist activities in the three cell types. The other direct modulators had low agonist activity in Ishikawa cells, no or inverse agonist activity in MCF‐7 cells, and more variable activity in HepG2 liver cells. While it was known that direct modulators such as tamoxifen drive cell‐specific signaling, these experiments reveal that indirect modulators also drive cell‐specific signaling, since eight of fourteen classes showed significant differences in average activity (Figs 3A and EV2A–C). Ligand‐specific signaling underlies ERα‐mediated cell proliferation (A) Ligand‐specific ERα activities in HepG2, Ishikawa and MCF‐7 cells. The ligand‐induced L‐Luc ERα‐WT and E‐Luc activities and GREB1 mRNA levels are shown by scaffold (mean + SD). (B) Ligand class analysis of the L‐Luc ERα‐WT and ERα‐ΔAB activities in HepG2 cells. Significant sensitivity to AB domain deletion was determined by Student's t‐test (n = number of ligands per scaffold in Fig 2). The average activities of ligands classes are shown (mean + SEM). Correlation and regression analyses in a large test set. The r\n2 values are plotted as a heat map. In cluster 1, the first three comparisons (rows) showed significant positive correlations (F‐test for nonzero slope, P ≤ 0.05). In cluster 2, only one of these comparisons revealed a significant positive correlation, while none was significant in cluster 3. +, statistically significant correlations gained by deletion of the AB or F domains. −, significant correlations lost upon deletion of AB or F domains. \nSource data are available online for this figure.\n Tamoxifen depends on AF‐1 for its cell‐specific activity (Sakamoto et al, 2002); therefore, we asked whether cell‐specific signaling observed here is due to a similar dependence on AF‐1 for activity (Fig EV1). To test this idea, we compared the average L‐Luc activities of each scaffold in HepG2 cells co‐transfected with wild‐type ERα or with ERα lacking the AB domain (Figs 1B and EV1). While E2 showed similar L‐Luc ERα‐WT and ERα‐ΔAB activities, tamoxifen showed complete loss of activity without the AB domain (Fig EV1B). Deletion of the AB domain significantly reduced the average L‐Luc activities of 14 scaffolds (Student's t‐test, P ≤ 0.05) (Fig 3B). These “AF‐1‐sensitive” activities were exhibited by both direct and indirect modulators, and were not limited to scaffolds that showed cell‐specific signaling (Fig 3A and B). Thus, the strength of AF‐1 signaling does not determine cell‐specific signaling. Identifying cell‐specific signaling clusters in ERα ligand classes As another approach to identifying cell‐specific signaling, we determined the degree of correlation between ligand‐induced activities in the different cell types. Here, we compared ligands within each class (Fig 3C), instead of comparing average activities (Fig 3A and B). For each ligand class or scaffold, we calculated the Pearson's correlation coefficient, r, for pairwise comparison of activity profiles in breast (GREB1), liver (L‐Luc), and endometrial cells (E‐Luc). The value of r ranges from −1 to 1, and it defines the extent to which the data fit a straight line when compounds show similar agonist/antagonist activity profiles between cell types (Fig EV3A). We also calculated the coefficient of determination, r 2, which describes the percentage of variance in a dependent variable such as proliferation that can be predicted by an independent variable such as GREB1 expression. We present both calculations as r 2 to readily compare signaling specificities using a heat map on which the red–yellow palette indicates significant positive correlations (P ≤ 0.05, F‐test for nonzero slope), while the blue palette denotes negative correlations (Fig 3C–F). The side chain of OBHS‐BSC analogs induces cell‐specific signaling Correlation analysis of OBHS versus OBHS‐BSC activity across cell types. Correlation analysis of L‐Luc ERα‐ΔAB activity versus endogenous ERα activity of OBHS analogs. In panel (D), L‐Luc ERα‐WT activity from panel (B) is shown for comparison. Correlation analysis of L‐Luc ERα‐ΔF activity versus endogenous ERα activities of OBHS analogs. Correlation analysis of MCF‐7 cell proliferation versus NCOA2/3 recruitment or GREB1 levels observed in response to (G) OBHS‐N and (H) OBHS‐BSC analogs. \nData information: In each panel, a data point indicates the activity of a distinct compound.Source data are available online for this figure.\n This analysis revealed diverse signaling specificities that we grouped into three clusters. Scaffolds in cluster 1 exhibited strongly correlated GREB1 levels, E‐Luc and L‐Luc activity profiles across the three cell types (Fig 3C lanes 1–4), suggesting these ligands use similar ERα signaling pathways in the breast, endometrial, and liver cell types. This cluster includes WAY‐C, OBHS, OBHS‐N, and triaryl‐ethylene analogs, all of which are indirect modulators. Cluster 2 contains scaffolds with activities that were positively correlated in only two of the three cell types, indicating cell‐specific signaling (Fig 3C lanes 5–12). This cluster includes two classes of direct modulators (cyclofenil‐ASC and WAY dimer), and six classes of indirect modulators (2,5‐DTP, 3,4‐DTP, S‐OBHS‐2 and S‐OBHS‐3, furan, and WAY‐D). In this cluster, the correlated activities varied by scaffold. For example, 3,4‐DTP, furan, and S‐OBHS‐2 drove positively correlated GREB1 levels and E‐Luc but not L‐Luc ERα‐WT activity (Fig 3C lanes 5–7). In contrast, WAY dimer and WAY‐D analogs drove positively correlated GREB1 levels and L‐Luc ERα‐WT but not E‐Luc activity (Fig 3C lanes 8 and 9). The last set of scaffolds, cluster 3, displayed cell‐specific activities that were not correlated in any of the three cell types (Fig 3C lanes 13–19). This cluster includes two direct modulator scaffolds (OBHS‐ASC and OBHS‐BSC), and five indirect modulator scaffolds (A‐CD, cyclofenil, 3,4‐DTPD, imine, and imidazopyridine). These results suggest that addition of an extended side chain to an ERα ligand scaffold is sufficient to induce cell‐specific signaling, where the relative activity profiles of the individual ligands change between cell types. This is demonstrated by directly comparing the signaling specificities of matched OBHS (indirect modulator, cluster 1) and OBHS‐BSC analogs (direct modulator, cluster 3), which differ only in the basic side chain (Fig 2E). The activities of OBHS analogs were positively correlated across the three cell types, but the side chain of OBHS‐BSC analogs was sufficient to abolish these correlations (Figs 3C lanes 1 and 19, and EV3A–C). The indirect modulator scaffolds in clusters 2 and 3 showed cell‐specific signaling patterns without the extended side chain typically viewed as the primary chemical and structural mechanism driving cell‐specific activity. Many of these scaffolds drove similar average activities of the ligand class in the different cell types (Fig 3A), but the individual ligands in each class had different cell‐specific activities (Fig EV2A–C). Thus, examining the correlated patterns of ERα activity within each scaffold demonstrates that an extended side chain is not required for cell‐specific signaling. Modulation of signaling specificity by AF‐1 To evaluate the role of AF‐1 and the F domain in ERα signaling specificity, we compared activity of truncated ERα constructs in HepG2 liver cells with endogenous ERα activity in the other cell types. The positive correlation between the L‐Luc and E‐Luc activities or GREB1 levels induced by scaffolds in cluster 1 was generally retained without the AB domain, or the F domain (Fig 3D lanes 1–4). This demonstrates that the signaling specificities underlying these positive correlations are not modified by AF‐1. OBHS analogs showed an average L‐Luc ERα‐ΔAB activity of 3.2% ± 3 (mean + SEM) relative to E2. Despite this nearly complete lack of activity, the pattern of L‐Luc ERα‐ΔAB activity was still highly correlated with the E‐Luc activity and GREB1 expression (Fig EV3D and E), demonstrating that very small AF‐2 activities can be amplified by AF‐1 to produce robust signals. Similarly, deletion of the F domain did not abolish correlations between the L‐Luc and E‐Luc or GREB1 levels induced by OBHS analogs (Fig EV3F). These similar patterns of ligand activity in the wild‐type and deletion mutants suggest that AF‐1 and the F domain purely amplify the AF‐2 activities of ligands in cluster 1. In contrast, AF‐1 was a determinant of signaling specificity for scaffolds in cluster 2. Deletion of the AB or F domain altered correlations for six of the eight scaffolds in this cluster (2,5‐DTP, 3,4‐DTP, S‐OBHS‐3, WAY‐D, WAY dimer, and cyclofenil‐ASC) (Fig 3D lanes 5–12). Comparing Fig 3C and D, the + and − signs indicate where the deletion mutant assays led to a gain or loss of statically significant correlation, respectively. Thus, in cluster 2, AF‐1 substantially modulated the specificity of ligands with cell‐specific activity (Fig 3D lanes 5–12). For ligands in cluster 3, we could not eliminate a role for AF‐1 in determining signaling specificity, since this cluster lacked positively correlated activity profiles (Fig 3C), and deletion of the AB or F domain rarely induced such correlations (Fig 3D), except for A‐CD and OBHS‐ASC analogs, where deletion of the AB domain or F domain led to positive correlations with E‐Luc activity and/or GREB1 levels (Fig 3D lanes 13 and 18). Thus, ligands in cluster 2 rely on AF‐1 for both activity (Fig 3B) and signaling specificity (Fig 3D). As discussed below, this cell specificity derives from alternate coactivator preferences. Ligand‐specific control of GREB1 expression To determine whether ligand classes control expression of native ERα target genes through the canonical linear signaling pathway, we performed pairwise linear regression analyses using ERα–NCOA1/2/3 interactions in M2H assay as independent predictors of GREB1 expression (the dependent variable) (Figs EV1 and EV2A, F–H). In cluster 1, the recruitment of NCOA1 and NCOA2 was highest for WAY‐C, followed by triaryl‐ethylene, OBHS‐N, and OBHS series, while for NCOA3, OBHS‐N compounds induced the most recruitment and OBHS ligands were inverse agonists (Fig EV2F–H). The average induction of GREB1 by cluster 1 ligands showed greater variance, with a range between ~25 and ~75% for OBHS and a range from full agonist to inverse agonist for the others in cluster 1 (Fig EV2A). GREB1 levels induced by OBHS analogs were determined by recruitment of NCOA1 but not NCOA2/3 (Fig 3E lane 1), suggesting that there may be alternate or preferential use of these coactivators by different classes. However, in cluster 1, NCOA1/2/3 recruitment generally predicted GREB1 levels (Fig 3E lanes 1–4), consistent with the canonical signaling model (Fig 1D). For clusters 2 and 3, GREB1 activity was generally not predicted by NCOA1/2/3 recruitment. Direct modulators showed low NCOA1/2/3 recruitment (Fig EV2F–H), but only OBHS‐ASC analogs had NCOA2 recruitment profiles that predicted a full range of effects on GREB1 levels (Figs 3E lanes 9, 11, 18–19, and EV2A). The indirect modulators in clusters 2 and 3 stimulated NCOA1/2/3 recruitment and GREB1 expression with substantial variance (Figs 3A and EV2F–H). However, ligand‐induced GREB1 levels were generally not determined by NCOA1/2/3 recruitment (Fig 3E lanes 5–19), consistent with an alternate causality model (Fig 1E). Out of 11 indirect modulator series in cluster 2 or 3, only the S‐OBHS‐3 class had NCOA1/2/3 recruitment profiles that predicted GREB1 levels (Fig 3E lane 12). These results suggest that compounds that show cell‐specific signaling do not activate GREB1, or use coactivators other than NCOA1/2/3 to control GREB1 expression (Fig 1E). Ligand‐specific control of cell proliferation To determine mechanisms for ligand‐dependent control of breast cancer cell proliferation, we performed linear regression analyses across the 19 scaffolds using MCF‐7 cell proliferation as the dependent variable, and the other activities as independent variables (Fig 3F). In cluster 1, E‐Luc and L‐Luc activities, NCOA1/2/3 recruitment, and GREB1 levels generally predicted the proliferative response (Fig 3F lanes 2–4). With the OBHS‐N compounds, NCOA3 and GREB1 showed near perfect prediction of proliferation (Fig EV3G), with unexplained variance similar to the noise in the assays. The lack of significant predictors for OBHS analogs (Fig 3F lane 1) reflects their small range of proliferative effects on MCF‐7 cells (Fig EV2I). The significant correlations with GREB1 expression and NCOA1/2/3 recruitment observed in this cluster are consistent with the canonical signaling model (Fig 1D), where NCOA1/2/3 recruitment determines GREB1 expression, which then drives proliferation. Ligands in cluster 2 and cluster 3 showed a wide range of proliferative effects on MCF‐7 cells (Fig EV2I). Despite this phenotypic variance, proliferation was not generally predicted by correlated NCOA1/2/3 recruitment and GREB1 induction (Figs 3F lanes 5–19, and EV3H). Out of 15 ligand series in these clusters, only 2,5‐DTP analogs induced a proliferative response that was predicted by GREB1 levels, which were not determined by NCOA1/2/3 recruitment (Fig 3E and F lane 10). 3,4‐DTP, cyclofenil, 3,4‐DTPD, and imidazopyridine analogs had NCOA1/3 recruitment profiles that predicted their proliferative effects, without determining GREB1 levels (Fig 3E and F, lanes 5 and 14–16). Similarly, S‐OBHS‐3, cyclofenil‐ASC, and OBHS‐ASC had positively correlated NCOA1/2/3 recruitment and GREB1 levels, but none of these activities determined their proliferative effects (Fig 3E and F lanes 11–12 and 18). For ligands that show cell‐specific signaling, ERα‐mediated recruitment of other coregulators and activation of other target genes likely determine their proliferative effects on MCF‐7 cells. NCOA3 occupancy at GREB1 did not predict the proliferative response We also questioned whether promoter occupancy by coactivators is statistically robust and reproducible for ligand class analysis using a chromatin immunoprecipitation (ChIP)‐based quantitative assay, and whether it has a better predictive power than the M2H assay. ERα and NCOA3 cycle on and off the GREB1 promoter (Nwachukwu et al, 2014). Therefore, we first performed a time‐course study, and found that E2 and the WAY‐C analog, AAPII‐151‐4, induced recruitment of NCOA3 to the GREB1 promoter in a temporal cycle that peaked after 45 min in MCF‐7 cells (Fig 4A). At this time point, other WAY‐C analogs also induced recruitment of NCOA3 at this site to varying degrees (Fig 4B). The Z’ for this assay was 0.6, showing statistical robustness (see Materials and Methods). We prepared biological replicates with different cell passage numbers and separately prepared samples, which showed r 2 of 0.81, demonstrating high reproducibility (Fig 4C). \nNCOA3 occupancy at GREB1 is statistically robust but does not predict transcriptional activity Kinetic ChIP assay examining recruitment of NCOA3 to the GREB1 gene in MCF‐7 cells stimulated with E2 or the indicated WAY‐C analog. The average of duplicate experiments (mean ± SEM) is shown. NCOA3 occupancy at GREB1 was compared by ChIP assay 45 min after stimulation with vehicle, E2, or the WAY‐C analogs. In panel (B), the average recruitment of two biological replicates are shown as mean + SEM, and the Z‐score is indicated. In panel (C), correlation analysis was performed for two biological replicates. Linear regression analyses comparing the ability of NCOA3 recruitment, measured by ChIP or M2H, to predict other agonist activities of WAY‐C analogs. *Significant positive correlation (F‐test for nonzero slope, P‐value). \nSource data are available online for this figure.\n The M2H assay for NCOA3 recruitment broadly correlated with the other assays, and was predictive for GREB1 expression and cell proliferation (Fig 3E). However, the ChIP assays for WAY‐C‐induced recruitment of NCOA3 to the GREB1 promoter did not correlate with any of the other WAY‐C activity profiles (Fig 4D), although the positive correlation between ChIP assays and NCOA3 recruitment via M2H assay showed a trend toward significance with r 2 = 0.36 and P = 0.09 (F‐test for nonzero slope). Thus, the simplified coactivator‐binding assay showed much greater predictive power than the ChIP assay for ligand‐specific effects on GREB1 expression and cell proliferation. ERβ activity is not an independent predictor of cell‐specific activity One difference between MCF‐7 breast cancer cells and Ishikawa endometrial cancer cells is the contribution of ERβ to estrogenic response, as Ishikawa cells may express ERβ (Bhat \u0026 Pezzuto, 2001). When overexpressed in MCF‐7 cells, ERβ alters E2‐induced expression of only a subset of ERα‐target genes (Wu et al, 2011), raising the possibility that ligand‐induced ERβ activity may contribute to E‐Luc activities, and thus underlie the lack of correlation between the E‐Luc and L‐Luc ERα‐WT activities or GREB1 levels induced by cell‐specific modulators in cluster 2 and cluster 3 (Fig 3C). To test this idea, we determined the L‐Luc ERβ activity profiles of the ligands (Fig EV1). All direct modulator and two indirect modulator scaffolds (OBHS and S‐OBHS‐3) lacked ERβ agonist activity. However, the other ligands showed a range of ERβ activities (Fig EV2J). For most scaffolds, L‐Luc ERβ and E‐Luc activities were not correlated, except for 2,5‐DTP and cyclofenil analogs, which showed moderate but significant correlations (Fig EV4A). Nevertheless, the E‐Luc activities of both 2,5‐DTP and cyclofenil analogs were better predicted by their L‐Luc ERα‐WT than L‐Luc ERβ activities (Fig EV4A and B). Thus, ERβ activity was not an independent determinant of the observed activity profiles. ERβ activity is not an independent predictor of E‐Luc activity ERβ activity in HepG2 cells rarely correlates with E‐Luc activity. ERα activity of 2,5‐DTP and cyclofenil analogs correlates with E‐Luc activity. \nData information: The r\n2 and P values for the indicated correlations are shown in both panels. *Significant positive correlation (F‐test for nonzero slope, P‐value) Structural features of consistent signaling across cell types To overcome barriers to crystallization of ERα LBD complexes, we developed a conformation‐trapping X‐ray crystallography approach using the ERα‐Y537S mutation (Nettles et al, 2008; Bruning et al, 2010; Srinivasan et al, 2013). To further validate this approach, we solved the structure of the ERα‐Y537S LBD in complex with diethylstilbestrol (DES), which bound identically in the wild‐type and ERα‐Y537S LBDs, demonstrating again that this surface mutation stabilizes h12 dynamics to facilitate crystallization without changing ligand binding (Appendix Fig S1A and B) (Nettles et al, 2008; Bruning et al, 2010; Delfosse et al, 2012). Using this approach, we solved 76 ERα LBD structures in the active conformation and bound to ligands studied here (Appendix Fig S1C). Eleven of these structures have been published, while 65 are new, including the DES‐bound ERα‐Y537S LBD. We present 57 of these new structures here (Dataset EV2), while the remaining eight new structures bound to OBHS‐N analogs will be published elsewhere (S. Srinivasan et al, in preparation). Examining many closely related structures allows us to visualize subtle structural differences, in effect using X‐ray crystallography as a systems biology tool. The indirect modulator scaffolds in cluster 1 did not show cell‐specific signaling (Fig 3C), but shared common structural perturbations that we designed to modulate h12 dynamics. Based on our original OBHS structure, the OBHS, OBHS‐N, and triaryl‐ethylene compounds were modified with h11‐directed pendant groups (Zheng et al, 2012; Zhu et al, 2012; Liao et al, 2014). Superposing the LBDs based on the class of bound ligands provides an ensemble view of the structural variance and clarifies what part of the ligand‐binding pocket is differentially perturbed or targeted. The 24 structures containing OBHS, OBHS‐N, or triaryl‐ethylene analogs showed structural diversity in the same part of the scaffolds (Figs 5A and EV5A), and the same region of the LBD—the C‐terminal end of h11 (Figs 5B and C, and EV5B), which in turn nudges h12 (Fig 5C and D). We observed that the OBHS‐N analogs displaced h11 along a vector away from Leu354 in a region of h3 that is unaffected by the ligands, and toward the dimer interface. For the triaryl‐ethylene analogs, the displacement of h11 was in a perpendicular direction, away from Ile424 in h8 and toward h12. Remarkably, these individual inter‐atomic distances showed a ligand class‐specific ability to significantly predict proliferative effects (Fig 5E and F), demonstrating the feasibility of developing a minimal set of activity predictors from crystal structures. Structural determinants of consistent signaling Structure‐class analysis of triaryl‐ethylene analogs. Triaryl‐ethylene analogs bound to the superposed crystal structures of the ERα LBD are shown. Arrows indicate chemical variance in the orientation of the different h11‐directed ligand side groups (PDB 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF and 5DP0). Triaryl‐ethylene analogs induce variance of ERα conformations at the C‐terminal region of h11. Panel (B) shows the crystal structure of a triaryl‐ethylene analog‐bound ERα LBD (PDB 5DLR). The h11–h12 interface (circled) includes the C‐terminal part of h11. This region was expanded in panel (C), where the 10 triaryl‐ethylene analog‐bound ERα LBD structures (see Datasets EV1 and EV2) were superposed to show variations in the h11 C‐terminus (PDB 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF, and 5DP0). ERα LBDs in complex with diethylstilbestrol (DES) or a triaryl‐ethylene analog were superposed to show that the ligand‐induced difference in h11 conformation is transmitted to the C‐terminus of h12 (PDB 4ZN7, 5DMC). Inter‐atomic distances predict the proliferative effects of specific ligand series. Ile424–His524 distance measured in the crystal structures correlates with the proliferative effect of triaryl‐ethylene analogs in MCF‐7 cells. In contrast, the Leu354–Leu525 distance correlates with the proliferative effects of OBHS‐N analogs in MCF‐7 cells. Structure‐class analysis of WAY‐C analogs. WAY‐C side groups subtly nudge h12 Leu540. ERα LBD structures bound to 4 distinct WAY‐C analogs were superposed (PDB 4 IU7, 4IV4, 4IVW, 4IW6) (see Datasets EV1 and EV2). \nSource data are available online for this figure.\n Structure‐class analysis of indirect modulators Structure‐class analysis of indirect modulators in cluster 1. Crystal structures of the ERα LBD bound to OBHS and OBHS‐N analogs were superposed. The bound ligands are shown in panel (A). Arrows indicate chemical variance in the orientation of the different h11‐directed ligand side groups. Panel (B) shows the ligand‐induced conformational variation at the C‐terminal region of h11 (OBHS: PDB 4ZN9, 4ZNH, 4ZNS, 4ZNT, 4ZNU, 4ZNV, and 4ZNW; OBHS‐N: PDB 4ZUB, 4ZUC, 4ZWH, 4ZWK, 5BNU, 5BP6, 5BPR, and 5BQ4). Structure‐class analysis of indirect modulators in clusters 2 and 3. Crystal structures of the ERα LBD bound to ligands with cell‐specific activities were superposed. The bound ligands are shown, and arrows indicate considerable variation in the orientation of the different h3‐, h8‐, h11‐, or h12‐directed ligand side groups. As visualized in four LBD structures (Srinivasan et al, 2013), WAY‐C analogs were designed with small substitutions that slightly nudge h12 Leu540, without exiting the ligand‐binding pocket (Fig 5G and H). Therefore, changing h12 dynamics maintains the canonical signaling pathway defined by E2 (Fig 1D) to support AF‐2‐driven signaling and recruit NCOA1/2/3 for GREB1‐stimulated proliferation. Ligands with cell‐specific activity alter the shape of the AF‐2 surface Direct modulators like tamoxifen drive AF‐1‐dependent cell‐specific activity by completely occluding AF‐2, but it is not known how indirect modulators produce cell‐specific ERα activity. Therefore, we examined another 50 LBD structures containing ligands in clusters 2 and 3. These structures demonstrated that cell‐specific activity derived from altering the shape of the AF‐2 surface without an extended side chain. Ligands in cluster 2 and cluster 3 showed conformational heterogeneity in parts of the scaffold that were directed toward multiple regions of the receptor including h3, h8, h11, h12, and/or the β‐sheets (Fig EV5C–G). For instance, S‐OBHS‐2 and S‐OBHS‐3 analogs (Fig 2) had similar ERα activity profiles in the different cell types (Fig EV2A–C), but the 2‐ versus 3‐methyl substituted phenol rings altered the correlated signaling patterns in different cell types (Fig 3B lanes 7 and 12). Structurally, the 2‐ versus 3‐methyl substitutions changed the binding position of the A‐ and E‐ring phenols by 1.0 Å and 2.2 Å, respectively (Fig EV5C). This difference in ligand positioning altered the AF‐2 surface via a shift in the N‐terminus of h12, which directly contacts the coactivator. This effect is evident in a single structure due to its 1 Å magnitude (Fig 6A and B). The shifts in h12 residues Asp538 and Leu539 led to rotation of the coactivator peptide (Fig 6C). Thus, cell‐specific activity can stem from perturbation of the AF‐2 surface without an extended side chain, which presumably alters the receptor–coregulator interaction profile. Structural correlates of cell‐specific signaling S‐OBHS‐2/3 analogs subtly distort the AF‐2 surface. Panel (A) shows the crystal structure of an S‐OBHS‐3‐bound ERα LBD (PDB 5DUH). The h3–h12 interface (circled) at AF‐2 (pink) was expanded in panels (B, C). The S‐OBHS‐2/3‐bound ERα LBDs were superposed to show shifts in h3 (panel B) and the NCOA2 peptide docked at the AF‐2 surface (panel C). Crystal structures show that 2,5‐DTP analogs shift h3 and h11 further apart compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DRM, 5DRJ). The 2F\no‐F\nc electron density map and F\no‐F\nc difference map of a 2,5‐DTP‐bound structure (PDB 5DRJ) were contoured at 1.0 sigma and ± 3.0 sigma, respectively. Average (mean + SEM) α‐carbon distance measured from h3 Thr347 to h11 Leu525 of A‐CD‐, 2,5‐DTP‐, and 3,4‐DTPD‐bound ERα LBDs. *Two‐tailed Student's t‐test, P = 0.002 (PDB A‐CD: 5DI7, 5DID, 5DIE, 5DIG, and 4PPS; 2,5‐DTP: 4IWC, 5DRM, and 5DRJ; 3,4‐DTPD: 5DTV and 5DU5). Crystal structures show that a 3,4‐DTPD analog shifts h3 (F) and the NCOA2 (G) peptide compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DTV). Hierarchical clustering of ligand‐specific binding of 154 interacting peptides to the ERα LBD was performed in triplicate by MARCoNI analysis. \nSource data are available online for this figure.\n The 2,5‐DTP analogs showed perturbation of h11, as well as h3, which forms part of the AF‐2 surface. These compounds bind the LBD in an unusual fashion because they have a phenol‐to‐phenol length of ~12 Å, which is longer than steroids and other prototypical ERα agonists that are ~10 Å in length. One phenol pushed further toward h3 (Fig 6D), while the other phenol pushed toward the C‐terminus of h11 to a greater extent than A‐CD‐ring estrogens (Nwachukwu et al, 2014), which are close structural analogs of E2 that lack a B‐ring (Fig 2). To quantify this difference, we compared the distance between α‐carbons at h3 Thr347 and h11 Leu525 in the set of structures containing 2,5‐DTP analogs (n = 3) or A‐CD‐ring analogs (n = 5) (Fig 6E). We observed a difference of 0.4 Å that was significant (two‐tailed Student's t‐test, P = 0.002) due to the very tight clustering of the 2,5‐DTP‐induced LBD conformation. The shifts in h3 suggest these compounds are positioned to alter coregulator preferences. The 2,5‐DTP and 3,4‐DTP scaffolds are isomeric, but with aryl groups at obtuse and acute angles, respectively (Fig 2). The crystal structure of ERα in complex with a 3,4‐DTP is unknown; however, we solved two crystal structures of ERα bound to 3,4‐DTPD analogs and one structure containing a furan ligand—all of which have a 3,4‐diaryl configuration (Fig 2; Datasets EV1 and EV2). In these structures, the A‐ring mimetic of the 3,4‐DTPD scaffold bound h3 Glu353 as expected, but the other phenol wrapped around h3 to form a hydrogen bond with Thr347, indicating a change in binding epitopes in the ERα ligand‐binding pocket (Fig 6F). The 3,4‐DTPD analogs also induced a shift in h3 positioning, which translated again into a shift in the bound coactivator peptide (Fig 6F). Therefore, these indirect modulators, including S‐OBHS‐2, S‐OBHS‐3, 2,5‐DTP, and 3,4‐DTPD analogs—all of which show cell‐specific activity profiles—induced shifts in h3 and h12 that were transmitted to the coactivator peptide via an altered AF‐2 surface. To test whether the AF‐2 surface shows changes in shape in solution, we used the microarray assay for real‐time coregulator–nuclear receptor interaction (MARCoNI) analysis (Aarts et al, 2013). Here, the ligand‐dependent interactions of the ERα LBD with over 150 distinct LxxLL motif peptides were assayed to define structural fingerprints for the AF‐2 surface, in a manner similar to the use of phage display peptides as structural probes (Connor et al, 2001). Despite the similar average activities of these ligand classes (Fig 3A and B), 2,5‐DTP and 3,4‐DTP analogs displayed remarkably different peptide recruitment patterns (Fig 6H), consistent with the structural analyses. Hierarchical clustering revealed that many of the 2,5‐DTP analogs recapitulated most of the peptide recruitment and dismissal patterns observed with E2 (Fig 6H). However, there was a unique cluster of peptides that were recruited by E2 but not the 2,5‐DTP analogs. In contrast, 3,4‐DTP analogs dismissed most of the peptides from the AF‐2 surface (Fig 6H). Thus, the isomeric attachment of diaryl groups to the thiophene core changed the AF‐2 surface from inside the ligand‐binding pocket, as predicted by the crystal structures. Together, these findings suggest that without an extended side chain, cell‐specific activity stems from different coregulator recruitment profiles, due to unique ligand‐induced conformations of the AF‐2 surface, in addition to differential usage of AF‐1. Indirect modulators in cluster 1 avoid this by perturbing the h11–h12 interface, and modulating the dynamics of h12 without changing the shape of AF‐2 when stabilized. Discussion Our goal was to identify a minimal set of predictors that would link specific structural perturbations to ERα signaling pathways that control cell‐specific signaling and proliferation. We found a very strong set of predictors, where ligands in cluster 1, defined by similar signaling across cell types, showed indirect modulation of h12 dynamics via the h11–12 interface or slight contact with h12. This perturbation determined proliferation that correlated strongly with AF‐2 activity, recruitment of NCOA1/2/3 family members, and induction of the GREB1 gene, consistent with the canonical ERα signaling pathway (Fig 1D). For ligands in cluster 1, deletion of AF‐1 reduced activity to varying degrees, but did not change the underlying signaling patterns established through AF‐2. In contrast, an extended side chain designed to directly reposition h12 and completely disrupt the AF‐2 surface results in cell‐specific signaling. This was demonstrated with direct modulators in clusters 2 and 3. Cluster 2 was defined by ligand classes that showed correlated activities in two of the three cell types tested, while ligand classes in cluster 3 did not show correlated activities among any of the three cell types. Compared to cluster 1, the structural rules are less clear in clusters 2 and 3, but a number of indirect modulator classes perturbed the LBD conformation at the intersection of h3, the h12 N‐terminus, and the AF‐2 surface. Ligands in these classes altered the shape of AF‐2 to affect coregulator preferences. For direct and indirect modulators in cluster 2 or 3, the canonical ERα signaling pathway involving recruitment of NCOA1/2/3 and induction of GREB1 did not generally predict their proliferative effects, indicating an alternate causal model (Fig 1E). These principles outlined above provide a structural basis for how the ligand–receptor interface leads to different signaling specificities through AF‐1 and AF‐2. It is noteworthy that regulation of h12 dynamics indirectly through h11 can virtually abolish AF‐2 activity, and yet still drive robust transcriptional activity through AF‐1, as demonstrated with the OBHS series. This finding can be explained by the fact that NCOA1/2/3 contain distinct binding sites for interaction with AF‐1 and AF‐2 (McInerney et al, 1996; Webb et al, 1998), which allows ligands to nucleate ERα–NCOA1/2/3 interaction through AF‐2, and reinforce this interaction with additional binding to AF‐1. Completely blocking AF‐2 with an extended side chain or altering the shape of AF‐2 changes the preference away from NCOA1/2/3 for determining GREB1 levels and proliferation of breast cancer cells. AF‐2 blockade also allows AF‐1 to function independently, which is important since AF‐1 drives tissue‐selective effects in vivo. This was demonstrated with AF‐1 knockout mice that show E2‐dependent vascular protection, but not uterine proliferation, thus highlighting the role of AF‐1 in tissue‐selective or cell‐specific signaling (Billon‐Gales et al, 2009; Abot et al, 2013). One current limitation to our approach is the identification of statistical variables that predict ligand‐specific activity. Here, we examined many LBD structures and tested several variables that were not predictive, including ERβ activity, the strength of AF‐1 signaling, and NCOA3 occupancy at the GREB1 gene. Similarly, we visualized structures to identify patterns. There are many systems biology approaches that could contribute to the unbiased identification of predictive variables for statistical modeling. For example, phage display was used to identify the androgen receptor interactome, which was cloned into an M2H library and used to identify clusters of ligand‐selective interactions (Norris et al, 2009). Also, we have used siRNA screening to identify a number of coregulators required for ERα‐mediated repression of the IL‐6 gene (Nwachukwu et al, 2014). However, the use of larger datasets to identify such predictor variables has its own limitations, one of the major ones being the probability of false positives from multiple hypothesis testing. If we calculated inter‐atomic distance matrices containing 4,000 atoms per structure × 76 ligand–receptor complexes, we would have 3 × 105 predictions. One way to address this issue is to use the cross‐validation concept, where hypotheses are generated on training sets of ligands and tested with another set of ligands. Based on this work, we propose several testable hypotheses for drug discovery. We have identified atomic vectors for the OBHS‐N and triaryl‐ethylene classes that predict ligand response (Fig 5E and F). These ligands in cluster 1 drive consistent, canonical signaling across cell types, which is desirable for generating full antagonists. Indeed, the most anti‐proliferative compound in the OBHS‐N series had a fulvestrant‐like profile across a battery of assays (S. Srinivasan et al, in preparation). Secondly, our finding that WAY‐C compounds do not rely of AF‐1 for signaling efficacy may derive from the slight contacts with h12 observed in crystal structures (Figs 3B and 5H), unlike other compounds in cluster 1 that dislocate h11 and rely on AF‐1 for signaling efficacy (Figs 3B and 5C, and EV5B). Thirdly, we found ligands that achieved cell‐specific activity without a prototypical extended side chain. Some of these ligands altered the shape of the AF‐2 surface by perturbing the h3–h12 interface, thus providing a route to new SERM‐like activity profiles by combining indirect and direct modulation of receptor structure. Incorporation of statistical approaches to understand relationships between structure and signaling variables moves us toward predictive models for complex ERα‐mediated responses such as in vivo uterine proliferation or tumor growth, and more generally toward structure‐based design for other allosteric drug targets including GPCRs and other nuclear receptors. Materials and Methods Statistical analysis Correlation and linear regression analyses were performed using GraphPad Prism software. For correlation analysis, the degree to which two datasets vary together was calculated with the Pearson correlation coefficient (r). However, we reported r 2 rather than r, to facilitate comparison with the linear regression results for which we calculated and reported r 2 (Fig 3C–F). Significance for r 2 was determined using the F‐test for nonzero slope. High‐throughput assays were considered statistically robust if they show Z’ \u003e 0.5, where Z’ = 1 − (3(σp+σn)/|μp−μn|), for the mean (σ) and standard deviations (μ) of the positive and negative controls (Fig EV1A and B). ERα ligand library The library of compounds examined includes both previously reported (Srinivasan et al, 2013) and newly synthesized compound series (see Dataset EV1 for individual compound information, and Appendix Supplementary Methods for synthetic protocols). Luciferase reporter assays Cells were transfected with FugeneHD reagent (Roche Applied Sciences, Indianapolis, IN) in 384‐well plates. After 24 h, cells were stimulated with 10 μM compounds dispensed using a 100‐nl pintool Biomeck NXP workstation (Beckman Coulter Inc.). Luciferase activity was measured 24 h later (see Appendix Supplementary Methods for more details). Mammalian 2‐hybrid (M2H) assays HEK293T cells were transfected with 5× UAS‐luciferase reporter, and wild‐type ERα‐VP16 activation domain plus full‐length NCOA1/2/3‐GAL4 DBD fusion protein expression plasmids, using the TransIT‐LT1 transfection reagent (Mirus Bio LLC, Madison, WI). The next day, cells were stimulated with 10 μM compounds using a 100‐nl pintool Biomeck NXP workstation (Beckman Coulter Inc.). Luciferase activity was measured after 24 h (see Appendix Supplementary Methods for more details). Cell proliferation assay MCF‐7 cells were plated on 384‐well plates in phenol red‐free media plus 10% FBS and stimulated with 10 μM compounds using 100‐nl pintool Biomeck NXP workstation (Beckman Coulter Inc.). Cell numbers determined 1 week later (see Appendix Supplementary Methods for more details). Quantitative RT–PCR MCF‐7 cells were steroid‐deprived and stimulated with compounds for 24 h. Total RNA was extracted and reverse‐transcribed. The cDNA was analyzed using TaqMan Gene Expression Master Mix (Life Technologies, Grand Island, NY), GREB1 and GAPDH (control) primers, and hybridization probes (see Appendix Supplementary Methods for more details). MARCoNI coregulator‐interaction profiling This assay was performed as previously described with the ERα LBD, 10 μM compounds, and a PamChiP peptide microarray (PamGene International) containing 154 unique coregulator peptides (Aarts et al, 2013) (see Appendix Supplementary Methods for more details). Protein production and X‐ray crystallography ERα protein was produced as previously described (Bruning et al, 2010). New ERα LBD structures (see Dataset EV2 for data collection and refinement statistics) were solved by molecular replacement using PHENIX (Adams et al, 2010), refined using ExCoR as previously described (Nwachukwu et al, 2013), and COOT (Emsley \u0026 Cowtan, 2004) for ligand‐docking and rebuilding. Data availability Crystal structures analyzed in this study include the following: 1GWR (Warnmark et al, 2002), 3ERD and 3ERT (Shiau et al, 1998), 4ZN9 (Zheng et al, 2012), 4IWC, 4 IU7, 4IV4, 4IVW, 4IW6, 4IUI, 4IV2, 4IVY and 4IW8 (Srinivasan et al, 2013), and 4PPS (Nwachukwu et al, 2014). New crystal structures analyzed in this study were deposited in the RCSB protein data bank (http://www.pdb.org): 4ZN7, 4ZNH, 4ZNS, 4ZNT, 4ZNU, 4ZNV, 4ZNW, 5DI7, 5DID, 5DIE, 5DIG, 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF, 5DP0, 5DRM, 5DRJ, 5DTV, 5DU5, 5DUE, 5DUG, 5DUH, 5DXK, 5DXM, 5DXP, 5DXQ, 5DXR, 5EHJ, 5DY8, 5DYB, 5DYD, 5DZ0, 5DZ1, 5DZ3, 5DZH, 5DZI, 5E0W, 5E0X, 5E14, 5E15, 5E19, 5E1C, 5DVS, 5DVV, 5DWE, 5DWG, 5DWI, 5DWJ, 5EGV, 5EI1, 5EIT. Author contributions JCN and SS contributed equally to this work. JCN and SS designed and performed experiments and wrote the manuscript; YZ, KEC, SW, JM, CD, ZL, VC, JN, NJW, JSJ, and RH performed experiments; HBZ designed experiments; and JAK and KWN designed experiments and wrote the manuscript. Conflict of Interest The authors declare that they have no conflict of interest. Supporting information References Robust array‐based coregulator binding assay predicting ERalpha‐agonist potency and generating binding profiles reflecting ligand structure The AF‐1 activation function of estrogen receptor alpha is necessary and sufficient for uterine epithelial cell proliferation in vivo PHENIX: a comprehensive Python‐based system for macromolecular structure solution Role of the two activating domains of the oestrogen receptor in the cell‐type and promoter‐context dependent agonistic activity of the anti‐oestrogen 4‐hydroxytamoxifen Resveratrol exhibits cytostatic and antiestrogenic properties with human endometrial adenocarcinoma (Ishikawa) cells The transactivating function 1 of estrogen receptor alpha is dispensable for the vasculoprotective actions of 17beta‐estradiol Coupling of receptor conformation and ligand orientation determine graded activity Circumventing tamoxifen resistance in breast cancers using antiestrogens that induce unique conformational changes in the estrogen receptor Analysis of estrogen receptor interaction with a repressor of estrogen receptor activity (REA) and the regulation of estrogen receptor transcriptional activity by REA Structural and mechanistic insights into bisphenols action provide guidelines for risk assessment and discovery of bisphenol A substitutes Regulation of GREB1 transcription by estrogen receptor alpha through a multipartite enhancer spread over 20 kb of upstream flanking sequences Coot: model‐building tools for molecular graphics Purification and identification of p68 RNA helicase acting as a transcriptional coactivator specific for the activation function 1 of human estrogen receptor alpha Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P‐1 Study Time‐dependent changes in biochemical bone markers and serum cholesterol in ovariectomized rats: effects of raloxifene HCl, tamoxifen, estrogen, and alendronate PDZK1 and GREB1 are estrogen‐regulated genes expressed in hormone‐responsive breast cancer Steroid receptor coactivators 1, 2, and 3: critical regulators of nuclear receptor activity and steroid receptor modulator (SRM)‐based cancer therapy Antiestrogens and selective estrogen receptor modulators as multifunctional medicines. 1. Receptor interactions Diversity and modularity of G protein‐coupled receptor structures Characterization of the pharmacophore properties of novel selective estrogen receptor downregulators (SERDs) Triaryl‐substituted Schiff bases are high‐affinity subtype‐selective ligands for the estrogen receptor Genome‐wide protein‐DNA binding dynamics suggest a molecular clutch for transcription factor function GREB1 functions as a growth promoter and is modulated by IL6/STAT3 in breast cancer Definition of the molecular and cellular mechanisms underlying the tissue‐selective agonist/antagonist activities of selective estrogen receptor modulators Analysis of estrogen receptor transcriptional enhancement by a nuclear hormone receptor coactivator Estrogen receptor‐alpha directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter Thiophene‐core estrogen receptor ligands having superagonist activity Bridged bicyclic cores containing a 1,1‐diarylethylene motif are high‐affinity subtype‐selective ligands for the estrogen receptor Ligand control of coregulator recruitment to nuclear receptors NFκB selectivity of estrogen receptor ligands revealed by comparative crystallographic analyses Differential presentation of protein interaction surfaces on the androgen receptor defines the pharmacological actions of bound ligands Improved crystallographic structures using extensive combinatorial refinement Resveratrol modulates the inflammatory response via an estrogen receptor‐signal integration network How many drug targets are there? GREB 1 is a critical regulator of hormone dependent breast cancer growth Estrogen receptor‐mediated effects of tamoxifen on human endometrial cancer cells Fluorine‐substituted cyclofenil derivatives as estrogen receptor ligands: synthesis and structure‐affinity relationship study of potential positron emission tomography agents for imaging estrogen receptors in breast cancer Molecular determinants for the tissue specificity of SERMs The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen Ligand‐binding dynamics rewire cellular signaling via estrogen receptor‐alpha Identification and structure‐activity relationships of a novel series of estrogen receptor ligands based on 7‐thiabicyclo[2.2.1]hept‐2‐ene‐7‐oxide Interaction of transcriptional intermediary factor 2 nuclear receptor box peptides with the coactivator binding site of estrogen receptor alpha Estrogen receptor activation function 1 works by binding p160 coactivator proteins Recent developments in biased agonism Gene‐specific patterns of coregulator requirements by estrogen receptor‐alpha in breast cancer cells Estrogen receptor‐beta sensitizes breast cancer cells to the anti‐estrogenic actions of endoxifen Structure of a biologically active estrogen receptor‐coactivator complex on DNA Development of selective estrogen receptor modulator (SERM)‐like activity through an indirect mechanism of estrogen receptor antagonism: defining the binding mode of 7‐oxabicyclo[2.2.1]hept‐5‐ene scaffold core ligands Bicyclic core estrogens as full antagonists: synthesis, biological evaluation and structure‐activity relationships of estrogen receptor ligands based on bridged oxabicyclic core 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+[{"sourceid":"4850273","sourcedb":"","project":"","target":"","text":"Molecular Dissection of Xyloglucan Recognition in a Prominent Human Gut Symbiont ABSTRACT Polysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as “dietary fiber.” However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. In particular, the molecular basis of complex polysaccharide recognition, an essential prerequisite to hydrolysis by cell surface glycosidases and subsequent metabolism, is generally poorly understood. Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus, which are integral to growth on this key dietary vegetable polysaccharide. Biochemical analysis reveals that these outer membrane-anchored proteins are in fact exquisitely specific for the highly branched xyloglucan (XyG) polysaccharide. The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the β-glucan backbone. The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain. Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes. IMPORTANCE The Bacteroidetes are dominant bacteria in the human gut that are responsible for the digestion of the complex polysaccharides that constitute “dietary fiber.” Although this symbiotic relationship has been appreciated for decades, little is currently known about how Bacteroidetes seek out and bind plant cell wall polysaccharides as a necessary first step in their metabolism. Here, we provide the first biochemical, crystallographic, and genetic insight into how two surface glycan-binding proteins from the complex Bacteroides ovatus xyloglucan utilization locus (XyGUL) enable recognition and uptake of this ubiquitous vegetable polysaccharide. Our combined analysis illuminates new fundamental aspects of complex polysaccharide recognition, cleavage, and import at the Bacteroidetes cell surface that may facilitate the development of prebiotics to target this phylum of gut bacteria. INTRODUCTION The human gut microbiota influences the course of human development and health, playing key roles in immune stimulation, intestinal cell proliferation, and metabolic balance. This microbial community is largely bacterial, with the Bacteroidetes, Firmicutes, and Actinobacteria comprising the dominant phyla. The ability to acquire energy from carbohydrates of dietary or host origin is central to the adaptation of human gut bacterial species to their niche. More importantly, this makes diet a tractable way to manipulate the abundance and metabolic output of the microbiota toward improved human health. However, there is a paucity of data regarding how the vast array of complex carbohydrate structures are selectively recognized and imported by members of the microbiota, a critical process that enables these organisms to thrive in the competitive gut environment. The human gut bacteria Bacteroidetes share a profound capacity for dietary glycan degradation, with many species containing \u003e250 predicted carbohydrate-active enzymes (CAZymes), compared to 50 to 100 within many Firmicutes and only 17 in the human genome devoted toward carbohydrate utilization. A remarkable feature of the Bacteroidetes is the packaging of genes for carbohydrate catabolism into discrete polysaccharide utilization loci (PUL), which are transcriptionally regulated by specific substrate signatures. The archetypal PUL-encoded system is the starch utilization system (Sus) (Fig. 1B) of Bacteroides thetaiotaomicron. The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. The importance of PUL as a successful evolutionary strategy is underscored by the observation that Bacteroidetes such as B. thetaiotaomicron and Bacteroides ovatus devote ~18% of their genomes to these systems. Moving beyond seminal genomic and transcriptomic analyses, the current state-of-the-art PUL characterization involves combined reverse-genetic, biochemical, and structural studies to illuminate the molecular details of PUL function. Xyloglucan and the Bacteroides ovatus xyloglucan utilization locus (XyGUL). (A) Representative structures of common xyloglucans using the Consortium for Functional Glycomics Symbol Nomenclature (http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml). Cleavage sites for BoXyGUL glycosidases (GHs) are indicated for solanaceous xyloglucan. (B) BtSus and BoXyGUL. (C) Localization of BoXyGUL-encoded proteins in cellular membranes and concerted modes of action in the degradation of xyloglucans to monosaccharides. The location of SGBP-A/B is presented in this work; the location of GH5 has been empirically determined, and the enzymes have been placed based upon their predicted cellular location. We recently reported the detailed molecular characterization of a PUL that confers the ability of the human gut commensal B. ovatus ATCC 8483 to grow on a prominent family of plant cell wall glycans, the xyloglucans (XyG). XyG variants (Fig. 1A) constitute up to 25% of the dry weight of common vegetables. Analogous to the Sus locus, the xyloglucan utilization locus (XyGUL) encodes a cohort of carbohydrate-binding, -hydrolyzing, and -importing proteins (Fig. 1B and C). The number of glycoside hydrolases (GHs) encoded by the XyGUL is, however, more expansive than that by the Sus locus (Fig. 1B), which reflects the greater complexity of glycosidic linkages found in XyG vis-à-vis starch. Whereas our previous study focused on the characterization of the linkage specificity of these GHs, a key outstanding question regarding this locus is how XyG recognition is mediated at the cell surface. In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. Bacteroidetes PUL ubiquitously encode homologs of SusC and SusD, as well as proteins whose genes are immediately downstream of susD, akin to susE/F, and these are typically annotated as “putative lipoproteins”. The genes coding for these proteins, sometimes referred to as “susE/F positioned,” display products with a wide variation in amino acid sequence and which have little or no homology to other PUL-encoded proteins or known carbohydrate-binding proteins. As the Sus SGBPs remain the only structurally characterized cohort to date, we therefore wondered whether such glycan binding and function are extended to other PUL that target more complex and heterogeneous polysaccharides, such as XyG. We describe here the detailed functional and structural characterization of the noncatalytic SGBPs encoded by Bacova_02651 and Bacova_02650 of the XyGUL, here referred to as SGBP-A and SGBP-B, to elucidate their molecular roles in carbohydrate acquisition in vivo. Combined biochemical, structural, and reverse-genetic approaches clearly illuminate the distinct, yet complementary, functions that these two proteins play in XyG recognition as it impacts the physiology of B. ovatus. These data extend our current understanding of the Sus-like glycan uptake paradigm within the Bacteroidetes and reveals how the complex dietary polysaccharide xyloglucan is recognized at the cell surface. RESULTS AND DISCUSSION SGBP-A and SGBP-B are cell-surface-localized, xyloglucan-specific binding proteins. SGBP-A, encoded by the XyGUL locus tag Bacova_02651 (Fig. 1B), shares 26% amino acid sequence identity (40% similarity) with its homolog, B. thetaiotaomicron SusD, and similar homology with the SusD-like proteins encoded within syntenic XyGUL identified in our earlier work. In contrast, SGBP-B, encoded by locus tag Bacova_02650, displays little sequence similarity to the products of similarly positioned genes in syntenic XyGUL nor to any other gene product among the diversity of Bacteroidetes PUL. Whereas sequence similarity among SusC/SusD homolog pairs often serves as a hallmark for PUL identification, the sequence similarities of downstream genes encoding SGBPs are generally too low to allow reliable bioinformatic classification of their products into protein families, let alone prediction of function. Hence, there is a critical need for the elucidation of detailed structure-function relationships among PUL SGBPs, in light of the manifold glycan structures in nature. Immunofluorescence of formaldehyde-fixed, nonpermeabilized cells grown in minimal medium with XyG as the sole carbon source to induce XyGUL expression, reveals that both SGBP-A and SGBP-B are presented on the cell surface by N-terminal lipidation, as predicted by signal peptide analysis with SignalP (Fig. 2). Here, the SGBPs very likely work in concert with the cell-surface-localized endo-xyloglucanase B. ovatus GH5 (BoGH5) to recruit and cleave XyG for subsequent periplasmic import via the SusC-like TBDT of the XyGUL (Fig. 1B and C). SGBP-A and SGBP-B visualized by immunofluorescence. Formalin-fixed, nonpermeabilized B. ovatus cells were grown in minimal medium plus XyG, probed with custom rabbit antibodies to SGBP-A or SGBP-B, and then stained with Alexa Fluor 488 goat anti-rabbit IgG. (A) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-A. (B) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-B. (C) Bright-field image of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. (D) FITC images of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. Cells lacking SGBP-A (ΔSGBP-A) do not grow on XyG and therefore could not be tested in parallel. In our initial study focused on the functional characterization of the glycoside hydrolases of the XyGUL, we reported preliminary affinity PAGE and isothermal titration calorimetry (ITC) data indicating that both SGBP-A and SGBP-B are competent xyloglucan-binding proteins (affinity constant [Ka] values of 3.74 × 105 M−1 and 4.98 × 104 M−1, respectively [23]). Additional affinity PAGE analysis (Fig. 3) demonstrates that SGBP-A also has moderate affinity for the artificial soluble cellulose derivative hydroxyethyl cellulose [HEC; a β(1 → 4)-glucan] and limited affinity for mixed-linkage β(1→3)/β(1→4)-glucan (MLG) and glucomannan (GM; mixed glucosyl and mannosyl backbone), which together indicate general binding to polysaccharide backbone residues and major contributions from side-chain recognition. In contrast, SGBP-B bound to HEC more weakly than SGBP-A and did not bind to MLG or GM. Neither SGBP recognized galactomannan (GGM), starch, carboxymethylcellulose, or mucin (see Fig. S1 in the supplemental material). Together, these results highlight the high specificities of SGBP-A and SGBP-B for XyG, which is concordant with their association with XyG-specific GHs in the XyGUL, as well as transcriptomic analysis indicating that B. ovatus has discrete PUL for MLG, GM, and GGM (11). Notably, the absence of carbohydrate-binding modules in the GHs encoded by the XyGUL implies that noncatalytic recognition of xyloglucan is mediated entirely by SGBP-A and -B. SGBP-A and SGBP-B preferentially bind xyloglucan. Affinity electrophoresis (10% acrylamide) of SGBP-A and SGBP-B with BSA as a control protein. All samples were loaded on the same gel next to the BSA controls; thin black lines indicate where intervening lanes were removed from the final image for both space and clarity. The percentage of polysaccharide incorporated into each native gel is displayed. The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig. 1A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig. 1A; n = 2). ITC demonstrates that SGBP-A binds to XyG polysaccharide and XyGO2 (based on a Glc8 backbone) with essentially equal affinities, while no binding of XyGO1 (Glc4 backbone) was detectable (Table 1; see Fig. S2 and S3 in the supplemental material). Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. Together, these data clearly suggest that polysaccharide binding of both SGBPs is fulfilled by a dimer of the minimal repeat, corresponding to XyGO2 (cf. Fig. 1A). The observation by affinity PAGE that these proteins specifically recognize XyG is further substantiated by their lack of binding for the undecorated oligosaccharide cellotetraose (Table 1; see Fig. S3). Furthermore, SGBP-A binds cellohexaose with ~770-fold weaker affinity than XyG, while SGBP-B displays no detectable binding to this linear hexasaccharide. To provide molecular-level insight into how the XyGUL SGBPs equip B. ovatus to specifically harvest XyG from the gut environment, we performed X-ray crystallography analysis of both SGBP-A and SGPB-B in oligosaccharide-complex forms. Summary of thermodynamic parameters for wild-type SGBP-A and SGBP-B obtained by isothermal titration calorimetry at 25°Ca Carbohydrate\tKa (M−1)\tΔG (kcal ⋅ mol−1)\tΔH (kcal ⋅ mol−1)\tTΔS (kcal ⋅ mol−1)\t \tSGBP-A\tSGBP-B\tSGBP-A\tSGBP-B\tSGBP-A\tSGBP-B\tSGBP-A\tSGBP-B\t \tXyGb\t(4.4 ± 0.1) × 105\t(5.7 ± 0.2) × 104\t−7.7\t−6.5\t−14 ± 3\t−14 ± 2\t−6.5\t−7.6\t \tXyGO2c\t3.0 × 105\t2.0 × 104\t−7.5\t−5.9\t−17.2\t−17.6\t−9.7\t−11.7\t \tXyGO1\tNBd\t(2.4 ± 0.1) × 103\tNB\t−4.6\tNB\t−4.4 ± 0.2\tNB\t0.2\t \tCellohexaose\t568.0 ± 291.0\tNB\t−3.8\tNB\t−16 ± 8\tNB\t−12.7\tNB\t \tCellotetraose\tNB\tNB\tNB\tNB\tNB\tNB\tNB\tNB\t \t Shown are average values ± standard errors from two independent titrations, unless otherwise indicated. Binding thermodynamics for XyG based on the concentration of the binding unit, XyGO2. Values from a single titration. NB, no binding observed. SGBP-A is a SusD homolog with an extensive glycan-binding platform. As anticipated by sequence similarity, the high-resolution tertiary structure of apo-SGBP-A (1.36 Å, Rwork = 14.7%, Rfree = 17.4%, residues 28 to 546) (Table 2) displays the canonical “SusD-like” protein fold dominated by four tetratrico-peptide repeat (TPR) motifs that cradle the rest of the structure (Fig. 4A). Specifically, SGBP-A overlays B. thetaiotaomicron SusD (BtSusD) with a root mean square deviation (RMSD) value of 2.2 Å for 363 Cα pairs, which is notable given the 26% amino acid identity (40% similarity) between these homologs (Fig. 4C). Cocrystallization of SGBP-A with XyGO2 generated a substrate complex structure (2.3 Å, Rwork = 21.8%, Rfree = 24.8%, residues 36 to 546) (Fig. 4A and B; Table 2) that revealed the distinct binding-site architecture of the XyG binding protein. The SGBP-A:XyGO2 complex superimposes closely with the apo structure (RMSD of 0.6 Å) and demonstrates that no major conformational change occurs upon substrate binding; small deviations in the orientation of several surface loops are likely the result of differential crystal packing. It is particularly notable that although the location of the ligand-binding site is conserved between SGBP-A and SusD, that of SGBP-A displays an ~29-Å-long aromatic platform to accommodate the extended, linear XyG chain (see reference for a review of XyG secondary structure), versus the shorter, ~18-Å-long, site within SusD that complements the helical conformation of amylose (Fig. 4C and D). Molecular structure of SGBP-A (Bacova_02651). (A) Overlay of SGBP-A from the apo (rainbow) and XyGO2 (gray) structures. The apo structure is color ramped from blue to red. An omit map (2σ) for XyGO2 (orange and red sticks) is displayed. (B) Close-up view of the omit map as in panel A, rotated 90° clockwise. (C) Overlay of the Cα backbones of SGBP-A (black) with XyGO2 (orange and red spheres) and BtSusD (blue) with maltoheptaose (pink and red spheres), highlighting the conservation of the glycan-binding site location. (D) Close-up of the SGBP-A (black and orange) and SusD (blue and pink) glycan-binding sites. The approximate length of each glycan-binding site is displayed, colored to match the protein structures. (E) Stereo view of the xyloglucan-binding site of SGBP-A, displaying all residues within 4 Å of the ligand. The backbone glucose residues are numbered from the nonreducing end; xylose residues are labeled X1 and X2. Potential hydrogen-bonding interactions are shown as dashed lines, and the distance is shown in angstroms. X-ray data collection and refinement statistics Parameter\tValue(s) fora:\t \tSGBP-A apo\tSGBP-A/XyGO2\tSGBP-B/XyGO2\tSGBP-B (CD)/XyGO2\t \tPDB ID no.\t5E75\t5E76\t5E7G\t5E7H\t \tResolution (Å)\t21.48–1.36 (1.409–1.36)\t56.13–2.3 (2.382–2.3)\t39.19–2.37 (2.455–2.37)\t30.69–1.57 (1.626–1.570)\t \tSpace group\tP21\tI422\tR32\tP6122\t \tUnit cell dimensions, a, b, c (Å)\t52.8, 81.4, 57.7; β = 107.85°\t131.5, 131.5, 188\t207.4, 207.4, 117.9\t87.1, 87.1, 201.6\t \tNo. of reflections\t\t\t\t\t \t    Total\t355,272 (26,772)\t1,068,014 (102,923)\t324,544 (32,355)\t1,366,812 (129,645)\t \t    Unique\t99,136 (9,762)\t36,775 (3,625)\t39,362 (3,898)\t62,808 (6,068)\t \tMultiplicity\t3.6 (2.7)\t29.0 (28.4)\t8.2 (8.3)\t21.8 (21.4)\t \tCompleteness (%)\t99.71 (98.82)\t99.63 (99.42)\t99.96 (100.00)\t98.4 (96.98)\t \tMean I/σ〈I〉\t15.57 (2.29)\t24.93 (6.71)\t20.98 (2.36)\t38.52 (5.03)\t \tWilson B-factor\t11.91\t31.14\t43.91\t17.86\t \tRmerge\t0.04759 (0.4513)\t0.1428 (0.7178)\t0.09159 (1.197)\t0.05559 (0.7748)\t \tCC1/2b\t0.999 (0.759)\t0.999 (0.982)\t0.999 (0.794)\t1.000 (0.933)\t \tCC*c\t1.000 (0.929)\t1.000 (0.995)\t1.000 (0.941)\t1.000 (0.982)\t \tRwork\t0.1468 (0.2597)\t0.2178 (0.2788)\t0.1975 (0.3018)\t0.1560 (0.2008)\t \tRfree\t0.1738 (0.2632)\t0.2482 (0.2978)\t0.2260 (0.3219)\t0.1712 (0.2019)\t \tNo. of non-hydrogen atoms\t\t\t\t\t \t    All\t4,562\t4,319\t3,678\t2,328\t \t    Macromolecules\t4,079\t3,974\t3,425\t1,985\t \t    Ligands\t39\t116\t127\t25\t \t    Water\t444\t229\t126\t318\t \tNo. of protein residues\t506\t492\t446\t260\t \tRMSD\t\t\t\t\t \t    Bond length (Å)\t0.008\t0.007\t0.005\t0.009\t \t    Bond angle (°)\t1.15\t0.96\t0.87\t1.18\t \tRamachandran statistics\t\t\t\t\t \t    Favored (%)\t98\t95\t97\t98\t \t    Outliers (%)\t0\t0.41\t0.23\t0\t \t    Clash score\t0.5\t2.13\t0.86\t1.27\t \tAvg B-factors\t\t\t\t\t \t    All\t16.1\t53.2\t53\t25.4\t \t    Macromolecules\t15.2\t53.5\t52.5\t22.9\t \t    Ligands\t24.7\t61\t71.1\t47\t \t    Solvent\t24.4\t42.9\t47.6\t39\t \t Numbers in parentheses are for the highest-resolution shell. CC1/2, Pearson correlation coefficient between the average intensities of each subset. CC*, Pearson correlation coefficient for correlation between the observed data set and true signal. Seven of the eight backbone glucosyl residues of XyGO2 could be convincingly modeled in the ligand electron density, and only two α(1→6)-linked xylosyl residues were observed (Fig. 4B; cf. Fig. 1). Indeed, the electron density for the ligand suggests some disorder, which may arise from multiple oligosaccharide orientations along the binding site. Three aromatic residues—W82, W283, W306—comprise the flat platform that stacks along the naturally twisted β-glucan backbone (Fig. 4E). The functional importance of this platform is underscored by the observation that the W82A W283A W306A mutant of SGBP-A, designated SGBP-A*, is completely devoid of XyG affinity (Table 3; see Fig. S4 in the supplemental material). Dissection of the individual contribution of these residues reveals that the W82A mutant displays a significant 4.9-fold decrease in the Ka value for XyG, while the W306A substitution completely abolishes XyG binding. Contrasting with the clear importance of these hydrophobic interactions, there are remarkably few hydrogen-bonding interactions with the ligand, which are provided by R65, N83, and S308, which are proximal to Glc5 and Glc3. Most surprising in light of the saccharide-binding data, however, was a lack of extensive recognition of the XyG side chains; only Y84 appeared to provide a hydrophobic interface for a xylosyl residue (Xyl1). Summary of thermodynamic parameters for site-directed mutants of SGBP-A and SGBP-B obtained by ITC with XyG at 25°Ca Protein name\tKa\tΔG (kcal ⋅ mol−1)\tΔH (kcal ⋅ mol−1)\tTΔS (kcal ⋅ mol−1)\t \tFold changeb\tM−1\t \tSGBP-A(W82A W283A W306A)\tND\tNB\tNB\tNB\tNB\t \tSGBP-A(W82A)c\t4.9\t9.1 × 104\t−6.8\t−6.3\t0.5\t \tSGBP-A(W306)\tND\tNB\tNB\tNB\tNB\t \tSGBP-B(230–489)\t0.7\t(8.6 ± 0.20) × 104\t−6.7\t−14.9 ± 0.1\t−8.2\t \tSGBP-B(Y363A)\t19.7\t(2.9 ± 0.10) × 103\t−4.7\t−18.1 ± 0.1\t−13.3\t \tSGBP-B(W364A)\tND\tWeak\tWeak\tWeak\tWeak\t \tSGBP-B(F414A)\t3.2\t(1.80 ± 0.03) × 104\t−5.8\t−11.4 ± 0.1\t−5.6\t \t Shown are average values ± standard deviations from two independent titrations, unless otherwise indicated. Binding thermodynamics are based on the concentration of the binding unit, XyGO2. Weak binding represents a Ka of \u003c500 M−1. ND, not determined; NB, no binding. Ka fold change = Ka of wild-type protein/Ka of mutant protein for xyloglucan binding. Values from a single titration. SGBP-B has a multimodular structure with a single, C-terminal glycan-binding domain. The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37 Å, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table 2) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig. 5A). Domains A, B, and C display similar β-sandwich folds; domains B (residues 134 to 230) and C (residues 231 to 313) can be superimposed onto domain A (residues 34 to 133) with RMSDs of 1.1 and 1.2 Å, respectively, for 47 atom pairs (23% and 16% sequence identity, respectively). These domains also display similarity to the C-terminal β-sandwich domains of many GH13 enzymes, including the cyclodextrin glucanotransferase of Geobacillus stearothermophilus (Fig. 5B). Such domains are not typically involved in carbohydrate binding. Indeed, visual inspection of the SGBP-B structure, as well as individual production of the A and B domains and affinity PAGE analysis (see Fig. S5 in the supplemental material), indicates that these domains do not contribute to XyG capture. On the other hand, production of the fused domains C and D in tandem (SGBP-B residues 230 to 489) retains complete binding of xyloglucan in vitro, with the observed slight increase in affinity likely arising from a reduced potential for steric hindrance of the smaller protein construct during polysaccharide interactions (Table 3). While neither the full-length protein nor domain D displays structural homology to known XyG-binding proteins, the topology of SGBP-B resembles the xylan-binding protein Bacova_04391 (PDB 3ORJ) encoded within a xylan-targeting PUL of B. ovatus (Fig. 5C). The structure-based alignment of these proteins reveals 17% sequence identity, with a core RMSD of 3.6 Å for 253 aligned residues. While there is no substrate-complexed structure of Bacova_04391 available, the binding site is predicted to include W241 and Y404, which are proximal to the XyGO binding site in SGBP-B. However, the opposing, clamp-like arrangement of these residues in Bacova_04391 is clearly distinct from the planar surface arrangement of the residues that interact with XyG in SGBP-B (described below). Multimodular structure of SGBP-B (Bacova_02650). (A) Full-length structure of SGBP-B, color coded by domain as indicated. Prolines between domains are indicated as spheres. An omit map (2σ) for XyGO2 is displayed to highlight the location of the glycan-binding site. (B) Overlay of SGBP-B domains A, B, and C (colored as in panel A), with a C-terminal Ig-like domain of the G. stearothermophilus cyclodextrin glucanotransferase (PDB 1CYG [residues 375 to 493]) in green. (C) Cα overlay of SGBP-B (gray) and Bacova_04391 (PDB 3ORJ) (pink). (D) Close-up omit map for the XyGO2 ligand, contoured at 2σ. (E) Stereo view of the xyloglucan-binding site of SGBP-B, displaying all residues within 4 Å of the ligand. The backbone glucose residues are numbered from the nonreducing end, xylose residues are shown as X1, X2, and X3, potential hydrogen-bonding interactions are shown as dashed lines, and the distance is shown in angstroms. Inspection of the tertiary structure indicates that domains C and D are effectively inseparable, with a contact interface of 396 Å2. Domains A, B, and C do not pack against each other. Moreover, the five-residue linkers between these first three domains all feature a proline as the middle residue, suggesting significant conformational rigidity (Fig. 5A). Despite the lack of sequence and structural conservation, a similarly positioned proline joins the Ig-like domains of the xylan-binding Bacova_04391 and the starch-binding proteins SusE and SusF. We speculate that this is a biologically important adaptation that serves to project the glycan binding site of these proteins far from the membrane surface. Any mobility of SGBP-B on the surface of the cell (beyond lateral diffusion within the membrane) is likely imparted by the eight-residue linker that spans the predicted lipidated Cys (C28) and the first β-strand of domain A. Other outer membrane proteins from various Sus-like systems possess a similar 10- to 20-amino-acid flexible linker between the lipidated Cys that tethers the protein to the outside the cell and the first secondary structure element. Analogously, the outer membrane-anchored endo-xyloglucanase BoGH5 of the XyGUL contains a 100-amino-acid, all-β-strand, N-terminal module and flexible linker that imparts conformational flexibility and distances the catalytic module from the cell surface. XyG binds to domain D of SGBP-B at the concave interface of the top β-sheet, with binding mediated by loops connecting the β-strands. Six glucosyl residues, comprising the main chain, and three branching xylosyl residues of XyGO2 can be modeled in the density (Fig. 5D; cf. Fig. 1A). The backbone is flat, with less of the “twisted-ribbon” geometry observed in some cello- and xylogluco-oligosaccharides. The aromatic platform created by W330, W364, and Y363 spans four glucosyl residues, compared to the longer platform of SGBP-A, which supports six glucosyl residues (Fig. 5E). The Y363A site-directed mutant of SGBP-B displays a 20-fold decrease in the Ka for XyG, while the W364A mutant lacks XyG binding (Table 3; see Fig. S6 in the supplemental material). There are no additional contacts between the protein and the β-glucan backbone and surprisingly few interactions with the side-chain xylosyl residues, despite that fact that ITC data demonstrate that SGBP-B does not measurably bind the cellohexaose (Table 1). F414 stacks with the xylosyl residue of Glc3, while Q407 is positioned for hydrogen bonding with the O4 of xylosyl residue Xyl1. Surprisingly, an F414A mutant of SGBP-B displays only a mild 3-fold decrease in the Ka value for XyG, again suggesting that glycan recognition is primarily mediated via contact with the β-glucan backbone (Table 3; see Fig. S6). Additional residues surrounding the binding site, including Y369 and E412, may contribute to the recognition of more highly decorated XyG, but precisely how this is mediated is presently unclear. Hoping to achieve a higher-resolution view of the SGBP-B–xyloglucan interaction, we solved the crystal structure of the fused CD domains in complex with XyGO2 (1.57 Å, Rwork = 15.6%, Rfree = 17.1%, residues 230 to 489) (Table 2). The CD domains of the truncated and full-length proteins superimpose with a 0.4-Å RMSD of the Cα backbone, with no differences in the position of any of the glycan-binding residues (see Fig. S7A in the supplemental material). While density is observed for XyGO2, the ligand could not be unambiguously modeled into this density to achieve a reasonable fit between the X-ray data and the known stereochemistry of the sugar (see Fig. S7B and C). While this may occur for a number of reasons in crystal structures, it is likely that the poor ligand density even at higher resolution is due to movement or multiple orientations of the sugar averaged throughout the lattice. SGBP-A and SGBP-B have distinct, coordinated functions in vivo. The similarity of the glycan specificity of SGBP-A and SGBP-B presents an intriguing conundrum regarding their individual roles in XyG utilization by B. ovatus. To disentangle the functions of SGBP-A and SGBP-B in XyG recognition and uptake, we created individual in-frame deletion and complementation mutant strains of B. ovatus. In these growth experiments, overnight cultures of strains grown on minimal medium plus glucose were back-diluted 1:100-fold into minimal medium containing 5 mg/ml of the reported carbohydrate. Growth on glucose displayed the shortest lag time for each strain, and so lag times were normalized for each carbohydrate by subtracting the lag time of that strain in glucose (Fig. 6; see Fig. S8 in the supplemental material). A strain in which the entire XyGUL is deleted displays a lag of 24.5 h during growth on glucose compared to the isogenic parental wild-type (WT) Δtdk strain, for which exponential growth lags for 19.8 h (see Fig. S8D). It is unknown whether this is because cultures were not normalized by the starting optical density (OD) or viable cells or reflects a minor defect for glucose utilization. The former seems more likely as the growth rates are nearly identical for these strains on glucose and xylose. The ΔXyGUL and WT Δtdk strains display normalized lag times on xylose within experimental error, and curiously some of the mutant and complemented strains display a nominally shorter lag time on xylose than the WT Δtdk strain. Complementation of the ΔSGBP-A strain (ΔSGBP-A::SGBP-A) restores growth to wild-type rates on xyloglucan and XyGO1, yet the calculated rate of the complemented strain is ~72% that of the WT Δtdk strain on XyGO2; similar results were obtained for the SGBP-B complemented strain despite the fact that the growth curves do not appear much different (see Fig. S8C and F). The reason for this observation on XyGO2 is unclear, as the ΔSGBP-B mutant does not have a significantly different growth rate from the WT on XyGO2. Therefore, we limit our discussion to those mutants that displayed the most obvious defects in growth on particular substrates. Growth of select XyGUL mutants on xyloglucan and oligosaccharides. B. ovatus mutants were created in a thymidine kinase deletion (Δtdk) mutant as described previously. SGBP-A* denotes the Bacova_02651 (W82A W283A W306A) allele, and the GH9 gene is Bacova_02649. Growth was measured over time in minimal medium containing (A) XyG, (B) XyGO2, (C) XyGO1, (D) glucose, and (E) xylose. In panel F, the growth rate of each strain on the five carbon sources is displayed, and in panel G, the normalized lag time of each culture, relative to its growth on glucose, is displayed. Solid bars indicate conditions that are not statistically significant from the WT Δtdk cultures grown on the indicated carbohydrate, while open bars indicate a P value of \u003c0.005 compared to the WT Δtdk strain. Conditions denoted by the same letter (b, c, or d) are not statistically significant from each other but are significantly different from the condition labeled “a.” Complementation of ΔSGBP-A and ΔSBGP-B was performed by allelic exchange of the wild-type genes back into the genome for expression via the native promoter: these growth curves, quantified rates and lag times are displayed in Fig. S8 in the supplemental material. The ΔSGBP-A (ΔBacova_02651) strain (cf. Fig. 1B) was completely incapable of growth on XyG, XyGO1, and XyGO2, indicating that SGBP-A is essential for XyG utilization (Fig. 6). This result mirrors our previous data for the canonical Sus of B. thetaiotaomicron, which revealed that a homologous ΔsusD mutant is unable to grow on starch or malto-oligosaccharides, despite normal cell surface expression of all other PUL-encoded proteins. More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of ΔsusD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B. thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition. Similarly, the function of SGBP-A extends beyond glycan binding. Complementation of ΔSGBP-A with the SGBP-A* (W82A W283A W306A) variant, which does not bind XyG, supports growth on XyG and XyGOs (Fig. 6; ΔSGBP-A::SGBP-A*), with growth rates that are ~70% that of the WT. In previous studies, we observed that carbohydrate binding by SusD enhanced the sensitivity of the cells to limiting concentrations of malto-oligosaccharides by several orders of magnitude, such that the addition of 0.5 g/liter maltose was required to restore growth of the ΔsusD::SusD* strain on starch, which nonetheless occurred following an extended lag phase. In contrast, the ΔSGBP-A::SGBP-A* strain does not display an extended lag time on any of the xyloglucan substrates compared to the WT (Fig. 6). The specific glycan signal that upregulates BoXyGUL is currently unknown. From our present data, we cannot eliminate the possibility that the glycan binding by SGBP-A enhances transcriptional activation of the XyGUL. However, the modest rate defect displayed by the SGBP-A::SGBP-A* strain suggests that recognition of XyG and product import is somewhat less efficient in these cells. Intriguingly, the ΔSGBP-B strain (ΔBacova_02650) (cf. Fig. 1B) exhibited a minor growth defect on both XyG and XyGO2, with rates 84.6% and 93.9% that of the WT Δtdk strain. However, growth of the ΔSGBP-B strain on XyGO1 was 54.2% the rate of the parental strain, despite the fact that SGBP-B binds this substrate ca. 10-fold more weakly than XyGO2 and XyG (Fig. 6; Table 1). As such, the data suggest that SGBP-A can compensate for the loss of function of SGBP-B on longer oligo- and polysaccharides, while SGBP-B may adapt the cell to recognize smaller oligosaccharides efficiently. Indeed, a double mutant, consisting of a crippled SGBP-A and a deletion of SGBP-B (ΔSGBP-A::SGBP-A*/ΔSGBP-B), exhibits an extended lag time on both XyG and XyGO2, as well as XyGO1. Taken together, the data indicate that SGBP-A and SGBP-B functionally complement each other in the capture of XyG polysaccharide, while SGBP-B may allow B. ovatus to scavenge smaller XyGOs liberated by other gut commensals. This additional role of SGBP-B is especially notable in the context of studies on BtSusE and BtSusF (positioned similarly in the archetypal Sus locus) (Fig. 1B), for which growth defects on starch or malto-oligosaccharides have never been observed. Beyond SGBP-A and SGBP-B, we speculated that the catalytically feeble endo-xyloglucanase GH9, which is expendable for growth in the presence of GH5, might also play a role in glycan binding to the cell surface. However, combined deletion of the genes encoding GH9 (encoded by Bacova_02649) and SGBP-B does not exacerbate the growth defect on XyGO1 (Fig. 6; ΔSGBP-B/ΔGH9). The necessity of SGBP-B is elevated in the SGBP-A* strain, as the ΔSGBP-A::SGBP-A*/ ΔSGBP-B mutant displays an extended lag during growth on XyG and xylogluco-oligosaccharides, while growth rate differences are more subtle. The precise reason for this lag is unclear, but recapitulating our findings on the role of SusD in malto-oligosaccharide sensing in B. thetaiotaomicron, this extended lag may be due to inefficient import and thus sensing of xyloglucan in the environment in the absence of glycan binding by essential SGBPs. Our previous work demonstrates that B. ovatus cells grown in minimal medium plus glucose express low levels of the XyGUL transcript. Thus, in our experiments, we presume that each strain, initially grown in glucose, expresses low levels of the XyGUL transcript and thus low levels of the XyGUL-encoded surface proteins, including the vanguard GH5. Presumably without glycan binding by the SGBPs, the GH5 protein cannot efficiently process xyloglucan, and/or the lack of SGBP function prevents efficient capture and import of the processed oligosaccharides. It may then be that only after a sufficient amount of glycan is processed and imported by the cell is XyGUL upregulated and exponential growth on the glycan can begin. We hypothesize that during exponential growth the essential role of SGBP-A extends beyond glycan recognition, perhaps due to a critical interaction with the TBDT. In the BtSus, SusD and the TBDT SusC interact, and we speculate that this interaction is necessary for glycan uptake, as suggested by the fact that a ΔsusD mutant cannot grow on starch, but a ΔsusD::SusD* strain regains this ability if a transcriptional activator of the sus operon is supplied. Likewise, such cognate interactions between homologous protein pairs such as SGBP-A and its TBDT may underlie our observation that a ΔSGBP-A mutant cannot grow on xyloglucan. However, unlike the Sus, in which elimination of SusE and SusF does not affect growth on starch, SGBP-B appears to have a dedicated role in growth on small xylogluco-oligosaccharides. Conclusions. The ability of gut-adapted microorganisms to thrive in the gastrointestinal tract is critically dependent upon their ability to efficiently recognize, cleave, and import glycans. The human gut, in particular, is a densely packed ecosystem with hundreds of species, in which there is potential for both competition and synergy in the utilization of different substrates. Recent work has elucidated that Bacteroidetes cross-feed during growth on many glycans; the glycoside hydrolases expressed by one species liberate oligosaccharides for consumption by other members of the community. Thus, understanding glycan capture at the cell surface is fundamental to explaining, and eventually predicting, how the carbohydrate content of the diet shapes the gut community structure as well as its causative health effects. Here, we demonstrate that the surface glycan binding proteins encoded within the BoXyGUL play unique and essential roles in the acquisition of the ubiquitous and abundant vegetable polysaccharide xyloglucan. Yet, a number of questions remain regarding the molecular interplay of SGBPs with their cotranscribed cohort of glycoside hydrolases and TonB-dependent transporters. A particularly understudied aspect of glycan utilization is the mechanism of import via TBDTs (SusC homologs) (Fig. 1), which are ubiquitous and defining components of all PUL. PUL-encoded TBDTs in Bacteroidetes are larger than the well-characterized iron-targeting TBDTs from many Proteobacteria and are further distinguished as the only known glycan-importing TBDTs coexpressed with an SGBP. A direct interaction between the BtSusC TBDT and the SusD SGBP has been previously demonstrated, as has an interaction between the homologous components encoded by an N-glycan-scavenging PUL of Capnocytophaga canimorsus. Our observation here that the physical presence of the SusD homolog SGBP-A, independent of XyG-binding ability, is both necessary and sufficient for XyG utilization further supports a model of glycan import whereby the SusC-like TBDTs and the SusD-like SGBPs must be intimately associated to support glycan uptake (Fig. 1C). It is yet presently unclear whether this interaction is static or dynamic and to what extent the association of cognate TBDT/SGBPs is dependent upon the structure of the carbohydrate to be imported. On the other hand, there is clear evidence for independent TBDTs in Bacteroidetes that do not require SGBP association for activity. For example, it was recently demonstrated that expression of nanO, which encodes a SusC-like TBDT as part of a sialic-acid-targeting PUL from B. fragilis, restored growth on this monosaccharide in a mutant strain of E. coli. In this instance, coexpression of the susD-like gene nanU was not required, nor did the expression of the nanU gene enhance growth kinetics. Similarly, the deletion of BT1762 encoding a fructan-targeting SusD-like protein in B. thetaiotaomicron did not result in a dramatic loss of growth on fructans. Thus, the strict dependence on a SusD-like SGBP for glycan uptake in the Bacteroidetes may be variable and substrate dependent. Furthermore, considering the broader distribution of TBDTs in PUL lacking SGBPs (sometimes known as carbohydrate utilization containing TBDT [CUT] loci; see reference and reviewed in reference) across bacterial phyla, it appears that the intimate biophysical association of these substrate-transport and -binding proteins is the result of specific evolution within the Bacteroidetes. Equally intriguing is the observation that while SusD-like proteins such as SGBP-A share moderate primary and high tertiary structural conservation, the genes for the SGBPs encoded immediately downstream (Fig. 1B [sometimes referred to as “susE positioned”]) encode glycan-binding lipoproteins with little or no sequence or structural conservation, even among syntenic PUL that target the same polysaccharide. Such is the case for XyGUL from related Bacteroides species, which may encode either one or two of these predicted SGBPs, and these proteins vary considerably in length. The extremely low similarity of these SGBPs is striking in light of the moderate sequence conservation observed among homologous GHs in syntenic PUL. This, together with the observation that these SGBPs, as exemplified by BtSusE and BtSusF and the XyGUL SGBP-B of the present study, are expendable for polysaccharide growth, implies a high degree of evolutionary flexibility to enhance glycan capture at the cell surface. Because the intestinal ecosystem is a dense consortium of bacteria that must compete for their nutrients, these multimodular SGBPs may reflect ongoing evolutionary experiments to enhance glycan uptake efficiency. Whether organisms that express longer SGBPs, extending further above the cell surface toward the extracellular environment, are better equipped to compete for available carbohydrates is presently unknown. However, the natural diversity of these proteins represents a rich source for the discovery of unique carbohydrate-binding motifs to both inform gut microbiology and generate new, specific carbohydrate analytical reagents. In conclusion, the present study further illuminates the essential role that surface-glycan binding proteins play in facilitating the catabolism of complex dietary carbohydrates by Bacteroidetes. The ability of our resident gut bacteria to recognize polysaccharides is the first committed step of glycan consumption by these organisms, a critical process that influences the community structure and thus the metabolic output (i.e., short-chain fatty acid and metabolite profile) of these organisms. A molecular understanding of glycan uptake by human gut bacteria is therefore central to the development of strategies to improve human health through manipulation of the microbiota. MATERIALS AND METHODS Protein production and purification. The gene fragments corresponding to Bacova_02650 (encoding SGBP-B residues 34 to 489) and Bacova_02651 (encoding SGBP-A residues 28 to 546) were amplified from Bacteroidetes ovatus ATCC 8483 genomic DNA by PCR using forward primers, including NdeI restriction sites, and reverse primers, including XhoI. The gene products were ligated into a modified version of pET-28a (EMD Biosciences) containing a recombinant tobacco etch virus (rTEV) protease recognition site (pET-28aTEV) preceding an N-terminal 6-His tag for affinity purification. The expression vector (pET-28TEV) containing SGBP-B was used for subsequent cloning of the domains A (residues 34 to 133), B (residues 134 to 229), and CD (residues 230 to 489). The pET-28TEV vector expressing residues 28 to 546 of SGBP-A was utilized for carbohydrate-binding experiments and crystallization of the apo structure. To obtain crystals of SGBP-A with XyGO2, the DNA sequence coding for residues 36 to 546 was PCR amplified from genomic DNA for ligation-independent cloning into the pETite N-His vector (Lucigen, Madison, WI) according to the manufacturer’s instructions. The N-terminal primer for pETite N-His insertion contained a TEV cleavage site immediately downstream of the complementary 18-bp overlap (encoding the His tag) to create a TEV-cleavable His-tagged protein. The site-directed mutants of SGBP-A and SGBP-B in pET-28TEV were created using the QuikChange II site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions. The sequences of all primers to generate these constructs are displayed in Table S1 in the supplemental material. The plasmids containing the SGBP-A and SGBP-B genes were transformed into Escherichia coli BL21(DE3) or Rosetta(DE3) cells. For native protein expression, cells were cultured in Terrific Broth containing kanamycin (50 µg/ml) and chloramphenicol (20 µg/ml) at 37°C to the mid-exponential phase (A600 of ≈0.6), induced by the addition of 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), and then incubated for 2 days at 16°C or 1 day at 20°C. Cells were harvested by centrifugation and frozen at −80°C prior to protein purification. For selenomethionine-substituted SGBP-B, the pET-28TEV-SGBP-B plasmid was transformed into E. coli Rosetta(DE3)/pLysS and plated onto LB supplemented with kanamycin (50 µg/ml) and chloramphenicol (20 µg/ml). After 16 h of growth at 37°C, colonies were harvested from the plates, used to inoculate 100 ml of M9 minimal medium supplemented with kanamycin (30 µg/ml) and chloramphenicol (20 µg/ml), and then grown at 37°C for 16 h. This overnight culture was used to inoculate a 2-liter baffled flask containing 1 liter of Molecular Dimensions SelenoMet premade medium supplemented with 50 ml of the recommended sterile nutrient mix, chloramphenicol, and kanamycin. Cultures were grown at 37°C to an A600 of ≈0.45 before adjusting the temperature to 20°C and supplementing each flask with 100 mg each of l-lysine, l-threonine, and l-phenylalanine and 50 mg each of l-leucine, l-isoleucine, l-valine, and l-selenomethionine. After 20 additional minutes of growth, the cells were induced with 0.5 mM IPTG, and cultures were grown for an additional 48 h. For the purification of native and selenomethionine-substituted protein, cells were thawed and lysed via sonication in His buffer (25 mM NaH2PO4, 500 mM NaCl, 20 mM imidazole, pH 7.5) and purified via immobilized nickel affinity chromatography (His-Trap; GE Healthcare) using a gradient of 20 to 300 mM imidazole, according to the manufacturer’s instructions. The His tag was removed by incubation with TEV protease (1:100 molar ratio relative to protein) at room temperature for 2 h and then overnight at 4°C while being dialyzed against His buffer. The cleaved protein was then repurified via nickel affinity chromatography to remove undigested target protein, the cleaved His tag, and His-tagged TEV protease. Purified proteins were dialyzed against 20 mM HEPES–100 mM NaCl (pH 7.0) prior to crystallization and concentrated using Vivaspin 15 (10,000-molecular-weight-cutoff [MWCO]) centrifugal concentrators (Vivaproducts, Inc.). Glycans. Xyloglucan from tamarind seed, β-glucan from barley, and konjac glucomannan were purchased from Megazyme. Starch, guar, and mucin were purchased from Sigma. Hydroxyethyl cellulose was purchased from AMRESCO. Carboxymethyl cellulose was purchased from Acros Organics. Xylogluco-oligosaccharides XyGO1 and XyGO2 for biophysical analyses were prepared from tamarind seed XyG according to the method of Martinez-Fleites et al. with minor modifications. XyGO2 for cocrystallization was purchased from Megazyme (O-XGHDP). Affinity gel electrophoresis. Affinity PAGE was performed as described previously, with minor modification. Various polysaccharides were used at a concentration of 0.05 to 0.1% (wt/vol), and electrophoresis was carried out for 90 min at room temperature in native 10% (wt/vol) polyacrylamide gels. BSA was used as noninteracting negative-control protein. ITC. Isothermal titration calorimetry (ITC) of glycan binding by the SGPB-A mutants was performed using the TA Nano isothermal titration calorimeter calibrated to 25°C. Proteins were dialyzed against 20 mM HEPES–100 mM NaCl (pH 7.0), and sugars were prepared using the dialysis buffer. The protein (45 to 50 µM) was placed in the sample cell, and the syringe was loaded with 2.5 to 4 mg/ml XyG polysaccharide. Following an initial injection of 0.5 µl, 26 subsequent injections of 2 µl were performed with stirring at 350 rpm, and the resulting heat of reaction was recorded. Data were analyzed using the Nano Analyze software. All other ITC experiments were performed using a MicroCal VP-ITC titration calorimeter calibrated to 25°C. Proteins were dialyzed into 20 mM HEPES–100 mM NaCl (pH 7.0), and polysaccharides were prepared using the dialysis buffer. Proteins (micromolar concentrations) were placed in the sample cell, and a first injection of 2 µl was performed followed by 24 subsequent injections of 10 µl of 2 to 20 mM oligosaccharide (cellotetraose, cellohexaose, XyGO1, or XyGO2) or 1 to 2.5 mg/ml XyG polysaccharide. The solution was stirred at 280 rpm, and the resulting heat of reaction was recorded. Data were analyzed using the Origin software program. DSC. Structural integrity of the SGBP-B mutants was verified by differential scanning calorimetry (DSC). DSC studies were performed on a MicroCal VP-DSC (Malvern Instruments). Experiments were carried out in 50 mM HEPES (pH 7.0) at a scan rate of 60°C/h. All samples (40 µM protein) were degassed for 7 min with gentle stirring under vacuum prior to being loaded into the calorimeter. Background excess thermal power scans were obtained with buffer in both the sample and reference cells and subtracted from the scans for each sample solution to generate excess heat capacity versus temperature thermograms. The melting temperature decreased from 57.8 ± 0.9°C for the wild-type SGBP-B protein to 54.6 ± 0.1°C for the Y363A mutant, 54.2 ± 0.1°C for the W364A mutant, and 52 ± 1°C for the F414A mutant. All proteins were therefore in their stable folded state for the ITC measurements (see Fig. S9 in the supplemental material). Bacteroides ovatus mutagenesis. Gene deletions and complementations were performed via allelic exchange in a Bacteroides ovatus thymidine kinase gene (Bacova_03071) deletion (Δtdk) derivative strain of ATCC 8483 using the vector pExchange-tdk, as previously described. Primers for the construction of B. ovatus mutants are listed in Table S1. The B. ovatus Δtdk strain and the B. ovatus ΔXyGUL mutant were a generous gift from Eric Martens, University of Michigan Medical School. Bacteroides growth experiments. All Bacteroides ovatus culturing was performed in a Coy anaerobic chamber (85% N2, 10% H2, 5% CO2) at 37°C. Prior to growth on minimal medium plus the carbohydrates indicated (Fig. 6; see Fig. S8 in the supplemental material), each strain was grown for 16 h from a freezer stock in tryptone-yeast extract-glucose (TYG) medium and then back diluted 1:100 into Bacteroides minimal medium supplemented with 5 mg/ml glucose, as previously described. After growth for 24 h, cultures were back-diluted 1:100 into Bacteroides minimal medium supplemented with 5 mg/ml of glucose, xylose, XyG, XyGO1, or XyGO2. Growth experiments were performed in replicates of 12 (glucose, xylose, and xyloglucan) or 5 (XyGO1 and XyGO2) as 200-µl cultures in 96-well plates. Plates were loaded into a Biostack automated plate handling device coupled to a Powerwave HT absorbance reader (both devices from Biotek Instruments, Winooski, VT). Absorbance at 600 nm (A600; i.e., optical density at 600 nm [OD600]) was measured for each well at 20-min intervals. Data were processed using Gen5 software (BioTek) and Microsoft Excel. Growth was quantified in each assay by first identifying a minimum time point (Amin) at which A600 had increased by 15% over a baseline reading taken during the first 500 min of incubation. Next, we identified the time point at which A600 reached its maximum (Amax) immediately after exponential growth. The growth rate for each well was defined by (Amax − Amin)/(Tmax − Tmin), where Tmax and Tmin are the corresponding time values for each absorbance. To account for variations in inoculum density, for each strain, the lag time (Tmin) on glucose was subtracted from the lag time for the substrate of interest; in all cases, cultures had shorter lag times on glucose than other glycans. Immunofluorescence. Custom rabbit antibodies to recombinant SGBP-A and SGBP-B were generated by Cocalico Biologicals, Inc. (Reamstown, PA). The B. ovatus ATCC 8483 Δtdk and ΔSGBP-B strains were grown in 1 ml minimal Bacteroides medium supplemented with 5 mg/ml tamarind xyloglucan to an A600 of ≈0.6 and then harvested via centrifugation (7,000 × g for 3 min) and washed twice with phosphate-buffered saline (PBS). Cells were resuspended in 0.25 ml PBS, and 0.75 ml of 6% formalin in PBS was added. Cells were incubated with rocking at 20°C for 1.5 h and then washed twice with PBS. Cells were resuspended in 0.5 to 1 ml blocking solution (2% goat serum, 0.02% NaN3 in PBS) and incubated for 16 h at 4°C. Cells were centrifuged and resuspended in 0.5 ml of a 1/100 dilution of custom rabbit antibody sera in blocking solution and incubated by rocking for 2 h at 20°C. Cells were washed with PBS and then resuspended in 0.4 ml of a 1/500 dilution of Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies) in blocking solution and incubated with rocking for 1 h at 20°C. Cells were washed three times with an excess of PBS and then resuspended in 20 µl of PBS plus 1 µl of ProLong Gold antifade (Life Technologies). Cells were spotted on 0.8% agarose pads and imaged at the Center for Live Cell Imaging at the University of Michigan Medical School, using an Olympus IX70 inverted confocal microscope. Images were processed with Metamorph Software. Crystallization and data collection. All X-ray diffraction data for both native and selenomethionine-substituted protein crystals were collected at the Life Science Consortium (LS-CAT) at the Advance Photon Source at Argonne National Laboratory in Argonne, IL. The native protein SGBP-B (residues 34 to 489) was concentrated to an A280 of 12.25 prior to crystallization and mixed with 10 mM XyGO2 (Megazyme, O-XGHDP). Hanging drop vapor diffusion was performed against mother liquor consisting of 1.1 to 1.5 M ammonium sulfate and 30 to 70 mM sodium cacodylate (pH 6.5). To decrease crystal nucleation, 0.3 ml of paraffin oil was overlaid on top of 0.5 ml of mother liquor yielding diffraction-quality crystals within 2 weeks. Selenomethionine-substituted crystals of SGBP-B were generated using the same conditions as the native crystals. Crystals of the truncated SGBP-B (domains CD, residues 230 to 489) were obtained by mixing concentrated protein (A600 of 20.6) with 10 mM XyGO2 for hanging drop vapor diffusion against a solution containing 2 M sodium formate and 0.1 M sodium acetate (pH 4.6). All SGBP-B crystals were flash-frozen prior to data collection by briefly soaking in a solution of 80% mother liquor–20% glycerol plus 10 mM xylogluco-oligosaccharide. Data were processed and scaled using HKL2000 and Scalepack. SAD phasing from a selenomethionine-substituted protein crystals was used to determine the structure of SGBP-B. The AutoSol and Autobuild algorithms within the Phenix suite of programs were used to locate and refine the selenium positions and automatically build an initial model of the protein structure, respectively. Successive rounds of manual model building and refinement in Coot and Phenix, respectively, were utilized to build a 2.7-Å model of the selenomethionine-substituted protein, which then was placed in the unit cell of the native data set. Additional rounds of manual model building and refinement were performed to complete the 2.37-Å structure of SGBP-B with XyGO2. The structure of the truncated protein (CD domains, residues 230 to 489) was solved via molecular replacement with Phaser using the CD domains of the full-length protein as a model. The native protein SGBP-A (residues 28 to 546) was concentrated to an A280 of 28.6 and crystallized via hanging drop vapor diffusion from the Morpheus crystal screen (Molecular Dimensions). Crystals formed in well A1 (30 mM MgCl2, 30 mM CaCl2, 20% polyethylene glycol [PEG 500], 10% PEG 20K, 0.1 M imidazole-MES [morpholinethanesulfonic acid], pH 6.5), and were flash-frozen in liquid nitrogen without additional cryoprotectant. The truncated SGBP-A (residues 36 to 546) concentrated to an A280 of 38.2 yielded crystals with 10 mM XyGO2 via hanging drop vapor diffusion against 1.2 to 1.8 M sodium citrate (pH 6.15 to 6.25), and were flash-frozen in a cryoprotectant solution of 80% mother liquor–20% ethylene glycol with the glycan. Data were processed and scaled using HKL2000 and Scalepack. The structure of the apo protein was solved via molecular replacement with BALBES using the homologous structure PDB 3JYS, followed by successive rounds of automatic and manual model building with Autobuild and Coot. The structure of SGBP-A with XyGO2 was solved via molecular replacement with Phaser and refined with Phenix. X-data collection and refinement statistics are presented in Table 2. SUPPLEMENTAL MATERIAL Citation Tauzin AS, Kwiatkowski KJ, Orlovsky NI, Smith CJ, Creagh AL, Haynes CA, Wawrzak Z, Brumer H, Koropatkin NM. 2016. Molecular dissection of xyloglucan recognition in a prominent human gut symbiont. mBio 7(2):e02134-15. doi:10.1128/mBio.02134-15. REFERENCES An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system Site-specific programming of the host epithelial transcriptome by the gut microbiota Gut microbiota from twins discordant for obesity modulate metabolism in mice Mechanisms underlying the resistance to diet-induced obesity in germ-free mice Diversity of the human intestinal microbial flora Dynamics and associations of microbial community types across the human body The abundance and variety of carbohydrate-active enzymes in the human gut microbiota Evolution of symbiotic bacteria in the distal human intestine A genomic view of the human-Bacteroides thetaiotaomicron symbiosis The devil lies in the details: how variations in polysaccharide fine-structure impact the physiology and evolution of gut microbes Recognition and degradation of plant cell wall polysaccharides by two human gut symbionts Location and characterization of genes involved in binding of starch to the surface of Bacteroides thetaiotaomicron Characterization of four outer membrane proteins involved in binding starch to the cell surface of Bacteroides thetaiotaomicron Physiological characterization of SusG, an outer membrane protein essential for starch utilization by Bacteroides thetaiotaomicron SusG: A unique cell-membrane-associated alpha-amylase from a prominent human gut symbiont targets complex starch molecules Starch catabolism by a prominent human gut symbiont is directed by the recognition of amylose helices A Bacteroides thetaiotaomicron outer membrane protein that is essential for utilization of maltooligosaccharides and starch Multidomain carbohydrate-binding proteins involved in Bacteroides thetaiotaomicron starch metabolism Contribution of a neopullulanase, a pullulanase, and an alpha-glucosidase to growth of Bacteroides thetaiotaomicron on starch Bacteria of the human gut microbiome catabolize red seaweed glycans with carbohydrate-active enzyme updates from extrinsic microbes Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism Glycan complexity dictates microbial resource allocation in the large intestine A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes Plant cell walls as dietary fibre: range, structure, processing and function Structural diversity and function of xyloglucan sidechain substituents Multifunctional nutrient-binding proteins adapt human symbiotic bacteria for glycan competition in the gut by separately promoting enhanced sensing and catalysis Automatic prediction of polysaccharide utilization loci in Bacteroidetes species Complex glycan catabolism by the human gut microbiota: the Bacteroidetes Sus-like paradigm Mechanistic insight into polysaccharide use within the intestinal microbiota Expansion of the protein repertoire in newly explored environments: human gut microbiome specific protein families Glycan recognition by the Bacteroidetes Sus-like systems Xyloglucan in cellulose modification Structure of a SusD homologue, BT1043, involved in mucin O-glycan utilization in a prominent human gut symbiont Carbohydrate binding module recognition of xyloglucan defined by polar contacts with branching xyloses and CH-Pi interactions Understanding how noncatalytic carbohydrate binding modules can display specificity for xyloglucan Recognition of cellooligosaccharides by a family 28 carbohydrate-binding module Biochemical analysis of interactions between outer membrane proteins that contribute to starch utilization by Bacteroides thetaiotaomicron An ecological network of polysaccharide utilization among human intestinal symbionts Preferential packing of acidic glycosidases and proteases into Bacteroides outer membrane vesicles Learning from microbial strategies for polysaccharide degradation TonB-dependent transporters: regulation, structure, and function The N-glycan glycoprotein deglycosylation complex (Gpd) from Capnocytophaga canimorsus deglycosylates human IgG Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species Specificity of polysaccharide use in intestinal Bacteroides species determines diet-induced microbiota alterations Plant carbohydrate scavenging through tonB-dependent receptors: a feature shared by phytopathogenic and aquatic bacteria Advances in understanding the molecular basis of plant cell wall polysaccharide recognition by carbohydrate-binding modules Honor thy gut symbionts redux Prebiotics: why definitions matter Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin A novel carbohydrate-binding protein is a component of the plant cell wall-degrading complex of Piromyces equi  Mucosal glycan foraging enhances fitness and transmission of a saccharolytic human gut bacterial symbiont Processing of X-ray diffraction data collected in oscillation mode Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard PHENIX: building new software for automated crystallographic structure determination Coot: model-building tools for molecular graphics Phaser crystallographic software BALBES: a molecular-replacement pipeline Generation and structural validation of a library of diverse xyloglucan-derived oligosaccharides, including an update on xyloglucan nomenclature Crystal structures of Clostridium thermocellum xyloglucanase, XGH74A, reveal the structural basis for xyloglucan recognition and 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diff --git a/annotated_BioC_JSON/PMC4850288_ann.json b/annotated_BioC_JSON/PMC4850288_ann.json
new file mode 100644
index 0000000000000000000000000000000000000000..f1e5a845ef93b881287c393d1875c9f49dfcef09
--- /dev/null
+++ b/annotated_BioC_JSON/PMC4850288_ann.json
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+[{"sourceid":"4850288","sourcedb":"","project":"","target":"","text":"Crystal Structure and Activity Studies of the C11 Cysteine Peptidase from Parabacteroides merdae in the Human Gut Microbiome* Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. However, for the clostripain family (denoted C11), little is currently known. Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/β/α sandwich and an unusual C-terminal domain. It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other families in the clan. These studies also revealed a well ordered break in the polypeptide chain at Lys147, resulting in a large conformational rearrangement close to the active site. Biochemical and kinetic analysis revealed Lys147 to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca2+ for activity. Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms. Introduction Cysteine peptidases play crucial roles in the virulence of bacterial and other eukaryotic pathogens. In the MEROPS peptidase database, clan CD contains groups (or families) of cysteine peptidases that share some highly conserved structural elements. Clan CD families are typically described using the name of their archetypal, or founding, member and also given an identification number preceded by a “C,” to denote cysteine peptidase. Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80). No structural information is available for clostripain (C11), separase (C50), or PrtH-peptidase (C85). Clan CD enzymes have a highly conserved His/Cys catalytic dyad and exhibit strict specificity for the P1 residue of their substrates. However, despite these similarities, clan CD forms a functionally diverse group of enzymes: the overall structural diversity between (and at times within) the various families provides these peptidases with a wide variety of substrate specificities and activation mechanisms. Several members are initially expressed as proenzymes, demonstrating self-inhibition prior to full activation. The archetypal and arguably most notable family in the clan is that of the mammalian caspases (C14a), although clan CD members are distributed throughout the entire phylogenetic kingdom and are often required in fundamental biological processes. Interestingly, little is known about the structure or function of the C11 proteins, despite their widespread distribution and its archetypal member, clostripain from Clostridium histolyticum, first reported in the literature in 1938. Clostripain has been described as an arginine-specific peptidase with a requirement for Ca2+ and loss of an internal nonapeptide for full activation; lack of structural information on the family appears to have prohibited further investigation. As part of an ongoing project to characterize commensal bacteria in the microbiome that inhabit the human gut, the structure of C11 peptidase, PmC11, from Parabacteroides merdae was determined using the Joint Center for Structural Genomics (JCSG)4 HTP structural biology pipeline. The structure was analyzed, and the enzyme was biochemically characterized to provide the first structure/function correlation for a C11 peptidase. Experimental Procedures Cloning, expression, purification, crystallization, and structure determination of PmC11 were carried out using standard JCSG protocols as follows. Cloning Clones were generated using the polymerase incomplete primer extension (PIPE) cloning method. The gene encoding PmC11 (SP5111E) was amplified by polymerase chain reaction (PCR) from P. merdae genomic DNA using PfuTurbo DNA polymerase (Stratagene), using I-PIPE primers that included sequences for the predicted 5′ and 3′ ends (shown below). The expression vector, pSpeedET, which encodes an amino-terminal tobacco etch virus protease-cleavable expression and purification tag (MGSDKIHHHHHHENLYFQ/G), was PCR amplified with V-PIPE (Vector) primers. V-PIPE and I-PIPE PCR products were mixed to anneal the amplified DNA fragments together. Escherichia coli GeneHogs (Invitrogen) competent cells were transformed with the I-PIPE/V-PIPE mixture and dispensed on selective LB-agar plates. The cloning junctions were confirmed by DNA sequencing. The plasmid encoding the full-length protein was deposited in the PSI:Biology Materials Repository at the DNASU plasmid repository (PmCD00547516). For structure determination, to obtain soluble protein using the PIPE, method the gene segment encoding residues Met1-Asn22 was deleted because these residues were predicted to correspond to a signal peptide using SignalP. Protein Expression and Selenomethionine Incorporation The expression plasmid for the truncated PmC11 construct was transformed into E. coli GeneHogs competent cells and grown in minimal media supplemented with selenomethionine and 30 μg ml−1 of kanamycin at 37 °C using a GNF fermentor. A methionine auxotrophic strain was not required as selenomethionine is incorporated via the inhibition of methionine biosynthesis. Protein expression was induced using 0.1% (w/v) l-arabinose and the cells were left to grow for a further 3 h at 37 °C. At the end of the cell culture, lysozyme was added to all samples to a final concentration of 250 μg ml−1 and the cells were harvested and stored at −20 °C, until required. Primers used in this section are as follows: I-PIPE (forward): CTGTACTTCCAGGGCGAGACTCCGGAACCCCGGACAACCCGC; I-PIPE (reverse): AATTAAGTCGCGTTATTCATAAACTGCCTTATACCAGCCGAC; V-PIPE (forward): TAACGCGACTTAATTAACTCGTTTAAACGGTCTCCAGC; and V-PIPE (reverse): GCCCTGGAAGTACAGGTTTTCGTGATGATGATGATGAT. Protein Purification for Crystallization Cells were resuspended, homogenized, and lysed by sonication in 40 mm Tris (pH 8.0), 300 mm NaCl, 10 mm imidazole, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Lysis Buffer 1) containing 0.4 mm MgSO4 and 1 μl of 250 unit/μl−1 of benzonase (Sigma). The cell lysate was then clarified by centrifugation (32,500 × g for 25 min at 4 °C) before being passed over Ni2+-chelating resin equilibrated in Lysis Buffer 1 and washed in the same buffer supplemented with 40 mm imidazole and 10% (v/v) glycerol. The protein was subsequently eluted in 20 mm Tris (pH 8.0), 150 mm NaCl, 10% (v/v) glycerol, 1 mm TCEP, and 300 mm imidazole, and the fractions containing the protein were pooled. To remove the His tag, PmC11 was exchanged into 20 mm Tris (pH 8.0), 150 mm NaCl, 30 mm imidazole, and 1 mm TCEP using a PD-10 column (GE Healthcare), followed by incubation with 1 mg of His-tagged tobacco etch virus protease per 15 mg of protein for 2 h at room temperature and subsequent overnight incubation at 4 °C. The sample was centrifuged to remove any precipitated material (13,000 × g for 10 min at 4 °C) and the supernatant loaded onto Ni2+-chelating resin equilibrated with 20 mm Tris (pH 8.0), 150 mm NaCl, 30 mm imidazole, and 1 mm TCEP and washed with the same buffer. The flow-through and wash fractions were collected and concentrated to 13.3 mg ml−1 using Amicon Ultra-15 5K centrifugal concentrators (Millipore). Crystallization and Data Collection PmC11 was crystallized using the nanodroplet vapor diffusion method using standard JCSG crystallization protocols. Drops were comprised of 200 nl of protein solution mixed with 200 nl of crystallization solution in 96-well sitting-drop plates, equilibrated against a 50-μl reservoir. Crystals of PmC11 were grown at 4 °C in mother liquor consisting of 0.2 m NH4H2PO4, 20% PEG-3350 (JCSG Core Suite I). Crystals were flash cooled in liquid nitrogen using 10% ethylene glycol as a cryoprotectant prior to data collection and initial screening for diffraction was carried out using the Stanford Automated Mounting system at the Stanford Synchrotron Radiation Lightsource (SSRL, Menlo Park, CA). Single wavelength anomalous dispersion data were collected using a wavelength of 0.9793 Å, at the Advanced Light Source (ALS, beamline 8.2.2, Berkeley, CA) on an ADSC Quantum 315 CCD detector. The data were indexed and integrated with XDS and scaled using XSCALE. The diffraction data were indexed in space group P21 with a = 39.11, b = 108.68, c = 77.97 Å, and β = 94.32°. The unit cell contained two molecules in the asymmetric unit resulting in a solvent content of 39% (Matthews' coefficient (Vm) of 2.4 Å3 Da−1). Structure Determination The PmC11 structure was determined by the single wavelength anomalous dispersion method using an x-ray wavelength corresponding to the peak of the selenium K edge. Initial phases were derived using the autoSHARP interface, which included density modification with SOLOMON. Good quality electron density was obtained at 1.7-Å resolution, allowing an initial model to be obtained by automated model building with ARP/wARP. Model completion and refinement were iteratively performed with COOT and REFMAC to produce a final model with an Rcryst and Rfree of 14.3 and 17.5%, respectively. The refinement included experimental phase restraints in the form of Hendrickson-Lattman coefficients, TLS refinement with one TLS group per molecule in the asymmetric unit, and NCS restraints. The refined structure contains residues 24–375 and 28–375 for the two molecules in the crystallographic asymmetric unit. Structural validation was carried using the JCSG Quality Control Server that analyzes both the coordinates and data using a variety of structural validation tools to confirm the stereochemical quality of the model (ADIT, MOLPROBITY, and WHATIF 5.0) and agreement between model and data (SGCHECK and RESOLVE). All of the main-chain torsion angles were in the allowed regions of the Ramachandran plot and the MolProbity overall clash score for the structure was 2.09 (within the 99th percentile for its resolution). The atomic coordinates and structure factors for PmC11 have been deposited in the Protein Data Bank (PDB) with the accession code 3UWS. Data collection, model, and refinement statistics are reported in Table 1. Crystallographic statistics for PDB code 3UWS Values in parentheses are for the highest resolution shell. Data collection\t\t \t    Wavelength (Å)\t0.9793\t \t    Space group\tP21\t \t    Unit cell dimensions a, b, c (Å); β°\t39.11, 108.68, 77.97; β = 94.32°\t \t    Resolution range (Å)\t28.73–1.70 (1.79–1.70)\t \t    Unique reflections\t70,913\t \t    Rmergea on I (%)\t10.2 (49.0)\t \t    Rmeasb on I (%)\t11.0 (52.7)\t \t    Rpimc on I (%)\t4.1 (19.2)\t \t    I/σI\t15.6 (4.6)\t \t    Wilson B (Å2)\t15.9\t \t    Completeness (%)\t99.6 (99.8)\t \t    Multiplicity\t7.3 (7.5)\t \t\t \tModel and refinement\t\t \t    Reflections (total/test)\t70,883/3,577\t \t    Rcryst/Rfreed (%)\t14.3/17.5\t \t    No. protein residues/atoms\t700/5612\t \t    No. of water/EDO molecules\t690/7\t \t    ESUe based on Rfree (Å)\t0.095\t \t    B-values (Å2)\t\t \t        Average isotropic B (overall)\t20.0\t \t        Protein overall\t18.8\t \t        All main/side chains\t16.7/20.8\t \t        Solvent/EDO\t29.4/35.6\t \t    RMSDg\t\t \t        Bond lengths (Å)\t0.01\t \t        Bond angles (°)\t1.6\t \t    Ramachandran analysis (%)\t\t \t        Favored regions\t97.0\t \t        Allowed regions\t3.0\t \t        Outliers\t0.0\t \t a Rmerge = ΣhklΣi|Ii(hkl) − 〈I(hkl)〉|/Σhkl Σi(hkl). b Rmeas = Σhkl[N/(N-1)]1/2Σi|Ii(hkl) − 〈I(hkl)〉|/ΣhklΣiIi(hkl). c Rpim (precision-indicating Rmerge) = Σhkl[(1/(N-1)]1/2 Σi|Ii (hkl) − 〈I(hkl)〉|/ΣhklΣi Ii(hkl), where n is the multiplicity of reflection hkl, and Ii(hkl) and 〈I(hkl)〉 are the intensity of the ith measurement and the average intensity of reflection hkl, respectively. d Rcryst and Rfree = Σ‖Fobs| − |Fcalc‖/Σ|Fobs| for reflections in the working and test sets, respectively, where Fobs and Fcalc are the observed and calculated structure-factor amplitudes, respectively. Rfree is the same as Rcryst but for 5% of the total reflections chosen at random and omitted from structural refinement. e ESU is the estimated standard uncertainties of atoms. f The average isotropic B includes TLS and residual B components. g RMSD, root-mean-square deviation. Structural Analysis The primary sequence alignment with assigned secondary structure was prepared using CLUSTAL OMEGA and ALINE. The topology diagram was produced with TOPDRAW and all three-dimensional structural figures were prepared with PyMol with the electrostatic surface potential calculated with APBS and contoured at ±5 kT/e. Architectural comparisons with known structures revealed that PmC11 was most structurally similar to caspase-7, gingipain-K, and legumain (PBD codes 4hq0, 4tkx, and 4aw9, respectively). The statistical significance of the structural alignment between PmC11 and both caspase-7 and gingipain-K is equivalent (Z-score of 9.2) with legumain giving a very similar result (Z-score of 9.1). Of note, the β-strand topology of the CDP domains of Clostridium difficile toxin B (family C80; TcdB; PDB code 3pee) is identical to that observed in the PmC11 β-sheet, but the Z-score from DaliLite was notably less at 7.6. It is possible that the PmC11 structure is more closely related to the C80 family than other families in clan CD, and appear to reside on the same branch of the phylogenetic tree based on structure. Protein Production for Biochemical Assays The PmCD00547516 plasmid described above was obtained from the PSI:Biology Materials Repository and used to generate a cleavage site mutant PmC11K147A and an active-site mutant PmC11C179A using the QuikChange Site-directed Mutagenesis kit (Stratagene) as per the manufacturer's instructions using the following primers: K147A mutant (forward): CAGAATAAGCTGGCAGCGTTCGGACAGGACG, and K147A mutant (reverse): CGTCCTGTCCGAACGCTGCCAGCTTATTCTG; C179A mutant (forward): CCTGTTCGATGCCGCCTACATGGCAAGC, and C179A mutant (reverse): GCTTGCCATGTAGGCGGCATCGAACAGG. The expression plasmids containing PmC11 were transformed into E. coli BL21 Star (DE3) and grown in Luria-Bertani media containing 30 μg ml−1 of kanamycin at 37 °C until an optical density (600 nm) of ∼0.6 was reached. l-Arabinose was added to a final concentration of 0.2% (w/v) and the cells incubated overnight at 25 °C. Compared with the protein production for crystallography, a slightly modified purification protocol was employed for biochemical assays. Initially, the cells were resuspended in 20 mm sodium phosphate (pH 7.5), 150 mm NaCl (Lysis Buffer 2) containing an EDTA-free protease inhibitor mixture (cOmplete, Roche Applied Science). Cells were disrupted by three passages (15 KPSI) through a One-Shot cell disruptor (Constant Systems) followed by centrifugation at 20,000 × g for 20 min at 4 °C. The supernatant was collected and sterile-filtered (0.2 μm) before being applied to a 5-ml HisTrap HP column (GE Healthcare) equilibrated in Lysis Buffer 2 containing 25 mm imidazole, and the protein was eluted in the same buffer containing 250 mm imidazole. The peak fractions were pooled and buffer exchanged into the assay buffer (20 mm Tris, 150 mm NaCl, pH 8.0) using a PD-10 column. When required, purified PmC11 was concentrated using Vivaspin 2 30-K centrifugal concentrators (Sartorius). Protein concentration was routinely measured using Bradford's reagent (Bio-Rad) with a BSA standard. Fluorogenic Substrate Activity Assays The release of the fluorescent group AMC (7-amino-4-methylcoumarin) from potential peptide substrates was used to assess the activity of PmC11. Peptidase activity was tested using 20 μg of PmC11 and 100 μm substrate (unless otherwise stated) in assay buffer to a final reaction volume of 200 μl and all samples were incubated (without substrate) at 37 °C for 16 h prior to carrying out the assay. The substrate and plate reader were brought to 37 °C for 20 min prior to the addition of the PmC11 and samples prepared without PmC11 were used as blanks (negative controls). The curves were plotted using the blank-corrected fluorescence units against the time of acquisition (in min). The assays were carried out in black 96-well flat-bottomed plates (Greiner). AMC fluorescence was measured using a PHERAstar FS plate reader (BMG Labtech) with excitation and emission wavelengths of 355 and 460 nm, respectively. To investigate the substrate specificity of PmC11, substrates Z-GGR-AMC, Bz-R-AMC, Z-GP-AMC, Z-HGP-AMC, Ac-DEVD-AMC (all Bachem), BOC-VLK-AMC, and BOC-K-AMC (both PeptaNova) were prepared at 100 mm in 100% dimethyl sulfoxide. The amount of AMC (micromoles) released was calculated by generating an AMC standard curve (as described in Ref.) and the specific activity of PmC11 was calculated as picomoles of AMC released per min per mg of the protein preparation. The reaction rates (Vmax) and Km values were determined for mutants PmC11K147A and PmC11C179A by carrying out the activity assay at varying concentrations of Bz-R-AMC between 0 and 600 μm. The blank-corrected relative fluorescence units were plotted against time (min) with ΔFU/T giving the reaction rate. The Km and Vmax of PmC11 and PmC11K147A against an R-AMC substrate were determined from the Lineweaver-Burk plot as described, calculated using GraphPad Prism6 software. All experiments were carried out in triplicate. Effect of VRPR-FMK on PmC11 To test the effect of the inhibitor on the activity of PmC11, 25 μm Z-VRPR-FMK (100 mm stock in 100% dimethyl sulfoxide, Enzo Life Sciences), 20 μg of PmC11, 100 μm R-AMC substrate, 1 mm EGTA were prepared in the assay buffer and the activity assay carried out as described above. A gel-shift assay, to observe Z-VRPR-FMK binding to PmC11, was also set up using 20 μg of PmC11, 25 μm inhibitor, 1 mm EGTA in assay buffer. The reactions were incubated at 37 °C for 20 min before being stopped by the addition SDS-PAGE sample buffer. Samples were analyzed by loading the reaction mixture on a 10% NuPAGE BisTris gel using MES buffer. Effect of Cations on PmC11 The enzyme activity of PmC11 was tested in the presence of various divalent cations: Mg2+, Ca2+, Mn2+, Co2+, Fe2+, Zn2+, and Cu2+. The final concentration of the salts (MgSO4, CaCl2, MnCl2, CoCl2, FeSO4, ZnCl2, and CuSO4) was 1 mm and the control was set up without divalent ions but with addition of 1 mm EGTA. The assay was set up using 20 mg of PmC11, 1 mm salts, 100 μm R-AMC substrate, and the assay buffer, and incubated at 37 °C for 16 h. The activity assay was carried out as described above. Size Exclusion Chromatography Affinity-purified PmC11 was loaded onto a HiLoad 16/60 Superdex 200 gel filtration column (GE Healthcare) equilibrated in the assay buffer. The apparent molecular weight of PmC11 was determined from calibration curves based on protein standards of known molecular weights. Autoprocessing Profile of PmC11 Autoprocessing of PmC11 was evaluated by incubating the enzyme at 37 °C and removing samples at 1-h intervals from 0 to 16 h and placing into SDS-PAGE loading buffer to stop the processing. Samples were then analyzed on a 4–12% NuPAGE (Thermo Fisher) Novex BisTris gel run in MES buffer. Autoprocessing Cleavage Site Analysis To investigate whether processing is a result of intra- or inter-molecular cleavage, the PmC11C179A mutant was incubated with increasing concentrations of activated PmC11 (0, 0.1, 0.2, 0.5, 1, 2, and 5 μg). The final assay volume was 40 μl and the proteins were incubated at 37 °C for 16 h in the PmC11 assay buffer. To stop the reaction, NuPAGE sample buffer was added to the protein samples and 20 μl was analyzed on 10% NuPAGE Novex BisTris gel using MES buffer. These studies revealed no apparent cleavage of PmC11C179A by the active enzyme at low concentrations of PmC11 and that only limited cleavage was observed when the ratio of active enzyme (PmC11: PmC11C179A) was increased to ∼1:10 and 1:4. Results Structure of PmC11 The crystal structure of the catalytically active form of PmC11 revealed an extended caspase-like α/β/α sandwich architecture comprised of a central nine-stranded β-sheet, with an unusual C-terminal domain (CTD), starting at Lys250. A single cleavage was observed in the polypeptide chain at Lys147 (Fig. 1, A and B), where both ends of the cleavage site are fully visible and well ordered in the electron density. The central nine-stranded β-sheet (β1–β9) of PmC11 consists of six parallel and three anti-parallel β-strands with 4↑3↓2↑1↑5↑6↑7↓8↓9↑ topology (Fig. 1A) and the overall structure includes 14 α-helices with six (α1–α2 and α4–α7) closely surrounding the β-sheet in an approximately parallel orientation. Helices α1, α7, and α6 are located on one side of the β-sheet with α2, α4, and α5 on the opposite side (Fig. 1A). Helix α3 sits at the end of the loop following β5 (L5), just preceding the Lys147 cleavage site, with both L5 and α3 pointing away from the central β-sheet and toward the CTD, which starts with α8. The structure also includes two short β-hairpins (βA–βB and βD–βE) and a small β-sheet (βC–βF), which is formed from two distinct regions of the sequence (βC precedes α11, α12 and β9, whereas βF follows the βD-βE hairpin) in the middle of the CTD (Fig. 1B). Crystal structure of a C11 peptidase from P. merdae.\nA, primary sequence alignment of PmC11 (Uniprot ID A7A9N3) and clostripain (Uniprot ID P09870) from C. histolyticum with identical residues highlighted in gray shading. The secondary structure of PmC11 from the crystal structure is mapped onto its sequence with the position of the PmC11 catalytic dyad, autocatalytic cleavage site (Lys147), and S1 binding pocket Asp (Asp177) highlighted by a red star, a red downturned triangle, and a red upturned triangle, respectively. Connecting loops are colored gray, the main β-sheet is in orange, with other strands in olive, α-helices are in blue, and the nonapeptide linker of clostripain that is excised upon autocleavage is underlined in red. Sequences around the catalytic site of clostripain and PmC11 align well. B, topology diagram of PmC11 colored as in A except that additional (non-core) β-strands are in yellow. Helices found on either side of the central β-sheet are shown above and below the sheet, respectively. The position of the catalytic dyad (H, C) and the processing site (Lys147) are highlighted. Helices (1–14) and β-strands (1–9 and A-F) are numbered from the N terminus. The core caspase-fold is highlighted in a box. C, tertiary structure of PmC11. The N and C termini (N and C) of PmC11 along with the central β-sheet (1–9), helix α5, and helices α8, α11, and α13 from the C-terminal domain, are all labeled. Loops are colored gray, the main β-sheet is in orange, with other β-strands in yellow, and α-helices are in blue. The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. Of the interacting secondary structure elements, α5 is perhaps the most interesting. This helix makes a total of eight hydrogen bonds with the CTD, including one salt bridge (Arg191-Asp255) and is surrounded by the CTD on one side and the main core of the enzyme on the other, acting like a linchpin holding both components together (Fig. 1C). Structural Comparisons PmC11 is, as expected, most structurally similar to other members of clan CD with the top hits in a search of known structures being caspase-7, gingipain-K, and legumain (PBD codes 4hq0, 4tkx, and 4aw9, respectively) (Table 2). The C-terminal domain is unique to PmC11 within clan CD and structure comparisons for this domain alone does not produce any hits in the PDB (DaliLite, PDBeFold), suggesting a completely novel fold. As the archetypal and arguably most well studied member of clan CD, the caspases were used as the basis to investigate the structure/function relationships in PmC11, with caspase-7 as the representative member. Six of the central β-strands in PmC11 (β1–β2 and β5–β8) share the same topology as the six-stranded β-sheet found in caspases, with strands β3, β4, and β9 located on the outside of this core structure (Fig. 1B, box). His133 and Cys179 were found at locations structurally homologous to the caspase catalytic dyad, and other clan CD structures, at the C termini of strands β5 and β6, respectively (Figs. 1, A and B, and 2A). A multiple sequence alignment of C11 proteins revealed that these residues are highly conserved (data not shown). Summary of PDBeFOLD superposition of structures found to be most similar to PmC11 in the PBD based on DaliLite  The results are ordered in terms of structural homology (QH), where %SSEPC-X is the percentage of the SSEs in the PmC11 that can be identified in the target X (where X = caspase-7, legumain, gingipain, and TcdB-CPD; % SSEX-PC is the percentage of SSEs in X (as above) that can be identified in PmC11 (as above); % sequence ID is the percentage sequence identity after structural alignment; Nalign is the number of matched residues; and r.m.s. deviation the root mean squared deviation on the Cα positions of the matched residues. Enzyme\tFamily\tPDB code\tQH\tZ-score\t%SSEPC-X\t%SSEX-PC\t% Seq. ID\tNalign\tRMSD (Å)\tNStrands\t \tPmC11\tC11\t3UWS\t1.00\t33.4\t100\t100\t100\t352\t0.00\t9\t \tCaspase-7\tC14A\t4HQ0\t0.16\t4.3\t38\t79\t14\t162\t3.27\t6\t \tLegumain\tC13\t4AW9\t0.13\t5.5\t31\t53\t13\t161\t2.05\t6\t \tTcdB-CPD\tC80\t3PEE\t0.10\t4.9\t28\t50\t12\t138\t3.18\t9\t \tGingipain\tC25\t4TKX\t0.07\t5.4\t28\t27\t12\t153\t2.97\t10\t \t Biochemical and structural characterization of PmC11.\nA, ribbon representation of the overall structure of PmC11 illustrating the catalytic site, cleavage site displacement, and potential S1 binding site. The overall structure of PmC11 is shown in gray, looking down into the catalytic site with the catalytic dyad in red. The two ends of the autolytic cleavage site (Lys147 and Ala148, green) are displaced by 19.5 Å (thin black line) from one another and residues in the potential substrate binding pocket are highlighted in blue. B, size exclusion chromatography of PmC11. PmC11 migrates as a monomer with a molecular mass around 41 kDa calculated from protein standards of known molecular weights. Elution fractions across the major peak (1–6) were analyzed by SDS-PAGE on a 4–12% gel in MES buffer. C, the active form of PmC11 and two mutants, PmC11C179A (C) and PmC11K147A (K), were examined by SDS-PAGE (lane 1) and Western blot analysis using an anti-His antibody (lane 2) show that PmC11 autoprocesses, whereas mutants, PmC11C179A and PmC11K147A, do not show autoprocessing in vitro. D, cysteine peptidase activity of PmC11. Km and Vmax of PmC11 and K147A mutant were determined by monitoring change in the fluorescence corresponding to AMC release from Bz-R-AMC. Reactions were performed in triplicate and representative values ± S.D. are shown. E, intermolecular processing of PmC11C179A by PmC11. PmC11C179A (20 μg) was incubated overnight at 37 °C with increasing amounts of processed PmC11 and analyzed on a 10% SDS-PAGE gel. Inactive PmC11C179A was not processed to a major extent by active PmC11 until around a ratio of 1:4 (5 μg of active PmC11). A single lane of 20 μg of active PmC11 (labeled 20) is shown for comparison. F, activity of PmC11 against basic substrates. Specific activity is shown ± S.D. from three independent experiments. G, electrostatic surface potential of PmC11 shown in a similar orientation, where blue and red denote positively and negatively charged surface potential, respectively, contoured at ±5 kT/e. The position of the catalytic dyad, one potential key substrate binding residue Asp177, and the ends of the cleavage site Lys147 and Ala148 are indicated. Five of the α-helices surrounding the β-sheet of PmC11 (α1, α2, α4, α6, and α7) are found in similar positions to the five structurally conserved helices in caspases and other members of clan CD, apart from family C80. Other than its more extended β-sheet, PmC11 differs most significantly from other clan CD members at its C terminus, where the CTD contains a further seven α-helices and four β-strands after β8. Autoprocessing of PmC11 Purification of recombinant PmC11 (molecular mass = 42.6 kDa) revealed partial processing into two cleavage products of 26.4 and 16.2 kDa, related to the observed cleavage at Lys147 in the crystal structure (Fig. 2A). Incubation of PmC11 at 37 °C for 16 h, resulted in a fully processed enzyme that remained as an intact monomer when applied to a size-exclusion column (Fig. 2B). The single cleavage site of PmC11 at Lys147 is found immediately after α3, in loop L5 within the central β-sheet (Figs. 1, A and B, and 2A). The two ends of the cleavage site are remarkably well ordered in the crystal structure and displaced from one another by 19.5 Å (Fig. 2A). Moreover, the C-terminal side of the cleavage site resides near the catalytic dyad with Ala148 being 4.5 and 5.7 Å from His133 and Cys179, respectively. Consequently, it appears feasible that the helix attached to Lys147 (α3) could be responsible for steric autoinhibition of PmC11 when Lys147 is covalently bonded to Ala148. Thus, the cleavage would be required for full activation of PmC11. To investigate this possibility, two mutant forms of the enzyme were created: PmC11C179A (a catalytically inactive mutant) and PmC11K147A (a cleavage-site mutant). Initial SDS-PAGE and Western blot analysis of both mutants revealed no discernible processing occurred as compared with active PmC11 (Fig. 2C). The PmC11K147A mutant enzyme had a markedly different reaction rate (Vmax) compared with WT, where the reaction velocity of PmC11 was 10 times greater than that of PmC11K147A (Fig. 2D). Taken together, these data reveal that PmC11 requires processing at Lys147 for optimum activity. To investigate whether processing is a result of intra- or intermolecular cleavage, the PmC11C179A mutant was incubated with increasing concentrations of processed and activated PmC11. These studies revealed that there was no apparent cleavage of PmC11C179A by the active enzyme at low concentrations of PmC11 and that only limited cleavage was observed when the ratio of active enzyme (PmC11:PmC11C179A) was increased to ∼1:10 and 1:4, with complete cleavage observed at a ratio of 1:1 (Fig. 2E). This suggests that cleavage of PmC11C179A was most likely an effect of the increasing concentration of PmC11 and intermolecular cleavage. Collectively, these data suggest that the pro-form of PmC11 is autoinhibited by a section of L5 blocking access to the active site, prior to intramolecular cleavage at Lys147. This cleavage subsequently allows movement of the region containing Lys147 and the active site to open up for substrate access. Substrate Specificity of PmC11 The autocatalytic cleavage of PmC11 at Lys147 (sequence KLK∧A) demonstrates that the enzyme accepts substrates with Lys in the P1 position. The substrate specificity of the enzyme was further tested using a variety of fluorogenic substrates. As expected, PmC11 showed no activity against substrates with Pro or Asp in P1 but was active toward substrates with a basic residue in P1 such as Bz-R-AMC, Z-GGR-AMC, and BOC-VLK-AMC. The rate of cleavage was ∼3-fold greater toward the single Arg substrate Bz-R-AMC than for the other two (Fig. 2F) and, unexpectedly, PmC11 showed no activity toward BOC-K-AMC. These results confirm that PmC11 accepts substrates containing Arg or Lys in P1 with a possible preference for Arg. The catalytic dyad of PmC11 sits near the bottom of an open pocket on the surface of the enzyme at a conserved location in the clan CD family. The PmC11 structure reveals that the catalytic dyad forms part of a large acidic pocket (Fig. 2G), consistent with a binding site for a basic substrate. This pocket is lined with the potential functional side chains of Asn50, Asp177, and Thr204 with Gly134, Asp207, and Met205 also contributing to the pocket (Fig. 2A). Interestingly, these residues are in regions that are structurally similar to those involved in the S1 binding pockets of other clan CD members (shown in Ref.). Because PmC11 recognizes basic substrates, the tetrapeptide inhibitor Z-VRPR-FMK was tested as an enzyme inhibitor and was found to inhibit both the autoprocessing and activity of PmC11 (Fig. 3A). Z-VRPR-FMK was also shown to bind to the enzyme: a size-shift was observed, by SDS-PAGE analysis, in the larger processed product of PmC11 suggesting that the inhibitor bound to the active site (Fig. 3B). A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). Asp177 is located near the catalytic cysteine and is conserved throughout the C11 family, suggesting it is the primary S1 binding site residue. In the structure of PmC11, Asp207 resides on a flexible loop pointing away from the S1 binding pocket (Fig. 3C). However, this loop has been shown to be important for substrate binding in clan CD and this residue could easily rotate and be involved in substrate binding in PmC11. Thus, Asn50, Asp177, and Asp207 are most likely responsible for the substrate specificity of PmC11. Asp177 is highly conserved throughout the clan CD C11 peptidases and is thought to be primarily responsible for substrate specificity of the clan CD enzymes, as also illustrated from the proximity of these residues relative to the inhibitor Z-VRPR-FMK when PmC11 is overlaid on the MALT1-P structure (Fig. 3C). PmC11 binds and is inhibited by Z-VRPR-FMK and does not require Ca2+ for activity.\nA, PmC11 activity is inhibited by Z-VRPR-FMK. Cleavage of Bz-R-AMC by PmC11 was measured in a fluorometric activity assay with (+, purple) and without (−, red) Z-VRPR-FMK. The relative fluorescence units of AMC released are plotted against time (min) (n = 3; ±S.D.). B, gel-shift assay reveals that Z-VRPR-FMK binds to PmC11. PmC11 was incubated with (+) or without (−) Z-VRPR-FMK and the samples analyzed on a 10% SDS-PAGE gel. A size shift can be observed in the larger processed product of PmC11 (26.1 kDa). C, PmC11 with the Z-VRPR-FMK from the MALT1-paracacaspase (MALT1-P) superimposed. A three-dimensional structural overlay of Z-VRPR-FMK from the MALT1-P complex onto PmC11. The position and orientation of Z-VRPR-FMK was taken from superposition of the PmC11 and MALTI_P structures and indicates the presumed active site of PmC11. Residues surrounding the inhibitor are labeled and represent potentially important binding site residues, labeled in black and shown in an atomic representation. Carbon atoms are shown in gray, nitrogen in blue, and oxygen in red. C, divalent cations do not increase the activity of PmC11. The cleavage of Bz-R-AMC by PmC11 was measured in the presence of the cations Ca2+, Mn2+, Zn2+, Co2+, Cu2+, Mg2+, and Fe3+ with EGTA as a negative control, and relative fluorescence measured against time (min). The addition of cations produced no improvement in activity of PmC11 when compared in the presence of EGTA, suggesting that PmC11 does not require metal ions for proteolytic activity. Furthermore, Cu2+, Fe2+, and Zn2+ appear to inhibit PmC11. Comparison with Clostripain Clostripain from C. histolyticum is the founding member of the C11 family of peptidases and contains an additional 149 residues compared with PmC11. A multiple sequence alignment revealed that most of the secondary structural elements are conserved between the two enzymes, although they are only ∼23% identical (Fig. 1A). Nevertheless, PmC11 may be a good model for the core structure of clostripain. The primary structural alignment also shows that the catalytic dyad in PmC11 is structurally conserved in clostripain (Fig. 1A). Unlike PmC11, clostripain has two cleavage sites (Arg181 and Arg190), which results in the removal of a nonapeptide, and is required for full activation of the enzyme (highlighted in Fig. 1A). Interestingly, Arg190 was found to align with Lys147 in PmC11. In addition, the predicted primary S1-binding residue in PmC11 Asp177 also overlays with the residue predicted to be the P1 specificity determining residue in clostripain (Asp229, Fig. 1A). As studies on clostripain revealed addition of Ca2+ ions are required for full activation, the Ca2+ dependence of PmC11 was examined. Surprisingly, Ca2+ did not enhance PmC11 activity and, furthermore, other divalent cations, Mg2+, Mn2+, Co2+, Fe2+, Zn2+, and Cu2+, were not necessary for PmC11 activity (Fig. 3D). In support of these findings, EGTA did not inhibit PmC11 suggesting that, unlike clostripain, PmC11 does not require Ca2+ or other divalent cations, for activity. Discussion The crystal structure of PmC11 now provides three-dimensional information for a member of the clostripain C11 family of cysteine peptidases. The enzyme exhibits all of the key structural elements of clan CD members, but is unusual in that it has a nine-stranded central β-sheet with a novel C-terminal domain. The structural similarity of PmC11 with its nearest structural neighbors in the PDB is decidedly low, overlaying better with six-stranded caspase-7 than any of the other larger members of the clan (Table 2). The substrate specificity of PmC11 is Arg/Lys and the crystal structure revealed an acidic pocket for specific binding of such basic substrates. In addition, the structure suggested a mechanism of self-inhibition in both PmC11 and clostripain and an activation mechanism that requires autoprocessing. PmC11 differs from clostripain in that is does not appear to require divalent cations for activation. Several other members of clan CD require processing for full activation including legumain, gingipain-R, MARTX-CPD, and the effector caspases, e.g. caspase-7. To date, the effector caspases are the only group of enzymes that require cleavage of a loop within the central β-sheet. This is also the case in PmC11, although the cleavage loop is structurally different to that found in the caspases and follows the catalytic His (Fig. 1A), as opposed to the Cys in the caspases. All other clan CD members requiring cleavage for full activation do so at sites external to their central sheets. The caspases and gingipain-R both undergo intermolecular (trans) cleavage and legumain and MARTX-CPD are reported to perform intramolecular (cis) cleavage. In addition, several members of clan CD exhibit self-inhibition, whereby regions of the enzyme block access to the active site. Like PmC11, these structures show preformed catalytic machinery and, for a substrate to gain access, movement and/or cleavage of the blocking region is required. The structure of PmC11 gives the first insight into this class of relatively unexplored family of proteins and should allow important catalytic and substrate binding residues to be identified in a variety of orthologues. Indeed, insights gained from an analysis of the PmC11 structure revealed the identity of the Trypanosoma brucei PNT1 protein as a C11 cysteine peptidase with an essential role in organelle replication. The PmC11 structure should provide a good basis for structural modeling and, given the importance of other clan CD enzymes, this work should also advance the exploration of these peptidases and potentially identify new biologically important substrates. Author Contributions K. M., J. S. G., D. D., I. A. W., and J. C. M. designed the research; K. M., J. S. G., and D. D. performed the research; K. M., J. S. G., D. D., G. H. C., A. S., M. A. E., and J. C. M. analyzed the data; A. G., S. A. L., A. M. D., M. A. E., and I. A. W. supervised various components of the JCSG structural genomics pipeline; M. K. G., A. G., S. A. L., A. M. D., and M. A. E. contributed reagents, materials, and analysis tools; and K. M., J. S. G., G. H. C., M. A. E., I. A. W., and J. C. M. wrote the paper. This work was supported by the Medical Research Council Grant MR/K019384, Wellcome Trust Grants 091790 and 104111, and National Institutes of Health Grant U54 GM094586 (JCSG). The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and National Institutes of Health (NIH), National Center for Research Resources, Biomedical Technology Program Grant P41RR001209, and the NIMGS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIGMS or NIH. The authors declare that they have no conflicts of interest with the contents of this article. The atomic coordinates and structure factors (code 3UWS) have been deposited in the Protein Data Bank (http://wwpdb.org/). JCSG Joint Center for Structural Genomics PIPE polymerase incomplete primer extension TCEP Tris(2-carboxyethyl)phosphine AMC 7-amino-4-methylcoumarin PDB Protein Data Bank BisTris 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol Z benzyloxycarbonyl FMK fluoromethyl ketone CTD C-terminal domain Bz-R-AMC benzoyl-l-Arg-4-methylcoumaryl-7-amide Z-GGR-AMC benzyloxycarbonyl-Gly-Gly-Arg-AMC BOC-VLK-AMC t-butyloxycarbonyl-Val-Leu-Lys. The abbreviations used are:  References MEROPS: the database of proteolytic enzymes, their substrates and inhibitors Comparative structural analysis of the caspase family with other clan CD cysteine peptidases Mechanistic and structural studies on legumain explain its zymogenicity, distinct activation pathways, and regulation Crystal structure of the cysteine protease interleukin-1β-converting enzyme: a (p20/p10)2 homodimer Crystal structure of the mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) paracaspase region Crystal structure of a Trypanosoma brucei metacaspase Crystal structure of gingipain R: an Arg-specific bacterial cysteine proteinase with a caspase-like fold Small molecule-induced allosteric activation of the Vibrio cholerae RTX cysteine protease domain Proteinase secretion and growth of Clostridium histolyticum Clostripain: characterization of the active site The JCSG high-throughput structural biology pipeline The Polymerase Incomplete Primer Extension (PIPE) method applied to high-throughput cloning and site-directed mutagenesis SignalP 4.0: discriminating signal peptides from transmembrane regions Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline Production of selenomethionyl proteins in prokaryotic and eukaryotic expression systems Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin An automated system to mount cryo-cooled protein crystals on a synchrotron beam line, using compact sample cassettes and a small-scale robot XDS Automated structure solution with autoSHARP Methods used in the structure determination of bovine mitochondrial F1 ATPase Automated macromolecular model building for x-ray crystallography using ARP/wARP version 7 Features and development of Coot Overview of the CCP4 suite and current developments REFMAC5 for the refinement of macromolecular crystal structures Automated and accurate deposition of structures solved by X-ray diffraction to the Protein Data Bank MolProbity: all-atom structure validation for macromolecular crystallography WHAT IF: a molecular modeling and drug design program SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model SOLVE and RESOLVE: automated structure solution, density modification and model building Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega ALINE: a WYSIWYG protein-sequence alignment editor for publication-quality alignments TopDraw: a sketchpad for protein structure topology cartoons  Purification, characterization, and crystallization of Trypanosoma metacaspases Structural determinants of MALT1 protease activity The specificity of clostripain from Clostridium histolyticum: mapping the S′ subsites via acyl transfer to amino acid amides and peptides Clostripain linker deletion variants yield active enzyme in Escherichia coli: a possible function of the linker peptide as intramolecular inhibitor of clostripain automaturation The structure-function relationship in the clostripain family of peptidases Multistep autoactivation of asparaginyl endopeptidase in vitro and in vivo Sequential autolytic processing activates the zymogen of Arg-gingipain Human caspase-7 activity and regulation by its N-terminal peptide PNT1 is a C11 cysteine peptidase essential for replication of the Trypanosome kinetoplast Two non-proline cis peptide bonds may be important for factor XIII function Improved R-factors for diffraction data analysis in macromolecular crystallography Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions DaliLite workbench for protein structure comparison Structural asymmetry of procaspase-7 bound to a specific inhibitor Structure of the lysine specific protease Kgp from Porphyromonas gingivalis, a target for improved oral health 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diff --git a/annotated_BioC_JSON/PMC4852598_ann.json b/annotated_BioC_JSON/PMC4852598_ann.json
new file mode 100644
index 0000000000000000000000000000000000000000..06bed7b267df3a431f4aadc00b06f2a039720cd5
--- /dev/null
+++ b/annotated_BioC_JSON/PMC4852598_ann.json
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+[{"sourceid":"4852598","sourcedb":"","project":"","target":"","text":"Structural basis for Mep2 ammonium transceptor activation by phosphorylation Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation.  Mep2 proteins are tightly regulated fungal ammonium transporters. Here, the authors report the crystal structures of closed states of Mep2 proteins and propose a model for their regulation by comparing them with the open ammonium transporters of bacteria. Transceptors are membrane proteins that function not only as transporters but also as receptors/sensors during nutrient sensing to activate downstream signalling pathways. A common feature of transceptors is that they are induced when cells are starved for their substrate. While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). One of the most important unresolved questions in the field is how the transceptors couple to downstream signalling pathways. One hypothesis is that downstream signalling is dependent on a specific conformation of the transporter. Mep2 (methylammonium (MA) permease) proteins are ammonium transceptors that are ubiquitous in fungi. They belong to the Amt/Mep/Rh family of transporters that are present in all kingdoms of life and they take up ammonium from the extracellular environment. Fungi typically have more than one Mep paralogue, for example, Mep1-3 in S. cerevisiae. Of these, only Mep2 proteins function as ammonium receptors/sensors in fungal development. Under conditions of nitrogen limitation, Mep2 initiates a signalling cascade that results in a switch from the yeast form to filamentous (pseudohyphal) growth that may be required for fungal pathogenicity. As is the case for other transceptors, it is not clear how Mep2 interacts with downstream signalling partners, but the protein kinase A and mitogen-activated protein kinase pathways have been proposed as downstream effectors of Mep2 (refs). Compared with Mep1 and Mep3, Mep2 is highly expressed and functions as a low-capacity, high-affinity transporter in the uptake of MA. In addition, Mep2 is also important for uptake of ammonium produced by growth on other nitrogen sources. With the exception of the human RhCG structure, no structural information is available for eukaryotic ammonium transporters. By contrast, several bacterial Amt orthologues have been characterized in detail via high-resolution crystal structures and a number of molecular dynamics (MD) studies. All the solved structures including that of RhCG are very similar, establishing the basic architecture of ammonium transporters. The proteins form stable trimers, with each monomer having 11 transmembrane (TM) helices and a central channel for the transport of ammonium. All structures show the transporters in open conformations. Intriguingly, fundamental questions such as the nature of the transported substrate and the transport mechanism are still controversial. Where earlier studies favoured the transport of ammonia gas, recent data and theoretical considerations suggest that Amt/Mep proteins are instead active, electrogenic transporters of either NH4+ (uniport) or NH3/H+ (symport). A highly conserved pair of channel-lining histidine residues dubbed the twin-His motif may serve as a proton relay system while NH3 moves through the channel during NH3/H+ symport. Ammonium transport is tightly regulated. In animals, this is due to toxicity of elevated intracellular ammonium levels, whereas for microorganisms ammonium is a preferred nitrogen source. In bacteria, amt genes are present in an operon with glnK, encoding a PII-like signal transduction class protein. By binding tightly to Amt proteins without inducing a conformational change in the transporter, GlnK sterically blocks ammonium conductance when nitrogen levels are sufficient. Under conditions of nitrogen limitation, GlnK becomes uridylated, blocking its ability to bind and inhibit Amt proteins. Importantly, eukaryotes do not have GlnK orthologues and have a different mechanism for regulation of ammonium transport activity. In plants, transporter phosphorylation and dephosphorylation are known to regulate activity. In S. cerevisiae, phosphorylation of Ser457 within the C-terminal region (CTR) in the cytoplasm was recently proposed to cause Mep2 opening, possibly via inducing a conformational change. To elucidate the mechanism of Mep2 transport regulation, we present here X-ray crystal structures of the Mep2 transceptors from S. cerevisiae and C. albicans. The structures are similar to each other but show considerable differences to all other ammonium transporter structures. The most striking difference is the fact that the Mep2 proteins have closed conformations. The putative phosphorylation site is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The channels of phosphorylation-mimicking mutants of C. albicans Mep2 are still closed but show large conformational changes within a conserved part of the CTR. Together with a structure of a C-terminal Mep2 variant lacking the segment containing the phosphorylation site, the results allow us to propose a structural model for phosphorylation-based regulation of eukaryotic ammonium transport. Results General architecture of Mep2 ammonium transceptors The Mep2 protein of S. cerevisiae (ScMep2) was overexpressed in S. cerevisiae in high yields, enabling structure determination by X-ray crystallography using data to 3.2 Å resolution by molecular replacement (MR) with the archaebacterial Amt-1 structure (see Methods section). Given that the modest resolution of the structure and the limited detergent stability of ScMep2 would likely complicate structure–function studies, several other fungal Mep2 orthologues were subsequently overexpressed and screened for diffraction-quality crystals. Of these, Mep2 from C. albicans (CaMep2) showed superior stability in relatively harsh detergents such as nonyl-glucoside, allowing structure determination in two different crystal forms to high resolution (up to 1.5 Å). Despite different crystal packing (Supplementary Table 1), the two CaMep2 structures are identical to each other and very similar to ScMep2 (Cα r.m.s.d. (root mean square deviation)=0.7 Å for 434 residues), with the main differences confined to the N terminus and the CTR (Fig. 1). Electron density is visible for the entire polypeptide chains, with the exception of the C-terminal 43 (ScMep2) and 25 residues (CaMep2), which are poorly conserved and presumably disordered. Both Mep2 proteins show the archetypal trimeric assemblies in which each monomer consists of 11 TM helices surrounding a central pore. Important functional features such as the extracellular ammonium binding site, the Phe gate and the twin-His motif within the hydrophobic channel are all very similar to those present in the bacterial transporters and RhCG. In the remainder of the manuscript, we will specifically discuss CaMep2 due to the superior resolution of the structure. Unless specifically stated, the drawn conclusions also apply to ScMep2. While the overall architecture of Mep2 is similar to that of the prokaryotic transporters (Cα r.m.s.d. with Amt-1=1.4 Å for 361 residues), there are large differences within the N terminus, intracellular loops (ICLs) ICL1 and ICL3, and the CTR. The N termini of the Mep2 proteins are ∼20–25 residues longer compared with their bacterial counterparts (Figs 1 and 2), substantially increasing the size of the extracellular domain. Moreover, the N terminus of one monomer interacts with the extended extracellular loop ECL5 of a neighbouring monomer. Together with additional, smaller differences in other extracellular loops, these changes generate a distinct vestibule leading to the ammonium binding site that is much more pronounced than in the bacterial proteins. The N-terminal vestibule and the resulting inter-monomer interactions likely increase the stability of the Mep2 trimer, in support of data for plant AMT proteins. However, given that an N-terminal deletion mutant (2-27Δ) grows as well as wild-type (WT) Mep2 on minimal ammonium medium (Fig. 3 and Supplementary Fig. 1), the importance of the N terminus for Mep2 activity is not clear. Mep2 channels are closed by a two-tier channel block The largest differences between the Mep2 structures and the other known ammonium transporter structures are located on the intracellular side of the membrane. In the vicinity of the Mep2 channel exit, the cytoplasmic end of TM2 has unwound, generating a longer ICL1 even though there are no insertions in this region compared to the bacterial proteins (Figs 2 and 4). ICL1 has also moved inwards relative to its position in the bacterial Amts. The largest backbone movements of equivalent residues within ICL1 are ∼10 Å, markedly affecting the conserved basic RxK motif (Fig. 4). The head group of Arg54 has moved ∼11 Å relative to that in Amt-1, whereas the shift of the head group of the variable Lys55 residue is almost 20 Å. The side chain of Lys56 in the basic motif points in an opposite direction in the Mep2 structures compared with that of, for example, Amt-1 (Fig. 4). In addition to changing the RxK motif, the movement of ICL1 has another, crucial functional consequence. At the C-terminal end of TM1, the side-chain hydroxyl group of the relatively conserved Tyr49 (Tyr53 in ScMep2) makes a strong hydrogen bond with the ɛ2 nitrogen atom of the absolutely conserved His342 of the twin-His motif (His348 in ScMep2), closing the channel (Figs 4 and 5). In bacterial Amt proteins, this Tyr side chain is rotated ∼4 Å away as a result of the different conformation of TM1, leaving the channel open and the histidine available for its putative role in substrate transport (Supplementary Fig. 2). Compared with ICL1, the backbone conformational changes observed for the neighbouring ICL2 are smaller, but large shifts are nevertheless observed for the conserved residues Glu140 and Arg141 (Fig. 4). Finally, the important ICL3 linking the pseudo-symmetrical halves (TM1-5 and TM6-10) of the transporter is also shifted up to ∼10 Å and forms an additional barrier that closes the channel on the cytoplasmic side (Fig. 5). This two-tier channel block likely ensures that very little ammonium transport will take place under nitrogen-sufficient conditions. The closed state of the channel might also explain why no density, which could correspond to ammonium (or water), is observed in the hydrophobic part of the Mep2 channel close to the twin-His motif. Significantly, this is also true for ScMep2, which was crystallized in the presence of 0.2 M ammonium ions (see Methods section). The final region in Mep2 that shows large differences compared with the bacterial transporters is the CTR. In Mep2, the CTR has moved away and makes relatively few contacts with the main body of the transporter, generating a more elongated protein (Figs 1 and 4). By contrast, in the structures of bacterial proteins, the CTR is docked tightly onto the N-terminal half of the transporters (corresponding to TM1-5), resulting in a more compact structure. This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2). These residues include those of the ‘ExxGxD' motif, which when mutated generate inactive transporters. In Amt-1 and other bacterial ammonium transporters, these CTR residues interact with residues within the N-terminal half of the protein. On one side, the Tyr390 hydroxyl in Amt-1 is hydrogen bonded with the side chain of the conserved His185 at the C-terminal end of loop ICL3. At the other end of ICL3, the backbone carbonyl groups of Gly172 and Lys173 are hydrogen bonded to the side chain of Arg370. Similar interactions were also modelled in the active, non-phosphorylated plant AtAmt-1;1 structure (for example, Y467-H239 and D458-K71). The result of these interactions is that the CTR ‘hugs' the N-terminal half of the transporters (Fig. 4). Also noteworthy is Asp381, the side chain of which interacts strongly with the positive dipole on the N-terminal end of TM2. This interaction in the centre of the protein may be particularly important to stabilize the open conformations of ammonium transporters. In the Mep2 structures, none of the interactions mentioned above are present. Phosphorylation target site is at the periphery of Mep2 Recently Boeckstaens et al. provided evidence that Ser457 in ScMep2 (corresponding to Ser453 in CaMep2) is phosphorylated by the TORC1 effector kinase Npr1 under nitrogen-limiting conditions. In the absence of Npr1, plasmid-encoded WT Mep2 in a S. cerevisiae mep1-3Δ strain (triple mepΔ) does not allow growth on low concentrations of ammonium, suggesting that the transporter is inactive (Fig. 3 and Supplementary Fig. 1). Conversely, the phosphorylation-mimicking S457D variant is active both in the triple mepΔ background and in a triple mepΔ npr1Δ strain (Fig. 3). Mutation of other potential phosphorylation sites in the CTR did not support growth in the npr1Δ background. Collectively, these data suggest that phosphorylation of Ser457 opens the Mep2 channel to allow ammonium uptake. Ser457 is located in a part of the CTR that is conserved in a subgroup of Mep2 proteins, but which is not present in bacterial proteins (Fig. 2). This segment (residues 450–457 in ScMep2 and 446–453 in CaMep2) was dubbed an autoinhibitory (AI) region based on the fact that its removal generates an active transporter in the absence of Npr1 (Fig. 3). Where is the AI region and the Npr1 phosphorylation site located? Our structures reveal that surprisingly, the AI region is folded back onto the CTR and is not located near the centre of the trimer as expected from the bacterial structures (Fig. 4). The AI region packs against the cytoplasmic ends of TM2 and TM4, physically linking the main body of the transporter with the CTR via main chain interactions and side-chain interactions of Val447, Asp449, Pro450 and Arg452 (Fig. 6). The AI regions have very similar conformations in CaMep2 and ScMep2, despite considerable differences in the rest of the CTR (Fig. 6). Strikingly, the Npr1 target serine residue is located at the periphery of the trimer, far away (∼30 Å) from any channel exit (Fig. 6). Despite its location at the periphery of the trimer, the electron density for the serine is well defined in both Mep2 structures and corresponds to the non-phosphorylated state (Fig. 6). This makes sense since the proteins were expressed in rich medium and confirms the recent suggestion by Boeckstaens et al. that the non-phosphorylated form of Mep2 corresponds to the inactive state. For ScMep2, Ser457 is the most C-terminal residue for which electron density is visible, indicating that the region beyond Ser457 is disordered. In CaMep2, the visible part of the sequence extends for two residues beyond Ser453 (Fig. 6). The peripheral location and disorder of the CTR beyond the kinase target site should facilitate the phosphorylation by Npr1. The disordered part of the CTR is not conserved in ammonium transporters (Fig. 2), suggesting that it is not important for transport. Interestingly, a ScMep2 457Δ truncation mutant in which a His-tag directly follows Ser457 is highly expressed but has low activity (Fig. 3 and Supplementary Fig. 1b), suggesting that the His-tag interferes with phosphorylation by Npr1. The same mutant lacking the His-tag has WT properties (Supplementary Fig. 1b), confirming that the region following the phosphorylation site is dispensable for function. Mep2 lacking the AI region is conformationally heterogeneous Given that Ser457/453 is far from any channel exit (Fig. 6), the crucial question is how phosphorylation opens the Mep2 channel to generate an active transporter. Boeckstaens et al. proposed that phosphorylation does not affect channel activity directly, but instead relieves inhibition by the AI region. The data behind this hypothesis is the observation that a ScMep2 449-485Δ deletion mutant lacking the AI region is highly active in MA uptake both in the triple mepΔ and triple mepΔ npr1Δ backgrounds, implying that this Mep2 variant has a constitutively open channel. We obtained a similar result for ammonium uptake by the 446Δ mutant (Fig. 3), supporting the data from Marini et al. We then constructed and purified the analogous CaMep2 442Δ truncation mutant and determined the crystal structure using data to 3.4 Å resolution. The structure shows that removal of the AI region markedly increases the dynamics of the cytoplasmic parts of the transporter. This is not unexpected given the fact that the AI region bridges the CTR and the main body of Mep2 (Fig. 6). Density for ICL3 and the CTR beyond residue Arg415 is missing in the 442Δ mutant, and the density for the other ICLs including ICL1 is generally poor with visible parts of the structure having high B-factors (Fig. 7). Interestingly, however, the Tyr49-His342 hydrogen bond that closes the channel in the WT protein is still present (Fig. 7 and Supplementary Fig. 2). Why then does this mutant appear to be constitutively active? We propose two possibilities. The first one is that the open state is disfavoured by crystallization because of lower stability or due to crystal packing constraints. The second possibility is that the Tyr–His hydrogen bond has to be disrupted by the incoming substrate to open the channel. The latter model would fit well with the NH3/H+ symport model in which the proton is relayed by the twin-His motif. The importance of the Tyr–His hydrogen bond is underscored by the fact that its removal in the ScMep2 Y53A mutant results in a constitutively active transporter (Fig. 3). Phosphorylation causes a conformational change in the CTR Do the Mep2 structures provide any clues regarding the potential effect of phosphorylation? The side-chain hydroxyl of Ser457/453 is located in a well-defined electronegative pocket that is solvent accessible (Fig. 6). The closest atoms to the serine hydroxyl group are the backbone carbonyl atoms of Asp419, Glu420 and Glu421, which are 3–4 Å away. We therefore predict that phosphorylation of Ser453 will result in steric clashes as well as electrostatic repulsion, which in turn might cause substantial conformational changes within the CTR. To test this hypothesis, we determined the structure of the phosphorylation-mimicking R452D/S453D protein (hereafter termed ‘DD mutant'), using data to a resolution of 2.4 Å. The additional mutation of the arginine preceding the phosphorylation site was introduced (i) to increase the negative charge density and make it more comparable to a phosphate at neutral pH, and (ii) to further destabilize the interactions of the AI region with the main body of the transporter (Fig. 6). The ammonium uptake activity of the S. cerevisiae version of the DD mutant is the same as that of WT Mep2 and the S453D mutant, indicating that the mutations do not affect transporter functionality in the triple mepΔ background (Fig. 3). Unexpectedly, the AI segment containing the mutated residues has only undergone a slight shift compared with the WT protein (Fig. 8 and Supplementary Fig. 3). By contrast, the conserved part of the CTR has undergone a large conformational change involving formation of a 12-residue-long α-helix from Leu427 to Asp438. In addition, residues Glu420-Leu423 including Glu421 of the ExxGxD motif are now disordered (Fig. 8 and Supplementary Fig. 3). Overall, ∼20 residues are affected by the introduced mutations. This is the first time a large conformational change has been observed in an ammonium transporter as a result of a mutation, and confirms previous hypotheses that phosphorylation causes structural changes in the CTR. To exclude the possibility that the additional R452D mutation is responsible for the observed changes, we also determined the structure of the ‘single D' S453D mutant. As shown in Supplementary Fig. 4, the consequence of the single D mutation is very similar to that of the DD substitution, with conformational changes and increased dynamics confined to the conserved part of the CTR (Supplementary Fig. 4). To supplement the crystal structures, we also performed modelling and MD studies of WT CaMep2, the DD mutant and phosphorylated protein (S453J). In the WT structure, the acidic residues Asp419, Glu420 and Glu421 are within hydrogen bonding distance of Ser453. After 200 ns of MD simulation, the interactions between these residues and Ser453 remain intact. The protein backbone has an average r.m.s.d. of only ∼3 Å during the 200-ns simulation, indicating that the protein is stable. There is flexibility in the side chains of the acidic residues so that they are able to form stable hydrogen bonds with Ser453. In particular, persistent hydrogen bonds are observed between the Ser453 hydroxyl group and the acidic group of Glu420, and also between the amine group of Ser453 and the backbone carbonyl of Glu420 (Supplementary Fig. 5). The DD mutant is also stable during the simulations, but the average backbone r.m.s.d of ∼3.6 Å suggests slightly more conformational flexibility than WT. As the simulation proceeds, the side chains of the acidic residues move away from Asp452 and Asp453, presumably to avoid electrostatic repulsion. For example, the distance between the Asp453 acidic oxygens and the Glu420 acidic oxygens increases from ∼7 to \u003e22 Å after 200 ns simulations, and thus these residues are not interacting. The protein is structurally stable throughout the simulation with little deviation in the other parts of the protein. Finally, the S453J mutant is also stable throughout the 200-ns simulation and has an average backbone deviation of ∼3.8 Å, which is similar to the DD mutant. The movement of the acidic residues away from Arg452 and Sep453 is more pronounced in this simulation in comparison with the movement away from Asp452 and Asp453 in the DD mutant. The distance between the phosphate of Sep453 and the acidic oxygen atoms of Glu420 is initially ∼11 Å, but increases to \u003e30 Å after 200 ns. The short helix formed by residues Leu427 to Asp438 unravels during the simulations to a disordered state. The remainder of the protein is not affected (Supplementary Fig. 5). Thus, the MD simulations support the notion from the crystal structures that phosphorylation generates conformational changes in the conserved part of the CTR. However, the conformational changes for the phosphomimetic mutants in the crystals are confined to the CTR (Fig. 8), and the channels are still closed (Supplementary Fig. 2). One possible explanation is that the mutants do not accurately mimic a phosphoserine, but the observation that the S453D and DD mutants are fully active in the absence of Npr1 suggests that the mutations do mimic the effect of phosphorylation (Fig. 3). The fact that the S453D structure was obtained in the presence of 10 mM ammonium ions suggests that the crystallization process favours closed states of the Mep2 channels. Discussion Knowledge about ammonium transporter structure has been obtained from experimental and theoretical studies on bacterial family members. In addition, a number of biochemical and genetic studies are available for bacterial, fungal and plant proteins. These efforts have advanced our knowledge considerably but have not yet yielded atomic-level answers to several important mechanistic questions, including how ammonium transport is regulated in eukaryotes and the mechanism of ammonium signalling. In Arabidopsis thaliana Amt-1;1, phosphorylation of the CTR residue T460 under conditions of high ammonium inhibits transport activity, that is, the default (non-phosphorylated) state of the plant transporter is open. Interestingly, phosphomimetic mutations introduced into one monomer inactivate the entire trimer, indicating that (i) heterotrimerization occurs and (ii) the CTR mediates allosteric regulation of ammonium transport activity via phosphorylation. Owing to the lack of structural information for plant AMTs, the details of channel closure and inter-monomer crosstalk are not yet clear. Contrasting with the plant transporters, the inactive states of Mep2 proteins under conditions of high ammonium are non-phosphorylated, with channels that are closed on the cytoplasmic side. The reason why similar transporters such as A. thaliana Amt-1;1 and Mep2 are regulated in opposite ways by phosphorylation (inactivation in plants and activation in fungi) is not known. In fungi, preventing ammonium entry via channel closure in ammonium transporters would be one way to alleviate ammonium toxicity, in addition to ammonium excretion via Ato transporters and amino-acid secretion. By determining the first structures of closed ammonium transporters and comparing those structures with the permanently open bacterial proteins, we demonstrate that Mep2 channel closure is likely due to movements of the CTR and ICL1 and ICL3. More specifically, the close interactions between the CTR and ICL1/ICL3 present in open transporters are disrupted, causing ICL3 to move outwards and block the channel (Figs 4 and 9a). In addition, ICL1 has shifted inwards to contribute to the channel closure by engaging His2 from the twin-His motif via hydrogen bonding with a highly conserved tyrosine hydroxyl group. Upon phosphorylation by the Npr1 kinase in response to nitrogen limitation, the region around the conserved ExxGxD motif undergoes a conformational change that opens the channel (Fig. 9). Importantly, the structural similarities in the TM parts of Mep2 and AfAmt-1 (Fig. 5a) suggest that channel opening/closure does not require substantial changes in the residues lining the channel. How exactly the channel opens and whether opening is intra-monomeric are still open questions; it is possible that the change in the CTR may disrupt its interactions with ICL3 of the neighbouring monomer (Fig. 9b), which could result in opening of the neighbouring channel via inward movement of its ICL3. Owing to the crosstalk between monomers, a single phosphorylation event might lead to opening of the entire trimer, although this has not yet been tested (Fig. 9b). Whether or not Mep2 channel opening requires, in addition to phosphorylation, disruption of the Tyr–His2 interaction by the ammonium substrate is not yet clear. Is our model for opening and closing of Mep2 channels valid for other eukaryotic ammonium transporters? Our structural data support previous studies and clarify the central role of the CTR and cytoplasmic loops in the transition between closed and open states. However, even the otherwise highly similar Mep2 proteins of S. cerevisiae and C. albicans have different structures for their CTRs (Fig. 1 and Supplementary Fig. 6). In addition, the AI region of the CTR containing the Npr1 kinase site is conserved in only a subset of fungal transporters, suggesting that the details of the structural changes underpinning regulation vary. Nevertheless, given the central role of absolutely conserved residues within the ICL1-ICL3-CTR interaction network (Fig. 4), we propose that the structural basics of fungal ammonium transporter activation are conserved. The fact that Mep2 orthologues of distantly related fungi are fully functional in ammonium transport and signalling in S. cerevisiae supports this notion. It should also be noted that the tyrosine residue interacting with His2 is highly conserved in fungal Mep2 orthologues, suggesting that the Tyr–His2 hydrogen bond might be a general way to close Mep2 proteins. With regards to plant AMTs, it has been proposed that phosphorylation at T460 generates conformational changes that would close the neighbouring pore via the C terminus. This assumption was based partly on a homology model for Amt-1;1 based on the (open) archaebacterial AfAmt-1 structure, which suggested that the C terminus of Amt-1;1 would extend further to the neighbouring monomer. Our Mep2 structures show that this assumption may not be correct (Fig. 4 and Supplementary Fig. 6). In addition, the considerable differences between structurally resolved CTR domains means that the exact environment of T460 in Amt-1;1 is also not known (Supplementary Fig. 6). Based on the available structural information, we consider it more likely that phosphorylation-mediated pore closure in Amt-1;1 is intra-monomeric, via disruption of the interactions between the CTR and ICL1/ICL3 (for example, Y467-H239 and D458-K71). There is generally no equivalent for CaMep2 Tyr49 in plant AMTs, indicating that a Tyr–His2 hydrogen bond as observed in Mep2 may not contribute to the closed state in plant transporters. We propose that intra-monomeric CTR-ICL1/ICL3 interactions lie at the basis of regulation of both fungal and plant ammonium transporters; close interactions generate open channels, whereas the lack of ‘intra-' interactions leads to inactive states. The need to regulate in opposite ways may be the reason why the phosphorylation sites are in different parts of the CTR, that is, centrally located close to the ExxGxD motif in AMTs and peripherally in Mep2. In this way, phosphorylation can either lead to channel closing (in the case of AMTs) or channel opening in the case of Mep2. Our model also provides an explanation for the observation that certain mutations within the CTR completely abolish transport activity. An example of an inactivating residue is the glycine of the ExxGxD motif of the CTR. Mutation of this residue (G393 in EcAmtB; G456 in AtAmt-1;1) inactivates transporters as diverse as Escherichia coli AmtB and A. thaliana Amt-1;1 (refs). Such mutations likely cause structural changes in the CTR that prevent close contacts between the CTR and ICL1/ICL3, thereby stabilizing a closed state that may be similar to that observed in Mep2. Regulation and modulation of membrane transport by phosphorylation is known to occur in, for example, aquaporins and urea transporters, and is likely to be a common theme for eukaryotic channels and transporters. Recently, phosphorylation was also shown to modulate substrate affinity in nitrate transporters. With respect to ammonium transport, phosphorylation has thus far only been shown for A. thaliana AMTs and for S. cerevisiae Mep2 (refs). However, the absence of GlnK proteins in eukaryotes suggests that phosphorylation-based regulation of ammonium transport may be widespread. Nevertheless, as discussed above, considerable differences may exist between different species. With respect to Mep2-mediated signalling to induce pseudohyphal growth, two models have been put forward as to how this occurs and why it is specific to Mep2 proteins. In one model, signalling is proposed to depend on the nature of the transported substrate, which might be different in certain subfamilies of ammonium transporters (for example, Mep1/Mep3 versus Mep2). For example, NH3 uniport or symport of NH3/H+ might result in changes in local pH, but NH4+ uniport might not, and this difference might determine signalling. In the other model, signalling is thought to require a distinct conformation of the Mep2 transporter occurring during the transport cycle. While the current study does not specifically address the mechanism of signalling underlying pseudohyphal growth, our structures do show that Mep2 proteins can assume different conformations. It is clear that ammonium transport across biomembranes remains a fascinating and challenging field in large part due to the unique properties of the substrate. Our Mep2 structural work now provides a foundation for future studies to uncover the details of the structural changes that occur during eukaryotic ammonium transport and signaling, and to assess the possibility to utilize small molecules to shut down ammonium sensing and downstream signalling pathways in pathogenic fungi. Methods Mep2 overexpression and purification Ammonium transporter genes were amplified from genomic DNA or cDNA by PCR (Phusion, New England Biolabs). In both ScMEP2 and CaMEP2, Asn4 was replaced by a glutamine to prevent glycosylation. In order to allow transformation of yeast by recombination, the following primer extensions were used: forward 5′-GAAAAAACCCCGGATTCTAGAACTAGTGGATCCTCC-3′ and reverse 5′-TGACTCGAGTTATGCACCGTGGTGGTGATGGTGATG-3′. These primers result in a construct that lacks the cleavable N- and C-terminal tags present in the original vector, and replaces these with a C-terminal hexa-histidine tag. Recombination in yeast strain W303 pep4Δ was carried out using ∼50–100 ng of SmaI-digested vector 83νΔ (ref.) and at least a fourfold molar excess of PCR product via the lithium acetate method. Transformants were selected on SCD -His plates incubated at 30 °C. Construction of mutant CaMEP2 genes was done using the Q5 site-directed mutagenesis kit (NEB) per manufacturer's instructions. Three CaMep2 mutants were made for crystallization: the first mutant is a C-terminal truncation mutant 442Δ, lacking residues 443–480 including the AI domain. The second mutant, R452D/S453D, mimics the protein phosphorylated at Ser453. Given that phosphate is predominantly charged −2 at physiological pH, we introduced the second aspartate residue for Arg452. However, we also constructed the ‘single D', S453D CaMep2 variant. For expression, cells were grown in shaker flasks at 30 °C for ∼24 h in synthetic minimal medium lacking histidine and with 1% (w/v) glucose to a typical OD600 of 6–8. Cells were subsequently spun down for 15 min at 4,000g and resuspended in YP medium containing 1.5% (w/v) galactose, followed by another 16–20 h growth at 30 °C/160 r.p.m. and harvesting by centrifugation. Final OD600 values typically reached 18–20. Cells were lysed by bead beating (Biospec) for 5 × 1 min with 1 min intervals on ice, or by 1–2 passes through a cell disrupter operated at 35,000 p.s.i. (TS-Series 0.75 kW; Constant Systems). Membranes were collected from the suspension by centrifugation at 200,000g for 90 min (45Ti rotor; Beckmann Coulter). Membrane protein extraction was performed by homogenization in a 1:1 (w/w) mixture of dodecyl-β-D-maltoside and decyl-β-D-maltoside (DDM/DM) followed by stirring at 4 °C overnight. Typically, 1 g (1% w/v) of total detergent was used for membranes from 2 l of cells. The membrane extract was centrifuged for 35 min at 200,000g and the supernatant was loaded onto a 10-ml Nickel column (Chelating Sepharose; GE Healthcare) equilibrated in 20 mM Tris/300 mM NaCl/0.2% DDM, pH 8. The column was washed with 15 column volumes buffer containing 30 mM imidazole and eluted in 3 column volumes with 250 mM imidazole. Proteins were purified to homogeneity by gel filtration chromatography in 10 mM HEPES/100 mM NaCl/0.05% DDM, pH 7–7.5. For polishing and detergent exchange, a second gel filtration column was performed using various detergents. In the case of ScMep2, diffracting crystals were obtained only with 0.05% decyl-maltose neopentyl glycol. For the more stable CaMep2 protein, we obtained crystals in, for example, nonyl-glucoside, decyl-maltoside and octyl-glucose neopentyl glycol. Proteins were concentrated to 7–15 mg ml−1 using 100 kDa cutoff centrifugal devices (Millipore), flash-frozen and stored at −80 °C before use. Crystallization and structure determination Crystallization screening trials by sitting drop vapour diffusion were set up at 4 and 20 °C using in-house screens and the MemGold 1 and 2 screens (Molecular Dimensions) with a Mosquito crystallization robot. Crystals were harvested directly from the initial trials or optimized by sitting or hanging drop vapour diffusion using larger drops (typically 2–3 μl total volume). Bar-shaped crystals for ScMep2 diffracting to 3.2 Å resolution were obtained from 50 mM 2-(N-morpholino)ethanesulfonic acid (MES)/0.2 M di-ammonium hydrogen phosphate/30% PEG 400, pH 6. They belong to space group P212121 and have nine molecules (three trimers) in the asymmetric unit (AU). Well-diffracting crystals for CaMep2 were obtained in space group P3 from 0.1 M MES/0.2 M lithium sulphate/20% PEG400, pH 6 (two molecules per AU). An additional crystal form in space group R3 was grown in 0.04 M Tris/0.04 M NaCl/27% PEG350 MME, pH 8 (one molecule per AU). Diffracting crystals for the phosporylation-mimicking CaMep2 DD mutant were obtained in space group P6322 from 0.1 M sodium acetate/15–20% PEG400, pH 5 (using decyl-maltoside as detergent; one molecule per AU), while S453D mutant crystals grew in 24% PEG400/0.05 M sodium acetate, pH 5.4/0.05 M magnesium acetate tetrahydrate/10 mM NH4Cl (space group R32; one molecule per AU). Finally, the 442Δ truncation mutant gave crystals under many different conditions, but most of these diffracted poorly or not at all. A reasonable low-resolution data set (3.4 Å resolution) was eventually obtained from a crystal grown in 24% PEG400/0.05 M sodium acetate/0.05 M magnesium acetate, pH 6.1 (space group R32). Diffraction data were collected at the Diamond Light Source and processed with XDS or HKL2000 (ref. ). For MR, a search model was constructed with Sculptor within Phenix, using a sequence alignment of ScMep2 with Archaeoglobus fulgidus Amt-1 (PDB ID 2B2H; ∼40% sequence identity to ScMep2). A clear solution with nine molecules (three trimers) in the AU was obtained using Phaser. The model was subsequently completed by iterative rounds of manual building within Coot followed by refinement within Phenix. The structures for WT CaMep2 were solved using the best-defined monomer of ScMep2 (60% sequence identity with CaMep2) in MR with Phaser, followed by automated model building within Phenix. Finally, the structures of the three mutant CaMep2 proteins were solved using WT CaMep2 as the search model. The data collection and refinement statistics for all six solved structures have been summarized in Supplementary Tables 1 and 2. Growth assays The S. cerevisiae haploid triple mepΔ strain (Σ1278b MATα mep1::LEU2 mep2::LEU2 mep3::G418 ura3-52) and triple mepΔ npr1Δ strain (Σ1278b MATα mep1::LEU2 mep2::LEU2 mep3::G418 npr1::NAT1 ura3-52) were generated by integrating the NAT1 resistance gene at one NPR1 locus in the diploid strain MLY131 (ref.), followed by isolation of individual haploid strains. Cells were grown in synthetic minimal medium with glucose (2%) as the carbon source and ammonium sulphate (1 mM) or glutamate (0.1%) as the nitrogen source. Yeast cells were transformed as described. All DNA sequences encoding epitope-tagged ScMep2 and its mutant derivatives were generated by PCR and homologous recombination using the vector pRS316 (ref. ). In each case, the ScMEP2 sequences included the ScMEP2 promoter (1 kb), the ScMEP2 terminator and sequences coding for a His-6 epitope at the C-terminal end of the protein. All Mep2-His fusions contain the N4Q mutation to prevent glycosylation of Mep2 (ref.). All newly generated plasmid inserts were verified by DNA sequencing. For growth assays, S. cerevisiae cells containing plasmids expressing ScMep2 or mutant derivatives were grown overnight in synthetic minimal glutamate medium, washed, spotted by robot onto solid agar plates and culture growth followed by time course photography. Images were then processed to quantify the growth of each strain over 3 days as described. Protein modelling The MODELLER (version 9.15) software package was used to build protein structures for MD simulations. This method was required to construct two complete protein models, the double mutant R452D/S453D (with the four missing residues from the X-ray structure added) and also the construct in which the mutation at position 452 is reverted to R, and D453 is replaced with a phosphoserine. The quality of these models was assessed using normalized Discrete Optimized Protein Energy (DOPE) values and the molpdf assessment function within the MODELLER package. The model R452D/S453D mutant has a molpdf assessment score of 1854.05, and a DOPE assessment score of -60920.55. The model of the S453J mutant has a molpdf assessment score of 1857.01 and a DOPE assessment score of −61032.15. MD simulations WT and model structures were embedded into a pre-equilibrated lipid bilayer composed of 512 dipalmitoylphosphatidylcholine lipids using the InflateGRO2 computer programme. The bilayers were then solvated with the SPC water model and counterions were added to achieve a charge neutral state. All simulations were performed with the GROMACS package (version 4.5.5), and the GROMOS96 43a1p force field. During simulation time, the temperature was maintained at 310 K using the Nosé-Hoover thermostat with a coupling constant of 0.5 ps. Pressure was maintained at 1 bar using semi-isotropic coupling with the Parrinello-Rahman barostat and a time constant of 5 ps. Electrostatic interactions were treated using the smooth particle mesh Ewald algorithm with a short-range cutoff of 0.9 nm. Van der Waals interactions were truncated at 1.4 nm with a long-range dispersion correction applied to energy and pressure. The neighbour list was updated every five steps. All bonds were constrained with the LINCS algorithm, so that a 2-fs time step could be applied throughout. The phospholipid parameters for the dipalmitoylphosphatidylcholine lipids were based on the work of Berger. The embedded proteins were simulated for 200 ns each; a repeat simulation was performed for each system with different initial velocities to ensure reproducibility. To keep the c.p.u. times within reasonable limits, all simulations were performed on Mep2 monomers. This is also consistent with previous simulations for E. coli AmtB. Additional information Accession codes: The atomic coordinates and the associated structure factors have been deposited in the Protein Data Bank (http:// www.pdbe.org) with accession codes 5AEX (ScMep2), 5AEZ(CaMep2; R3), 5AF1(CaMep2; P3), 5AID(CaMep2; 442D), 5AH3 (CaMep2; R452D/S453D) and 5FUF (CaMep2; S453D). How to cite this article: van den Berg, B. et al. Structural basis for Mep2 ammonium transceptor activation by phosphorylation. Nat. Commun. 7:11337 doi: 10.1038/ncomms11337 (2016). Supplementary Material The eukaryotic plasma membrane as a nutrient-sensing device Nutrient sensing and signaling in the yeast Saccharomyces cerevisiae The transporter classification database A family of ammonium transporters in Saccharomyces cerevisiae A split-ubiquitin two-hybrid screen for proteins physically interacting with the yeast amino acid transceptor Gap1 and ammonium transceptor Mep2 The MEP2 ammonium permease regulates pseudohyphal differentiation in Saccharomyces cerevisiae Differential activation of ammonium transporters during the accumulation of ammonia by Colletotrichum gloeosporioides and its effect on appressoria formation andpathogenicity A Mep2-dependent transcriptional profile links permease function to gene expression during pseudohyphal growth in Saccharomyces cerevisiae The Mep2p ammonium permease controls nitrogen starvation-induced filamentous growth in Candida albicans Distinct transport mechanisms in yeast ammonium transport/sensor proteins of the Mep/Amt/Rh family and impact on filamentation The yeast ammonium transport protein Mep2 and its positive regulator, the Npr1 kinase, play an important role in normal and pseudohyphal growth on various nitrogen media through retrieval of excreted ammonium Function of human Rh based on structure of RhCG at 2.1A Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35A Crystal structure of the archaeal ammonium transporter Amt-1 from Archaeoglobus fulgidus An unusual twin-his arrangement in the pore of ammonia channels is essential for substrate conductance Ammonium transporters achieve charge transfer by fragmenting their substrate Ammonium transport proteins with changes in one of the conserved pore histidines have different performance in ammonia and methylamine conduction Molecular dynamics simulations on the Escherichia coli ammonia channel protein AmtB: mechanism of ammonia/ammonium transport Periplasmic vestibule plays an important role for solute recruitment, selectivity, and gating in the Rh/Amt/MEP superfamily The mechanism of ammonia transport based on the crystal structure of AmtB of Escherichia coli Different hydration patterns in the pores of AmtB and RhCG could determine their transport mechanisms Nitrogen assimilation in Escherichia coli: putting molecular data into a systems perspective Direct observation of electrogenic NH4(+) transport in ammonium transport (Amt) proteins Uncoupling of ionic currents from substrate transport in the plant ammonium transporter AtAMT1;2 Molecular mechanism of acute ammonia toxicity: role of NMDA receptors The glnKamtB operon. A conserved gene pair in prokaryotes Inhibitory complex of the transmembrane ammonia channel, AmtB, and the cytosolic regulatory protein, GlnK, at 1.96A In vitro analysis of the Escherichia coli AmtB-GlnK complex reveals a stoichiometric interaction and sensitivity to ATP and 2-oxoglutarate Adjusting ammonium uptake via phosphorylation The TORC1 effector kinase Npr1 fine tunes the inherent activity of the Mep2 ammonium transport protein N- terminal cysteines affect oligomer stability of the allosterically regulated ammonium transporter LeAMT1;1 The conserved carboxy-terminal region of the ammonia channel AmtB plays a critical role in channel function A cytosolic trans-activation domain essential for ammonium uptake Allosteric regulation of transport activity by heterotrimerization of Arabidopsis ammonium transporter complexes in vivo Ammonium toxicity and potassium limitation in yeast Isolation and characterization from pathogenic fungi of genes encoding ammonium permeases and their roles in dimorphism Impact of ammonium permeases mepA, mepB, and mepC on nitrogen-regulated secondary metabolism in Fusarium fujikuroi Molecular characterization, function and regulation of ammonium transporters (Amt) and ammonium-metabolizing enzymes (GS, NADP-GDH) in the ectomycorrhizal fungus Hebeloma cylindrosporum Structural insights into eukaryotic aquaporin regulation Expression of urea transporters and their regulation Molecular basis of nitrate uptake by the plant nitrate transporter NRT1.1 Overexpression and purification of integral membrane proteins in yeast XDS Processing of X-ray diffraction data collected in oscillation mode Towards automated crystallographic structure refinement with phenix.refine Likelihood-enhanced fast translation functions Coot: model-building tools for molecular graphics High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier Plasmid construction by homologous recombination in yeast In vivo N-glycosylation of the Mep2 high-affinity ammonium transporter of Saccharomyces cerevisiae reveals an extracytosolic N-terminus Quantitative fitness analysis shows that NMD proteins and many other protein complexes suppress or enhance distinct telomere cap defects Comparative protein structure modeling of genes and genomes LAMBADA and InflateGRO2: efficient membrane alignment and insertion of membrane proteins for molecular dynamics simulations  GROMACS: fast, flexible, and free A unified formulation of the constant temperature molecular dynamics methods Canonical dynamics: equilibrium phase-space distributions Polymorphic transitions in single crystals: a new molecular dynamics method A smooth particle mesh Ewald method LINCS: a linear constraint solver for molecular simulations Molecular dynamics simulations of a fluid bilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature  The PyMOL Molecular Graphics System. version 1.7.4 (Schrödinger, LLC). Author contributions B.v.d.B. performed the experiments related to Mep2 structure determination, designed research and wrote the paper. A.C. performed ammonium growth experiments of Mep variants. D.J. and S.K. performed modelling studies and MD simulations. A.B. collected the X-ray synchrotron data and maintained the Newcastle Structural Biology Laboratory. J.C.R. designed research related to the S. cerevisiae growth assays. X-ray crystal structures of Mep2 transceptors. (a) Monomer cartoon models viewed from the side for (left) A.\nfulgidus Amt-1 (PDB ID 2B2H), S. cerevisiae Mep2 (middle) and\nC. albicans Mep2 (right). The cartoons are in rainbow\nrepresentation. The region showing ICL1 (blue), ICL3 (green) and the CTR\n(red) is boxed for comparison. (b) CaMep2 trimer viewed from the\nintracellular side (right). One monomer is coloured as in a and one\nmonomer is coloured by B-factor (blue, low; red; high). The CTR is boxed.\n(c) Overlay of ScMep2 (grey) and CaMep2 (rainbow), illustrating\nthe differences in the CTRs. All structure figures were generated with\nPymol. Sequence conservation in ammonium transporters. ClustalW alignment of CaMep2, ScMep2, A. fulgidus Amt-1, E.\ncoli AmtB and A. thaliana Amt-1;1. The secondary structure\nelements observed for CaMep2 are indicated, with the numbers corresponding\nto the centre of the TM segment. Important regions are labelled. The\nconserved RxK motif in ICL1 is boxed in blue, the ER motif in ICL2 in cyan,\nthe conserved ExxGxD motif of the CTR in red and the AI region in yellow.\nColoured residues are functionally important and correspond to those of the\nPhe gate (blue), the binding site Trp residue (magenta) and the twin-His\nmotif (red). The Npr1 kinase site in the AI region is highlighted pink. The\ngrey sequences at the C termini of CaMep2 and ScMep2 are not visible in the\nstructures and are likely disordered. Growth of ScMep2 variants on low ammonium medium. (a) The triple mepΔ strain (black) and triple\nmepΔ npr1Δ strain (grey) containing plasmids\nexpressing WT and variant ScMep2 were grown on minimal medium containing\n1 mM ammonium sulphate. The quantified cell density reflects\nlogarithmic growth after 24 h. Error bars are the s.d. for three\nreplicates of each strain (b) The strains used in a were also\nserially diluted and spotted onto minimal agar plates containing glutamate\n(0.1%) or ammonium sulphate (1 mM), and grown for 3 days at\n30 °C. Structural differences between Mep2 and bacterial ammonium\ntransporters. (a) ICL1 in AfAmt-1 (light blue) and CaMep2 (dark blue), showing\nunwinding and inward movement in the fungal protein. (b) Stereo\ndiagram viewed from the cytosol of ICL1, ICL3 (green) and the CTR (red) in\nAfAmt-1 (light colours) and CaMep2 (dark colours). The side chains of\nresidues in the RxK motif as well as those of Tyr49 and His342 are labelled.\nThe numbering is for CaMep2. (c) Conserved residues in ICL1-3 and the\nCTR. Views from the cytosol for CaMep2 (left) and AfAmt-1, highlighting the\nlarge differences in conformation of the conserved residues in ICL1 (RxK\nmotif; blue), ICL2 (ER motif; cyan), ICL3 (green) and the CTR (red). The\nlabelled residues are analogous within both structures. In b and\nc, the centre of the trimer is at top. Channel closures in Mep2. (a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of\nthe Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1\nthat forms a hydrogen bond with His2 in CaMep2. (b) Surface views\nfrom the side in rainbow colouring, showing the two-tier channel block\n(indicated by the arrows) in CaMep2. The Npr1 kinase target Ser453 is dephosphorylated and located in an\nelectronegative pocket. (a) Stereoviews of CaMep2 showing 2Fo–Fc\nelectron density (contoured at 1.0 σ) for CTR residues\nAsp419-Met422 and for Tyr446-Thr455 of the AI region. For clarity, the\nresidues shown are coloured white, with oxygen atoms in red and nitrogen\natoms in blue. The phosphorylation target residue Ser453 is labelled in\nbold. (b) Overlay of the CTRs of ScMep2 (grey) and CaMep2 (green),\nshowing the similar electronegative environment surrounding the\nphosphorylation site (P). The AI regions are coloured magenta. (c)\nCytoplasmic view of the Mep2 trimer indicating the large distance between\nSer453 and the channel exits (circles; Ile52 lining the channel exit is\nshown). Effect of removal of the AI region on Mep2 structure. (a) Side views for WT CaMep2 (left) and the truncation mutant\n442Δ (right). The latter is shown as a putty model according to\nB-factors to illustrate the disorder in the protein on the cytoplasmic side.\nMissing regions are labelled. (b) Stereo superpositions of WT CaMep2\nand the truncation mutant. 2Fo–Fc electron\ndensity (contoured at 1.0 σ) for residues Tyr49 and His342 is\nshown for the truncation mutant. Phosphorylation causes conformational changes in the CTR. (a) Cytoplasmic view of the DD mutant trimer, with WT CaMep2\nsuperposed in grey for one of the monomers. The arrow indicates the\nphosphorylation site. The AI region is coloured magenta. (b) Monomer\nside-view superposition of WT CaMep2 and the DD mutant, showing the\nconformational change and disorder around the ExxGxD motif. Side chains for\nresidues 452 and 453 are shown as stick models. Schematic model for phosphorylation-based regulation of Mep2 ammonium\ntransporters. (a) In the closed, non-phosphorylated state (i), the CTR (magenta) and\nICL3 (green) are far apart with the latter blocking the intracellular\nchannel exit (indicated with a hatched circle). Upon phosphorylation and\nmimicked by the CaMep2 S453D and DD mutants (ii), the region around the\nExxGxD motif undergoes a conformational change that results in the CTR\ninteracting with the inward-moving ICL3, opening the channel (full circle)\n(iii). The arrows depict the movements of important structural elements. The\nopen-channel Mep2 structure is represented by archaebacterial Amt-1 and\nshown in lighter colours consistent with Fig. 4. As\ndiscussed in the text, similar structural arrangements may occur in plant\nAMTs. In this case however, the open channel corresponds to the\nnon-phosphorylated state; phosphorylation breaks the CTR–ICL3\ninteractions leading to channel closure. (b) Model based on AMT\ntransporter analogy showing how phosphorylation of a\nMep2 monomer might allosterically open channels in the entire trimer via\ndisruption of the interactions between the CTR and ICL3 of a neighbouring\nmonomer 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+[{"sourceid":"4854314","sourcedb":"","project":"","target":"","text":"RNA protects a nucleoprotein complex against radiation damage Systematic analysis of radiation damage within a protein–RNA complex over a large dose range (1.3–25 MGy) reveals significant differential susceptibility of RNA and protein. A new method of difference electron-density quantification is presented. Radiation damage during macromolecular X-ray crystallographic data collection is still the main impediment for many macromolecular structure determinations. Even when an eventual model results from the crystallographic pipeline, the manifestations of radiation-induced structural and conformation changes, the so-called specific damage, within crystalline macromolecules can lead to false interpretations of biological mechanisms. Although this has been well characterized within protein crystals, far less is known about specific damage effects within the larger class of nucleoprotein complexes. Here, a methodology has been developed whereby per-atom density changes could be quantified with increasing dose over a wide (1.3–25.0 MGy) range and at higher resolution (1.98 Å) than the previous systematic specific damage study on a protein–DNA complex. Specific damage manifestations were determined within the large trp RNA-binding attenuation protein (TRAP) bound to a single-stranded RNA that forms a belt around the protein. Over a large dose range, the RNA was found to be far less susceptible to radiation-induced chemical changes than the protein. The availability of two TRAP molecules in the asymmetric unit, of which only one contained bound RNA, allowed a controlled investigation into the exact role of RNA binding in protein specific damage susceptibility. The 11-fold symmetry within each TRAP ring permitted statistically significant analysis of the Glu and Asp damage patterns, with RNA binding unexpectedly being observed to protect these otherwise highly sensitive residues within the 11 RNA-binding pockets distributed around the outside of the protein molecule. Additionally, the method enabled a quantification of the reduction in radiation-induced Lys and Phe disordering upon RNA binding directly from the electron density. Introduction    With the wide use of high-flux third-generation synchrotron sources, radiation damage (RD) has once again become a dominant reason for the failure of structure determination using macromolecular crystallography (MX) in experiments conducted both at room temperature and under cryocooled conditions (100 K). Significant progress has been made in recent years in understanding the inevitable manifestations of X-ray-induced RD within protein crystals, and there is now a body of literature on possible strategies to mitigate the effects of RD (e.g. Zeldin, Brockhauser et al., 2013; Bourenkov \u0026 Popov, 2010). However, there is still no general consensus within the field on how to minimize RD during MX data collection, and debates on the dependence of RD progression on incident X-ray energy (Shimizu et al., 2007; Liebschner et al., 2015) and the efficacy of radical scavengers (Allan et al., 2013) have yet to be resolved. RD manifests in two forms. Global radiation damage is observed within reciprocal space as the overall decay of the summed intensity of reflections detected within the diffraction pattern as dose increases (Garman, 2010; Murray \u0026 Garman, 2002). Dose is defined as the absorbed energy per unit mass of crystal in grays (Gy; 1 Gy = 1 J kg−1), and is the metric against which damage progression should be monitored during MX data collection, as opposed to time. At 100 K, an experimental dose limit of 30 MGy has been recommended as an upper limit beyond which the biological information derived from any macromolecular crystal may be compromised (Owen et al., 2006).  Specific radiation damage (SRD) is observed in the real-space electron density, and has been detected at much lower doses than any observable decay in the intensity of reflections. Indeed, the C—Se bond in selenomethionine, the stability of which is key for the success of experimental phasing methods, can be cleaved at a dose as low as 2 MGy for a crystal maintained at 100 K (Holton, 2007). SRD has been well characterized in a large range of proteins, and is seen to follow a reproducible order: metallo-centre reduction, disulfide-bond cleavage, acidic residue decarboxylation and methionine methylthio cleavage (Ravelli \u0026 McSweeney, 2000; Burmeister, 2000; Weik et al., 2000; Yano et al., 2005). Furthermore, damage susceptibility within each residue type follows a preferential ordering influenced by a combination of local environment factors (solvent accessibility, conformational strain, proximity to active sites/high X-ray cross-section atoms; Holton, 2009). Deconvoluting the individual roles of these parameters has been surprisingly challenging, with factors such as solvent accessibility currently under active investigation (Weik et al., 2000; Fioravanti et al., 2007; Gerstel et al., 2015). There are a number of cases where SRD manifestations have compromised the biological information extracted from MX-determined structures at much lower doses than the recommended 30 MGy limit, leading to false structural interpretations of protein mechanisms. Active-site residues appear to be particularly susceptible, particularly for photosensitive proteins and in instances where chemical strain is an intrinsic feature of the reaction mechanism. For instance, structure determination of the purple membrane protein bacterio­rhodopsin required careful corrections for radiation-induced structural changes before the correct photosensitive intermediate states could be isolated (Matsui et al., 2002). The significant chemical strain required for catalysis within the active site of phosphoserine aminotransferase has been observed to diminish during X-ray exposure (Dubnovitsky et al., 2005). Since the majority of SRD studies to date have focused on proteins, much less is known about the effects of X-ray irradiation on the wider class of crystalline nucleoprotein complexes or how to correct for such radiation-induced structural changes. Understanding RD to such complexes is crucial, since DNA is rarely naked within a cell, instead dynamically interacting with proteins, facilitating replication, transcription, modification and DNA repair. As of early 2016, \u003e5400 nucleoprotein complex structures have been deposited within the PDB, with 91% solved by MX. It is essential to understand how these increasingly complex macromolecular structures are affected by the radiation used to solve them. Nucleoproteins also represent one of the main targets of radiotherapy, and an insight into the damage mechanisms induced by X-ray irradiation could inform innovative treatments. When a typical macromolecular crystal is irradiated with ionizing X-rays, each photoelectron produced via interactions with both the macromolecule (direct damage) and solvent (indirect damage) can induce cascades of up to 500 secondary low-energy electrons (LEEs) that are capable of inducing further ionizations. Investigations on sub-ionization-level LEEs (0–15 eV) interacting with both dried and aqueous oligonucleotides (Alizadeh \u0026 Sanche, 2014; Simons, 2006) concluded that resonant electron attachment to DNA bases and the sugar-phosphate backbone could lead to the preferential cleavage of strong (∼4 eV, 385 kJ mol−1) sugar-phosphate C—O covalent bonds within the DNA backbone and then base-sugar N1—C bonds, eventually leading to single-strand breakages (SSBs; Ptasińska \u0026 Sanche, 2007). Electrons have been shown to be mobile at 77 K by electron spin resonance spectroscopy studies (Symons, 1997; Jones et al., 1987), with rapid electron quantum tunnelling and positive hole migration along the protein backbone and through stacked DNA bases indicated as a dominant mechanism by which oxidative and reductive damage localizes at distances from initial ionization sites at 100 K (O’Neill et al., 2002). The investigation of naturally forming nucleoprotein complexes circumvents the inherent challenges in making controlled comparisons of damage mechanisms between protein and nucleic acids crystallized separately. Recently, for a well characterized bacterial protein–DNA complex (C.Esp1396I; PDB entry 3clc; resolution 2.8 Å; McGeehan et al., 2008) it was concluded that over a wide dose range (2.1–44.6 MGy) the protein was far more susceptible to SRD than the DNA within the crystal (Bury et al., 2015). Only at doses above 20 MGy were precursors of phosphodiester-bond cleavage observed within AT-rich regions of the 35-mer DNA. For crystalline complexes such as C.Esp1396I, whether the protein is intrinsically more susceptible to X-ray-induced damage or whether the protein scavenges electrons to protect the DNA remains unclear in the absence of a non-nucleic acid-bound protein control obtained under exactly the same crystallization and data-collection conditions. To monitor the effects of nucleic acid binding on protein damage susceptibility, a crystal containing two protein molecules per asymmetric unit, only one of which was bound to RNA, is reported here (Fig. 1 ▸). Using newly developed methodology, we present a controlled SRD investigation at 1.98 Å resolution using a large (∼91 kDa) crystalline protein–RNA complex: trp RNA-binding attenuation protein (TRAP) bound to a 53 bp RNA sequence (GAGUU)10GAG (PDB entry 1gtf; Hopcroft et al., 2002). TRAP consists of 11 identical subunits assembled into a ring with 11-fold rotational symmetry. It binds with high affinity (K d ≃ 1.0 nM) to RNA segments containing 11 GAG/UAG triplets separated by two or three spacer nucleotides (Elliott et al., 2001) to regulate the transcription of tryptophan biosynthetic genes in Bacillus subtilis (Antson et al., 1999). In this structure, the bases of the G1-A2-G3 nucleotides form direct hydrogen bonds to the protein, unlike the U4-U5 nucleotides, which appear to be more flexible. Ten successive 1.98 Å resolution MX data sets were collected from the same TRAP–RNA crystal to analyse X-ray-induced structural changes over a large dose range (d 1 = 1.3 MGy to d 10 = 25.0 MGy). To avoid the previous necessity for visual inspection of electron-density maps to detect SRD sites, a computational approach was designed to quantify the electron-density change for each refined atom with increasing dose, thus providing a rapid systematic method for SRD study on such large multimeric complexes. By employing the high 11-fold structural symmetry within each TRAP macromolecule, this approach permitted a thorough statistical quantification of the RD effects of RNA binding to TRAP. Materials and methods    RNA synthesis and protein preparation    As previously described (Hopcroft et al., 2002), the 53-base RNA (GAGUU)10GAG was synthesized by in vitro transcription with T7 RNA polymerase and gel-purified. TRAP from B. stearothermophilus was overexpressed in Escherichia coli and purified. Crystallization    TRAP–RNA crystals were prepared using a previously established hanging-drop crystallization protocol (Antson et al., 1999). By using a 2:1 molar ratio of TRAP to RNA, crystals successfully formed from the protein–RNA complex (∼15 mg ml−1) in a solution containing 70 mM potassium phosphate pH 7.8 and 10 mM l-tryptophan. The reservoir consisted of 0.2 M potassium glutamate, 50 mM triethanol­amine pH 8.0, 10 mM MgCl2, 8–11% monomethyl ether PEG 2000. In order to accelerate crystallization, a further gradient was induced by adding 0.4 M KCl to the reservoir after 1.5 µl protein solution had been mixed with an equal volume of the reservoir solution. Wedge-shaped crystals of approximate length 70 µm (longest dimension) grew within 3 d and were vitrified and stored in liquid nitrogen immediately after growth. The cryosolution consisted of 12% monomethyl ether PEG 2000, 30 mM triethanolamine pH 8.0, 6 mM l-tryptophan, 0.1 M potassium glutamate, 35 mM potassium phosphate pH 7.8, 5 mM MgCl2 with 25% 2-methyl-2,4-pentanediol (MPD) included as a cryoprotectant. X-ray data collection    Data were collected at 100 K from a wedge-shaped TRAP–RNA crystal of approximate dimensions 70 × 20 × 40 µm (see Supplementary Fig. S2) on beamline ID14-4 at the ESRF using an incident wavelength of 0.940 Å (13.2 keV) and an ADSC Q315R mosaic CCD detector at 304.5 mm from the crystal throughout the data collection. The beam size was slitted to 0.100 mm (vertical) × 0.160 mm (horizontal), with a uniformly distributed profile, such that the crystal was completely bathed within the beam throughout data collection. Ten successive (1.98 Å resolution) 180° data sets (with Δφ = 1°) were collected over the same angular range from a TRAP–RNA crystal at 28.9% beam transmission. The TRAP–RNA macromolecule crystallized in space group C2, with unit-cell parameters a = 140.9, b = 110.9, c = 137.8 Å, α = γ = 90, β = 137.8° (the values quoted are for the first data set; see Supplementary Table S1 for subsequent values). For the first nine data sets the attenuated flux was recorded to be ∼5 × 1011 photons s−1. A beam refill took place immediately before data set 10, requiring a flux-scale factor increase of 1.42 to be applied, based on the ratio of observed relative intensity I D/I 1 at data set 10 to that extrapolated from data set 9. Dose calculation     RADDOSE-3D (Zeldin, Gerstel et al., 2013) was used to calculate the absorbed dose distribution during each data set (see input file; Supplementary Figs. S1 and S2). The crystal composition was calculated from the deposited TRAP–RNA structure (PDB entry 1gtf; Hopcroft et al., 2002). Crystal absorption coefficients were calculated in RADDOSE-3D using the concentration (mmol l−1) of solvent heavy elements from the crystallization conditions. The beam-intensity profile was modelled as a uniform (‘top-hat’) distribution. The diffraction-weighted dose (DWD) values (Zeldin, Brock­hauser et al., 2013) are given in Supplementary Table S1. Data processing and model refinement    Each data set was integrated using iMosflm (Leslie \u0026 Powell, 2007) and was scaled using AIMLESS (Evans \u0026 Murshudov, 2013; Winn et al., 2011) using the same 5% R free set of test reflections for each data set. To phase the structure obtained from the first data set, molecular replacement was carried out with Phaser (McCoy et al., 2007), using an identical TRAP–RNA structure (PDB entry 1gtf; resolution 1.75 Å; Hopcroft et al., 2002) as a search model. The resulting TRAP–RNA structure (TR1) was refined using REFMAC5 (Murshudov et al., 2011), initially using rigid-body refinement, followed by repeated cycles of restrained, TLS and isotropic B-factor refinement, coupled with visual inspection in Coot (Emsley et al., 2010). TR1 was refined to 1.98 Å resolution, with a dimeric assembly of non-RNA-bound and RNA-bound TRAP rings within the asymmetric unit. Consistent with previous structures of the TRAP–RNA complex, the RNA sequence termini were not observed within the 2F o − F c map; the first spacer (U4) was then modelled at all 11 repeats around the TRAP ring and the second spacer (U5) was omitted from the final refined structure. For the later data sets, the observed structure-factor amplitudes from each separately scaled data set (output from AIMLESS) were combined with the phases of TR1 and the resulting higher-dose model was refined with phenix.refine (Adams et al., 2010) using only rigid-body and isotropic B-factor refinement. During this refinement, the TRAP–RNA complex and nonbound TRAP ring were treated as two separate rigid bodies within the asymmetric unit. Supplementary Table S1 shows the relevant summary statistics.  D loss metric calculation    The CCP4 program CAD was used to create a series of nine merged .mtz files combining observed structure-factor amplitudes for the first data set F obs(d 1) with each later data set F obs(d n) (for n = 2, …, 10). All later data sets were scaled against the initial low-dose data set in SCALEIT. For each data set an atom-tagged .map file was generated using the ATMMAP mode in SFALL (Winn et al., 2011). A full set of nine Fourier difference maps F obs(d n) − F obs(d 1) were calculated using FFT (Ten Eyck, 1973) over the full TRAP–RNA unit-cell dimensions, with the same grid-sampling dimensions as the atom-tagged .map file. All maps were cropped to the TRAP asymmetric unit in MAPMASK. Comparing the atom-tagged .map file and F obs(d n) − F obs(d 1) difference map at each dose, each refined atom was assigned a set of density-change values X. The maximum density-loss metric, D loss (units of e Å−3), was calculated to quantify the per-atom electron-density decay at each dose, assigned as the absolute magnitude of the most negative Fourier difference map voxel value in a local volume around each atom as defined by the set X. Model system calculation    Model calculations were run for the simple amino acids glutamate and aspartate. In order to avoid decarboxylation at the C-terminus instead of the side chain on the Cα atom, the C-terminus of each amino acid was methylated. While the structures of the closed shell acids are well known, the same is not true of those in the oxidized state. The quantum-chemical calculations employed were chosen to provide a satisfactory description of the structure of such radical species and also provide a reliable estimation of the relative C—C(O2) bond strengths, which are otherwise not available. Structures of methyl-terminated (at the N- and C-termini) carboxylates were determined using analytic energy gradients with density functional theory (B3LYP functional; Becke, 1993) and a flexible basis set of polarized valence triple-zeta size with diffuse functions on the non-H atoms [6-311+G(d,p)] in the Gaussian 09 computational chemistry package (Frisch et al., 2009). The stationary points obtained were characterized as at least local minima by examination of the associated analytic Hessian. Effects of the medium were modelled using a dielectric cavity approach (Tomasi et al., 1999) parameterized for water. Results    Per-atom quantification of electron density    To quantify the exact effects of nucleic acid binding to a protein on SRD susceptibility, a high-throughput and automated pipeline was created to systematically calculate the electron-density change for every refined atom within the TRAP–RNA structure as a function of dose. This provides an atom-specific quantification of density–dose dynamics, which was previously lacking within the field. Previous studies have characterized SRD sites by reporting magnitudes of F obs(d n) − F obs(d 1) Fourier difference map peaks in terms of the sigma (σ) contour level (the number of standard deviations from the mean map electron-density value) at which peaks become visible. However, these σ levels depend on the standard deviation values of the map, which can deviate between data sets, and are thus unsuitable for quantitative comparison of density between different dose data sets. Instead, we use here a maximum density-loss metric (D loss), which is the per-atom equivalent of the magnitude of these negative Fourier difference map peaks in units of e Å−3. Large positive D loss values indicate radiation-induced atomic disordering reproducibly throughout the unit cells with respect to the initial low-dose data set. For each TRAP–RNA data set, the D loss metric successfully identified the recognized forms of protein SRD (Fig. 2 ▸ a), with clear Glu and Asp side-chain decarboxylation even in the first difference map calculated (3.9 MGy; Fig. 3 ▸ a). The main sequence of TRAP does not contain any Trp and Cys residues (and thus contains no disulfide bonds). The substrate Trp amino-acid ligands also exhibited disordering of the free terminal carboxyl groups at higher doses (Fig. 2 ▸ a); however, no clear Fourier difference peaks could be observed visually. Even for radiation-insensitive residues (e.g. Gly) the average D loss increases with dose: this is the effect of global radiation damage, since as dose increases the electron density associated with each refined atom becomes weaker as the atomic occupancy decreases (Fig. 2 ▸ b). Only Glu and Asp residues exhibit a rate of D loss increase that consistently exceeds the average decay (Fig. 2 ▸ b, dashed line) at each dose. Additionally, the density surrounding ordered solvent molecules was determined to significantly diminish with increasing dose (Fig. 2 ▸ b). The rate of D loss (attributed to side-chain decarboxylation) was consistently larger for Glu compared with Asp residues over the large dose range (Fig. 2 ▸ b and Supplementary Fig. S3); this observation is consistent with our calculations on model systems (see above) that suggest that, without considering differential hydrogen-bonding environments, CO2 loss is more exothermic by around 8 kJ mol−1 from oxidized Glu residues than from their Asp counterparts. RNA is less susceptible to electron-density loss than protein within the TRAP–RNA complex    Visual inspection of Fourier difference maps illustrated the clear lack of RNA electron-density degradation with increasing dose compared with the obvious protein damage manifestations (Figs. 3 ▸ b and 3 ▸ c). Only at the highest doses investigated (\u003e20 MGy) was density loss observed at the RNA phosphate and C—O bonds of the phosphodiester backbone. However, the median D loss was lower by a factor of \u003e2 for RNA P atoms than for Glu and Asp side-chain groups at 25.0 MGy (Supplementary Fig. S4), and furthermore could not be numerically distinguished from Gly Cα atoms within TRAP, which are not radiation-sensitive at the doses tested here (Supplementary Fig. S3). RNA binding protects radiation-sensitive residues    For the large number of acidic residues per TRAP ring (four Asp and six Glu residues per protein monomer), a strong dependence of decarboxylation susceptibility on local environment was observed (Fig. 4 ▸). For each Glu Cδ or Asp Cγ atom, D loss provided a direct measure of the rate of side-chain carboxyl-group disordering and subsequent decarboxylation. For acidic residues with no differing interactions between nonbound and bound TRAP (Fig. 4 ▸ a), similar damage was apparent between the two rings within the asymmetric unit, as expected. However, TRAP residues directly on the RNA-binding interfaces exhibited greater damage accumulation in nonbound TRAP (Fig. 4 ▸ b), and for residues at the ring–ring interfaces (where crystal contacts were detected) bound TRAP exhibited enhanced SRD accumulation (Fig. 4 ▸ c). Three acidic residues (Glu36, Asp39 and Glu42) are involved in RNA interactions within each of the 11 TRAP ring subunits, and Fig. 5 ▸ shows their density changes with increasing dose. Hotelling’s T-squared test (the multivariate counterpart of Student’s t-test) was used to reject the null hypothesis that the means of the D loss metric were equal for the bound and nonbound groups in Fig. 5 ▸. A significant reduction in D loss is seen for Glu36 in RNA-bound compared with nonbound TRAP, indicative of a lower rate of side-chain decarboxylation (Fig. 5 ▸ a; p = 6.06 × 10−5). For each TRAP ring subunit, the Glu36 side-chain carboxyl group accepts a pair of hydrogen bonds from the two N atoms of the G3 RNA base. In our analysis, Asp39 in the TRAP–(GAGUU)10GAG structure appears to exhibit two distinct hydrogen bonds to the G1 base within each of the 11 TRAP–RNA interfaces, as does Glu36 to G3; however, the reduction in density disordering upon RNA binding is far less significant for Asp39 than for Glu36 (Fig. 5 ▸ b, p = 0.0925). RNA binding reduces radiation-induced disorder on the atomic scale    One oxygen (O∊1) of Glu42 appears to form a hydrogen bond to a nearby water within each TRAP RNA-binding pocket, with the other (O∊2) being involved in a salt-bridge interaction with Arg58 (Hopcroft et al., 2002; Antson et al., 1999). Salt-bridge interactions have previously been suggested to reduce the glutamate decarboxylation rate within the large (∼62.4 kDa) myrosinase protein structure (Burmeister, 2000). A significant difference was observed between the D loss dynamics for the nonbound/bound Glu42 O∊1 atoms (Fig. 5 ▸ c; p = 0.007) but not for the Glu42 O∊2 atoms (Fig. 5 ▸ d; p = 0.239), indicating that the stabilizing strength of this salt-bridge interaction was conserved upon RNA binding and that the water-mediated hydrogen bond had a greater relative susceptibility to atomic disordering in the absence of RNA. The density-change dynamics were statistically indistinguishable between bound and nonbound TRAP for each Glu42 carboxyl group Cδ atom (p = 0.435), indicating that upon RNA binding the conserved salt-bridge interaction ultimately dictated the overall Glu42 decarboxylation rate. The RNA-stabilizing effect was not restricted to radiation-sensitive acidic residues. The side chain of Phe32 stacks against the G3 base within the 11 TRAP RNA-binding interfaces (Antson et al., 1999). With increasing dose, the D loss associated with the Phe32 side chain was significantly reduced upon RNA binding (Fig. 5 ▸ e; Phe32 Cζ; p = 0.0014), an indication that radiation-induced conformation disordering of Phe32 had been reduced. The extended aliphatic Lys37 side chain stacks against the nearby G1 base, making a series of nonpolar contacts within each RNA-binding interface. The D loss for Lys37 side-chain atoms was also reduced when stacked against the G1 base (Fig. 5 ▸ f; p = 0.0243 for Lys37 C∊ atoms). Representative Phe32 and Lys37 atoms were selected to illustrate these trends. Discussion    Here, MX radiation-induced specific structural changes within the large TRAP–RNA assembly over a large dose range (1.3–25.0 MGy) have been analysed using a high-throughput quantitative approach, providing a measure of the electron-density distribution for each refined atom with increasing dose, D loss. Compared with previous studies, the results provide a further step in the detailed characterization of SRD effects in MX. Our method­ology, which eliminated tedious and error-prone visual inspection, permitted the determination on a per-atom basis of the most damaged sites, as characterized by F obs(d n) − F obs(d 1) Fourier difference map peaks between successive data sets collected from the same crystal. Here, it provided the precision required to quantify the role of RNA in the damage susceptibilities of equivalent atoms between RNA-bound and nonbound TRAP, but it is applicable to any MX SRD study. The RNA was found to be substantially more radiation-resistant than the protein, even at the highest doses investigated (∼25.0 MGy), which is in strong concurrence with our previous SRD investigation of the C.Esp1396I protein–DNA complex (Bury et al., 2015). Consistent with that study, at high doses of above ∼20 MGy, F obs(d n) − F obs(d 1) map density was detected around P, O3′ and O5′ atoms of the RNA backbone, with no significant difference density localized to RNA ribose and basic subunits. RNA backbone disordering thus appears to be the main radiation-induced effect in RNA, with the protein–base interactions maintained even at high doses (\u003e20 MGy). The U4 phosphate exhibited marginally larger D loss values above 20 MGy than G1, A2 and G3 (Supplementary Fig. S4). Since U4 is the only refined nucleotide not to exhibit significant base–protein interactions around TRAP (with a water-mediated hydrogen bond detected in only three of the 11 subunits and a single Arg58 hydrogen bond suggested in a further four subunits), this increased U4 D loss can be explained owing to its greater flexibility. At 25.0 MGy, the magnitude of the RNA backbone D loss was of the same order as for the radiation-insensitive Gly Cα atoms and on average less than half that of the acidic residues of the protein (Supplementary Fig. S3). Consequently, no clear single-strand breaks could be located, and since RNA-binding within the current TRAP–(GAGUU)10GAG complex is mediated predominantly through base–protein interactions, the biological integrity of the RNA complex was dictated by the rate at which protein decarboxylation occurred. RNA interacting with TRAP was shown to offer significant protection against radiation-induced structural changes. Both Glu36 and Asp39 bind directly to RNA, each through two hydrogen bonds to guanine bases (G3 and G1, respectively). However, compared with Asp39, Glu36 is strikingly less decarboxylated when bound to RNA (Fig. 4 ▸). This is in good agreement with previous mutagenesis and nucleoside analogue studies (Elliott et al., 2001), which indicated that the G1 nucleotide does not bind to TRAP as strongly as do A2 and G3, and plays little role in the high RNA-binding affinity of TRAP (K d ≃ 1.1 ± 0.4 nM). For Glu36 and Asp39, no direct quantitative correlation could be established between hydrogen-bond length and D loss (linear R 2 of \u003c0.23 for all doses; Supplementary Fig. S5). Thus, another factor must be responsible for this clear reduction in Glu36 CO2 decarboxyl­ation in RNA-bound TRAP. The Glu36 carboxyl side chain also potentially forms hydrogen bonds to His34 and Lys56, but since these interactions are conserved irrespective of G3 nucleotide binding, this cannot directly account for the stabilization effect on Glu36 in RNA-bound TRAP. Radiation-induced decarboxylation has been proposed to be mediated by preferential positive-hole migration to the side-chain carboxyl group, with rapid proton transfer trapping the hole at the carboxyl group (Burmeister, 2000; Symons, 1997):where the forward rate is K 1 and the backward rate is K −1, where the forward rate is K 2. When bound to RNA, the average solvent-accessible area of the Glu36 side-chain O atoms is reduced from ∼15 to 0 Å2. We propose that with no solvent accessibility Glu36 decarboxylation is inhibited, since the CO2-formation rate K 2 is greatly reduced, and suggest that steric hindrance prevents each radicalized Glu36 CO2 group from achieving the planar conformation required for complete dissociation from TRAP. The electron-recombination rate K −1 remains high, however, owing to rapid electron migration through the protein–RNA complex to refill the Glu36 positive hole (the precursor for Glu decarboxylation). Upon RNA binding, the Asp39 side-chain carboxyl group solvent-accessible area changes from ∼75 to 35 Å2, still allowing a high CO2-formation rate K 2. Previous studies have reported inconsistent results concerning the dependence of the acidic residue decarboxylation rate on solvent accessibility (Weik et al., 2000; Fioravanti et al., 2007; Gerstel et al., 2015). The prevalence of radical attack from solvent channels surrounding the protein in the crystal is a questionable cause, considering previous observations indicating that the strongly oxidizing hydroxyl radical is immobile at 100 K (Allan et al., 2013; Owen et al., 2012). Furthermore, the suggested electron hole-trapping mechanism which induces decarboxylation within proteins at 100 K has no clear mechanistic dependence on the solvent-accessible area of each carboxyl group. By comparing equivalent acidic residues with and without RNA, we have now deconvoluted the role of solvent accessibility from other local protein environment factors, and thus propose a suitable mechanism by which exceptionally low solvent accessibility can reduce the rate of decarboxylation. Overall, no direct correlation between solvent accessibility and decarboxylation susceptibility was observed, but it is very clear that inaccessible residues are protected. Apart from these RNA-binding interfaces, RNA binding was seen to enhance decarboxylation for residues Glu50, Glu71 and Glu73, all of which are involved in crystal contacts between TRAP rings (Fig. 4 ▸ c). However, for each of these residues the exact crystal contacts are not preserved between bound and nonbound TRAP or even between monomers within one TRAP ring. For example, in bound TRAP, Glu73 hydrogen-bonds to a nearby lysine on each of the 11 subunits, whereas in nonbound TRAP no such interaction exists and Glu73 interacts with a variable number of refined waters in each subunit. Thus, the dependence of decarboxylation rates on these interactions could not be established. Radiation-induced side-chain conformational changes have been poorly characterized in previous SRD investigations owing to their strong dependence on packing density and geometric strain. Such structural changes are known to have significant roles within enzymatic pathways, and experimenters must be aware of these possible confounding factors when assigning true functional mechanisms using MX. Our results show that RNA binding to TRAP physically stabilizes non-acidic residues within the TRAP macromolecule, most notably Lys37 and Phe32, which stack against the G1 and G3 bases, respectively. It has been suggested (Burmeister, 2000) that Tyr residues can lose their aromatic –OH group owing to radiation-induced effects; however, no energetically favourable pathway for –OH cleavage exists and this has not been detected in aqueous radiation-chemistry studies. In TRAP, D loss increased at a similar rate for both the Tyr O atoms and aromatic ring atoms, suggesting that full ring conformational disordering is more likely. Indeed, no convincing reproducible Fourier difference peaks above the background map noise were observed around any Tyr terminal –OH groups. The RNA-stabilization effects on protein are observed at short ranges and are restricted to within the RNA-binding interfaces around the TRAP ring. For example, Asp17 is located ∼6.8 Å from the G1 base, outside the RNA-binding interfaces, and has indistinguishable Cγ atom D loss dose-dynamics between RNA-bound and nonbound TRAP (p \u003e 0.9). An increase in the dose at which functionally important residues remain intact has biological ramifications for understanding the mechanisms at which ionizing radiation damage is mitigated within naturally forming DNA–protein and RNA–protein complexes. Observations of lower protein radiation-sensitivity in DNA-bound forms have been recorded in solution at RT at much lower doses (∼1 kGy) than those used for typical MX experiments [e.g. an oestrogen response element–receptor complex (Stísová et al., 2006) and a DNA glycosylase and its abasic DNA target site (Gillard et al., 2004)]. In these studies, the main damaging species is predicted to be the oxidizing hydroxyl radical produced through solvent irradiation, which is known to add to double covalent bonds within both DNA and RNA bases to induce strand breaks and base modification (Spotheim-Maurizot \u0026 Davídková, 2011; Chance et al., 1997). It was suggested that physical screening of DNA by protein shielded the DNA–protein interaction sites from radical damage, yielding an extended life-dose for the nucleoprotein complex compared with separate protein and DNA constituents at RT. However, in the current MX study at 100 K, the main damaging species are believed to be migrating LEEs and holes produced directly within the protein–RNA components or in closely associated solvent. The results presented here suggest that biologically relevant nucleoprotein complexes also exhibit prolonged life-doses under the effect of LEE-induced structural changes, involving direct physical protection of key RNA-binding residues. Such reduced radiation-sensitivity in this case ensures that the interacting protein remains bound long enough to the RNA to complete its function, even whilst exposed to ionizing radiation. Within the nonbound TRAP macromolecule, the acidic residues within the unoccupied RNA-binding interfaces (Asp39, Glu36, Glu42) are notably amongst the most susceptible residues within the asymmetric unit (Fig. 4 ▸). When exposed to X-rays, these residues will be preferentially damaged by X-rays and subsequently reduce the affinity with which TRAP binds to RNA. Within the cellular environment, this mechanism could reduce the risk that radiation-damaged proteins might bind to RNA, thus avoiding the detrimental introduction of incorrect DNA-repair, transcriptional and base-modification pathways. The Python scripts written to calculate the per atom D loss metric are available from the authors on request. Related literature    The following references are cited in the Supporting Information for this article: Chen et al. (2010). Supplementary Material References Adams, P. D. et al. (2010). Acta Cryst. D66, 213–221. Alizadeh, E. \u0026 Sanche, L. (2014). Eur. Phys. J. D, 68, 97. Allan, E. G., Kander, M. C., Carmichael, I. \u0026 Garman, E. F. (2013). J. Synchrotron Rad. 20, 23–36. Antson, A. A., Dodson, E. J., Dodson, G., Greaves, R. B., Chen, X. \u0026 Gollnick, P. (1999). Nature (London), 401, 235–242. Becke, A. D. (1993). J. Chem. Phys. 98, 5648–5652. Bourenkov, G. P. \u0026 Popov, A. N. (2010). Acta Cryst. D66, 409–419. Burmeister, W. P. (2000). Acta Cryst. D56, 328–341. Bury, C., Garman, E. F., Ginn, H. M., Ravelli, R. B. G., Carmichael, I., Kneale, G. \u0026 McGeehan, J. E. (2015). J. Synchrotron Rad. 22, 213–224. Chance, M. R., Sclavi, B., Woodson, S. A. \u0026 Brenowitz, M. (1997). Structure, 5, 865–869. Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S. \u0026 Richardson, D. C. (2010). Acta Cryst. D66, 12–21. Dubnovitsky, A. P., Ravelli, R. B. G., Popov, A. N. \u0026 Papageorgiou, A. C. (2005). Protein Sci. 14, 1498–1507. Elliott, M. B., Gottlieb, P. A. \u0026 Gollnick, P. (2001). RNA, 7, 85–93. Emsley, P., Lohkamp, B., Scott, W. G. \u0026 Cowtan, K. (2010). Acta Cryst. D66, 486–501. Evans, P. R. \u0026 Murshudov, G. N. (2013). Acta Cryst. D69, 1204–1214. Fioravanti, E., Vellieux, F. M. D., Amara, P., Madern, D. \u0026 Weik, M. (2007). J. Synchrotron Rad. 14, 84–91. Frisch, M. J. et al. (2009). Gaussian 09. Gaussian Inc., Wallingford, Connecticut, USA. Garman, E. F. (2010). Acta Cryst. D66, 339–351. Gerstel, M., Deane, C. M. \u0026 Garman, E. F. (2015). J. Synchrotron Rad. 22, 201–212. Gillard, N., Begusova, M., Castaing, B. \u0026 Spotheim-Maurizot, M. (2004). Radiat. Res. 162, 566–571. Holton, J. M. (2007). J. Synchrotron Rad. 14, 51–72. Holton, J. M. (2009). J. Synchrotron Rad. 16, 133–142. Hopcroft, N. H., Wendt, A. L., Gollnick, P. \u0026 Antson, A. A. (2002). Acta Cryst. D58, 615–621. Jones, G. D., Lea, J. S., Symons, M. C. \u0026 Taiwo, F. A. (1987). Nature (London), 330, 772–773. Leslie, A. G. W. \u0026 Powell, H. R. (2007). Evolving Methods for Macromolecular Crystallography, edited by R. J. Read \u0026 J. L. Sussman, pp. 41–51. Dordrecht: Springer. Liebschner, D., Rosenbaum, G., Dauter, M. \u0026 Dauter, Z. (2015). Acta Cryst. D71, 772–778. Matsui, Y., Sakai, K., Murakami, M., Shiro, Y., Adachi, S., Okumura, H. \u0026 Kouyama, T. (2002). J. Mol. Biol. 324, 469–481. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. \u0026 Read, R. J. (2007). J. Appl. Cryst. 40, 658–674. McGeehan, J. E., Streeter, S. D., Thresh, S. J., Ball, N., Ravelli, R. B. G. \u0026 Kneale, G. G. (2008). Nucleic Acids Res. 36, 4778–4787. Murray, J. \u0026 Garman, E. (2002). J. Synchrotron Rad. 9, 347–354. Murshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F. \u0026 Vagin, A. A. (2011). Acta Cryst. D67, 355–367. O’Neill, P., Stevens, D. L. \u0026 Garman, E. (2002). J. Synchrotron Rad. 9, 329–332. Owen, R. L., Axford, D., Nettleship, J. E., Owens, R. J., Robinson, J. I., Morgan, A. W., Doré, A. S., Lebon, G., Tate, C. G., Fry, E. E., Ren, J., Stuart, D. I. \u0026 Evans, G. (2012). Acta Cryst. D68, 810–818. Owen, R. L., Rudiño-Piñera, E. \u0026 Garman, E. F. (2006). Proc. Natl Acad. Sci. USA, 103, 4912–4917. Ptasińska, S. \u0026 Sanche, L. (2007). Phys. Rev. E, 75, 031915. Ravelli, R. B. G. \u0026 McSweeney, S. M. (2000). Structure, 8, 315–328. Shimizu, N., Hirata, K., Hasegawa, K., Ueno, G. \u0026 Yamamoto, M. (2007). J. Synchrotron Rad. 14, 4–10. Simons, J. (2006). Acc. Chem. Res. 39, 772–779. Spotheim-Maurizot, M. \u0026 Davídková, M. (2011). Mutat. Res. 711, 41–48. Stísová, V., Goffinont, S., Spotheim-Maurizot, M. \u0026 Davídková, M. (2006). Radiat. Prot. Dosimetry, 122, 106–109. Symons, M. C. R. (1997). Free Radical Biol. Med. 22, 1271–1276. Ten Eyck, L. F. (1973). Acta Cryst. A29, 183–191. Tomasi, J., Mennucci, B. \u0026 Cancès, E. (1999). J. Mol. Struct. 464, 211–226. Weik, M., Ravelli, R. B. G., Kryger, G., McSweeney, S., Raves, M. L., Harel, M., Gros, P., Silman, I., Kroon, J. \u0026 Sussman, J. L. (2000). Proc. Natl Acad. Sci. USA, 97, 623–628. Winn, M. D. et al. (2011). Acta Cryst. D67, 235–242. Yano, J., Kern, J., Irrgang, K. D., Latimer, M. J., Bergmann, U., Glatzel, P., Pushkar, Y., Biesiadka, J., Loll, B., Sauer, K., Messinger, J., Zouni, A. \u0026 Yachandra, V. K. (2005). Proc. Natl Acad. Sci. USA, 102, 12047–12052. Zeldin, O. B., Brockhauser, S., Bremridge, J., Holton, J. M. \u0026 Garman, E. F. (2013). Proc. Natl Acad. Sci. USA, 110, 20551–20556. Zeldin, O. B., Gerstel, M. \u0026 Garman, E. F. (2013). J. Appl. Cryst. 46, 1225–1230. The TRAP–(GAGUU)10GAG complex asymmetric unit (PDB entry 1gtf; Hopcroft et al., 2002). Bound tryptophan ligands are represented as coloured spheres. RNA is shown is yellow. (a) Electron-density loss sites as indicated by D\nloss in the TRAP–RNA complex crystal by residue/nucleotide type for five doses [sites determined above the 4× average D\nloss threshold, calculated over the TRAP–RNA structure for the first difference map: F\nobs(d\n2) − F\nobs(d\n1)]. Cumulative frequencies are normalized to both the total number of non-H atoms per residue/nucleotide and the total number of that residue/nucleotide type present. (b) Average D\nloss for each residue/nucleotide type with respect to the DWD (diffraction-weighted dose; Zeldin, Brock­hauser et al., 2013). 95% confidence intervals (CI) are shown. Only a subset of key TRAP residue types are included. The average D\nloss (calculated over the whole TRAP asymmetric unit) is shown at each dose (dashed line). \nF\nobs(d\nn) − F\nobs(d\n1) Fourier difference maps for (a) n = 2 (3.9 MGy), (b) n = 3 (6.5 MGy) and (c) n = 7 (16.7 MGy) contoured at ±4σ (a) and ±3.5σ (b, c). In (a) clear difference density is observed around the Glu42 carboxyl side chain in chain H, within the lowest dose difference map at d\n2 = 3.9 MGy. Radiation-induced protein disordering is evident across the large dose range (b, c); in comparison, no clear deterioration of the RNA density was observed. \nD\nloss calculated for all side-chain carboxyl group Glu Cδ and Asp Cγ atoms within the TRAP–RNA complex for a dose of 19.3 MGy (d\n8). Residues have been grouped by amino-acid number, and split into bound and nonbound groupings, with each bar representing the mean calculated over 11 equivalent atoms around a TRAP ring. Whiskers indicate 95% CI. The D\nloss behaviour shown here was consistently exhibited across the entire investigated dose range. \nD\nloss against dose for (a) Glu36 Cδ, (b) Asp39 Cγ, (c) Glu42 O∊1, (d) Glu42 O∊2, (e) Phe32 Cζ and (f) Lys37 C∊ atoms. 95% CI are included for each set of 11 equivalent atoms grouped as bound/nonbound. RNA-binding interface interactions are shown for TRAP chain N, with the F\nobs(d\n7) − F\nobs(d\n1) Fourier difference map (dose 16.7 MGy) overlaid and contoured at a ±4σ 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diff --git a/annotated_BioC_JSON/PMC4871749_ann.json b/annotated_BioC_JSON/PMC4871749_ann.json
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index 0000000000000000000000000000000000000000..718cfc3320cab5924206f9cf7a6ec34bdb45669a
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+[{"sourceid":"4871749","sourcedb":"","project":"","target":"","text":"The Taf14 YEATS domain is a reader of histone crotonylation The discovery of new histone modifications is unfolding at startling rates, however, the identification of effectors capable of interpreting these modifications has lagged behind. Here we report the YEATS domain as an effective reader of histone lysine crotonylation – an epigenetic signature associated with active transcription. We show that the Taf14 YEATS domain engages crotonyllysine via a unique π-π-π-stacking mechanism and that other YEATS domains have crotonyllysine binding activity. Crotonylation of lysine residues (crotonyllysine, Kcr) has emerged as one of the fundamental histone post-translational modifications (PTMs) found in mammalian chromatin. This epigenetic PTM is widespread and enriched at active gene promoters and potentially enhancers. The crotonyllysine mark on histone H3K18 is produced by p300, a histone acetyltransferase also responsible for acetylation of histones. Owing to some differences in their genomic distribution, the crotonyllysine and acetyllysine (Kac) modifications have been linked to distinct functional outcomes. p300-catalyzed histone crotonylation, which is likely metabolically regulated, stimulates transcription to a greater degree than p300-catalyzed acetylation. The discovery of individual biological roles for the crotonyllysine and acetyllysine marks suggests that these PTMs can be read by distinct readers. While a number of acetyllysine readers have been identified and characterized, a specific reader of the crotonyllysine mark remains unknown (reviewed in). A recent survey of bromodomains (BDs) demonstrates that only one BD associates very weakly with a crotonylated peptide, however it binds more tightly to acetylated peptides, inferring that bromodomains do not possess physiologically relevant crotonyllysine binding activity. The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. The acetyllysine binding function of the AF9 YEATS domain is essential for the recruitment of the histone methyltransferase DOT1L to H3K9ac-containing chromatin and for DOT1L-mediated H3K79 methylation and transcription. Similarly, activation of a subset of genes and DNA damage repair in yeast require the acetyllysine binding activity of the Taf14 YEATS domain. Consistent with its role in gene regulation, Taf14 was identified as a core component of the transcription factor complexes TFIID and TFIIF. However, Taf14 is also found in a number of chromatin-remodeling complexes (i.e., INO80, SWI/SNF and RSC) and the histone acetyltransferase complex NuA3, indicating a multifaceted role of Taf14 in transcriptional regulation and chromatin biology. In this study, we identified the Taf14 YEATS domain as a reader of crotonyllysine that binds to histone H3 crotonylated at lysine 9 (H3K9cr) via a distinctive binding mechanism. We found that H3K9cr is present in yeast and is dynamically regulated. To elucidate the molecular basis for recognition of the H3K9cr mark, we obtained a crystal structure of the Taf14 YEATS domain in complex with H3K9cr5-13 (residues 5–13 of H3) peptide (Fig. 1, Supplementary Results, Supplementary Fig. 1 and Supplementary Table 1). The Taf14 YEATS domain adopts an immunoglobin-like β sandwich fold containing eight anti-parallel β strands linked by short loops that form a binding site for H3K9cr (Fig. 1b). The H3K9cr peptide lays in an extended conformation in an orientation orthogonal to the β strands and is stabilized through an extensive network of direct and water-mediated hydrogen bonds and a salt bridge (Fig. 1c). The most striking feature of the crotonyllysine recognition mechanism is the unique coordination of crotonylated lysine residue. The fully extended side chain of K9cr transverses the narrow tunnel, crossing the β sandwich at right angle in a corkscrew-like manner (Fig. 1b and Supplementary Figure 1b). The planar crotonyl group is inserted between Trp81 and Phe62 of the protein, the aromatic rings of which are positioned strictly parallel to each other and at equal distance from the crotonyl group, yielding a novel aromatic-amide/aliphatic-aromatic π-π-π-stacking system that, to our knowledge, has not been reported previously for any protein-protein interaction (Fig. 1d and Supplementary Fig. 1c). The side chain of Trp81 appears to adopt two conformations, one of which provides maximum π-stacking with the alkene functional group while the other rotamer affords maximum π-stacking with the amide π electrons (Supplementary Fig. 1c). The dual conformation of Trp81 is likely due to the conjugated nature of the C=C and C=O π-orbitals within the crotonyl functional group. In addition to π-π-π stacking, the crotonyl group is stabilized by a set of hydrogen bonds and electrostatic interactions. The π bond conjugation of the crotonyl group gives rise to a dipole moment of the alkene moiety, resulting in a partial positive charge on the β-carbon (Cβ) and a partial negative charge on the α-carbon (Cα). This provides the capability for the alkene moiety to form electrostatic contacts, as Cα and Cβ lay within electrostatic interaction distances of the carbonyl oxygen of Gln79 and of the hydroxyl group of Thr61, respectively. The hydroxyl group of Thr61 also participates in a hydrogen bond with the amide nitrogen of the K9cr side chain (Fig. 1d). The fixed position of the Thr61 hydroxyl group, which facilitates interactions with both the amide and Cα of K9cr, is achieved through a hydrogen bond with imidazole ring of His59. Extra stabilization of K9cr is attained by a hydrogen bond formed between its carbonyl oxygen and the backbone nitrogen of Trp81, as well as a water-mediated hydrogen bond with the backbone carbonyl group of Gly82 (Fig 1d). This distinctive mechanism was corroborated through mapping the Taf14 YEATS-H3K9cr binding interface in solution using NMR chemical shift perturbation analysis (Supplementary Fig. 2a, b). Binding of the Taf14 YEATS domain to H3K9cr is robust. The dissociation constant (Kd) for the Taf14 YEATS-H3K9cr5-13 complex was found to be 9.5 μM, as measured by fluorescence spectroscopy (Supplementary Fig. 2c). This value is in the range of binding affinities exhibited by the majority of histone readers, thus attesting to the physiological relevance of the H3K9cr recognition by Taf14. To determine whether H3K9cr is present in yeast, we generated whole cell extracts from logarithmically growing yeast cells and subjected them to Western blot analysis using antibodies directed towards H3K9cr, H3K9ac and H3 (Fig. 2a, b, Supplementary Fig. 3 and Supplementary Table 2). Both H3K9cr and H3K9ac were detected in yeast histones; to our knowledge, this is the first report of H3K9cr occurring in yeast. We next asked if H3K9cr is regulated by the actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Towards this end, we probed extracts derived from yeast cells in which major yeast HATs (HAT1, Gcn5, and Rtt109) or HDACs (Rpd3, Hos1, and Hos2) were deleted. As shown in Figure 2a, b and Supplementary Fig. 3e, H3K9cr levels were abolished or reduced considerably in the HAT deletion strains, whereas they were dramatically increased in the HDAC deletion strains. Furthermore, fluctuations in the H3K9cr levels were more substantial than fluctuations in the corresponding H3K9ac levels. Together, these results reveal that H3K9cr is a dynamic mark of chromatin in yeast and suggest an important role for this modification in transcription as it is regulated by HATs and HDACs. We have previously shown that among acetylated histone marks, the Taf14 YEATS domain prefers acetylated H3K9 (also see Supplementary Fig. 3b), however it binds to H3K9cr tighter. The selectivity of Taf14 towards crotonyllysine was substantiated by 1H,15N HSQC experiments, in which either H3K9cr5-13 or H3K9ac5-13 peptide was titrated into the 15N-labeled Taf14 YEATS domain (Fig. 2c and Supplementary Fig. 4a, b). Binding of H3K9cr induced resonance changes in slow exchange regime on the NMR time scale, indicative of strong interaction. In contrast, binding of H3K9ac resulted in an intermediate exchange, which is characteristic of a weaker association. Furthermore, crosspeaks of Gly80 and Trp81 of the YEATS domain were uniquely perturbed by H3K9cr and H3K9ac, indicating a different chemical environment in the respective crotonyllysine and acetyllysine binding pockets (Supplementary Fig. 4a). These differences support our model that Trp81 adopts two conformations upon complex formation with the H3K9cr mark as compared to H3K9ac (Supplementary Figs. 1c, d and 4c). One of the conformations, characterized by the π stacking involving two aromatic residues and the alkene group, is observed only in the YEATS-H3K9cr complex. To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1–20 of H3). As shown in Figure 2d and Supplementary Fig. 5a, the Taf14 YEATS domain binds more strongly to H3K9cr1-20, as compared to other acylated histone peptides. The preference for H3K9cr over H3K9ac, H3K9pr and H3K9bu was supported by 1H,15N HSQC titration experiments. Addition of H3K9ac1-20, H3K9pr1-20, and H3K9bu1-20 peptides caused chemical shift perturbations in the Taf14 YEATS domain in intermediate exchange regime, implying that these interactions are weaker compared to the interaction with the H3K9cr1-20 peptide (Supplementary Fig. 5b). We concluded that H3K9cr is the preferred target of this domain. From comparative structural analysis of the YEATS complexes, Gly80 emerged as candidate residue potentially responsible for the preference for crotonyllysine. In attempt to generate a mutant capable of accommodating a short acetyl moiety but discriminating against a longer, planar crotonyl moiety, we mutated Gly80 to more bulky residues, however all mutants of Gly80 lost their binding activities towards either acylated peptide, suggesting that Gly80 is absolutely required for the interaction. In contrast, mutation of Val24, a residue located on another side of Trp81, had no effect on binding (Fig. 2d and Supplementary Fig. 5a, c). To determine if the binding to crotonyllysine is conserved, we tested human YEATS domains by pull-down experiments using singly and multiply acetylated, propionylated, butyrylated, and crotonylated histone peptides (Supplementary Fig. 6). We found that all YEATS domains tested are capable of binding to crotonyllysine peptides, though they display variable preferences for the acyl moieties. While YEATS2 and ENL showed selectivity for the crotonylated peptides, GAS41 and AF9 bound acylated peptides almost equally well. Unlike the YEATS domain, a known acetyllysine reader, bromodomain, does not recognize crotonyllysine. We assayed a large set of BDs in pull-down experiments and found that this module is highly specific for acetyllysine and propionyllysine containing peptides (Supplementary Fig. 7). However, bromodomains did not interact (or associated very weakly) with longer acyl modifications, including crotonyllysine, as in the case of BDs of TAF1 and BRD2, supporting recent reports. These results demonstrate that the YEATS domain is currently the sole reader of crotonyllysine. In conclusion, we have identified the YEATS domain of Taf14 as the first reader of histone crotonylation. The unique and previously unobserved aromatic-amide/aliphatic-aromatic π-π-π-stacking mechanism facilitates the specific recognition of the crotonyl moiety. We further demonstrate that H3K9cr exists in yeast and is dynamically regulated by HATs and HDACs. As we previously showed the importance of acyllysine binding by the Taf14 YEATS domain for the DNA damage response and gene transcription, it will be essential in the future to define the physiological role of crotonyllysine recognition and to differentiate the activities of Taf14 that are due to binding to crotonyllysine and acetyllysine modifications. Furthermore, the functional significance of crotonyllysine recognition by other YEATS proteins will be of great importance to elucidate and compare. ONLINE METHODS Protein expression and purification The Taf14 YEATS constructs (residues 1–132 or 1–137) were expressed in E. coli BL21 (DE3) RIL in either Luria Broth or M19 minimal media supplemented with 15NH4Cl and purified as N-terminal GST fusion proteins. Cells were harvested by centrifugation and resuspended in 50 mM HEPES (pH 7.5) supplemented with 150 mM NaCl and 1 mM TCEP. Cells are lysed by freeze-thaw followed by sonication. Proteins were purified on glutathione Sepharose 4B beads and the GST tag was cleaved with PreScission protease. X-ray data collection and structure determination Taf14 YEATS (residues 1–137) was concentrated to 9 mg/mL in 25 mM MES (pH 6.5) and incubated with 2 molar equivalence of the H3K9cr5-13 at RT for 30 mins prior to crystallization. Crystals were obtain via sitting drop diffusion method at 18°C by mixing 800 nL of protein/peptide solution with 800 nL of well solution composed of 44% PEG600 (v/v) and 0.2 M citric acid (pH 6.0). X-ray diffraction data was collected at a wavelength of 1.54 Å at 100 K from a single crystal on the UC Denver Biophysical Core home source composed of a Rigaku Micromax 007 high frequency microfocus X-ray generator with a Pilatus 200K 2D area detector. HKL3000 was used for indexing, scaling, and data reduction. Solution was solved via molecular replacement with Phaser using the Taf14 YEATS domain (PDB 5D7E) as search model with waters, ligands, and peptide removed. Phenix was used for refinement of structure and waters were manually placed by inception of difference maps in Coot. Ramachandran plot indicates good stereochemistry of the three-dimensional structure with 100% of all residues falling within the favored (98%) and allowed (2%) regions. The crystallographic statistics are shown in Supplementary Table 1. NMR spectroscopy NMR spectroscopy was carried out on a Varian INOVA 600 MHz spectrometer outfitted with a cryogenic probe. Chemical shift perturbation (CSP) analysis was performed using uniformly 15N-labeled Taf14 (1–132). 1H,15N heteronuclear single quantum coherence (HSQC) spectra of the Taf14 YEATS domain were collected in the presence of increasing concentrations of either H3K9cr5-13, H3K9ac5-13, H3K9cr1-20, H3K9ac1-20 H3K9pr1-20, H3K9bu1-20 or free Kcr in PBS buffer pH 6.8, 8% D2O. Fluorescence binding assays Tryptophan fluorescence measurements were performed on a Fluorolog spectrofluorometer at room temperature as described. The samples containing 2 μM of Taf14 YEATS in PBS (pH 7.4) and increasing concentrations of H3K9cr5-13 were excited at 295 nm. Emission spectra were recorded from 310 to 340 nm with a 1 nm step size and a 0.5 sec integration time. The Kd value was determined using a nonlinear least-squares analysis and the equation:   where [L] is the concentration of the peptide, [P] is the concentration of the protein, ΔI is the observed change of signal intensity, and ΔImax is the difference in signal intensity of the free and bound states. The Kd values were averaged over 3 separate experiments, with error calculated as the standard deviation (SD). Peptide pull-downs YEATS domains in pGEX vectors were expressed in SoluBL21 cells (Amsbio) by induction with 1 mM IPTG at 16–18°C overnight with shaking. Cells were lysed by freeze-thaw and sonication then purified over glutathione agarose (Pierce) in a buffer containing 50 mM Tris pH 8.0, 500 mM NaCl, 20% glycerol (v/v) and 1 mM dithiothreitol (DTT). Peptide pull-downs were performed essentially as described except that the assay buffer contained 50 mM Tris pH 8.0, 500 mM NaCl, and 0.1% NP-40, and 500 pmols of biotinylated histone peptides were loaded onto streptavidin coated magnetic beads before incubation with 40 pmols of protein. Bound proteins were detected with rabbit GST antibody (Sigma, G7781). Point mutants were generated by site-directed mutagenesis and purified/assayed as described above. The YEATS domains of Taf14, AF9, ENL, and GAS41 were previously described. Western blotting Yeast cultures were grown in YPD media at 30°C to mid-log phase and extracts were prepared as previously described. Proteins from cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane. Anti-H3K9ac (Millipore, 07-352) and anti-H3K9cr (PTM Biolabs, PTM-516) were diluted to 1:2000 and 1:1000, respectively, in 1x Superblock (ThermoScientific). An HRP-conjugated anti-rabbit (GE Healthcare) was used for detection. Bands were quantified using the ImageJ program. Dot blotting Increasing concentrations of biotinylated histone peptides (0.06–1.5 μg) were spotted onto a PVDF membrane then probed with the anti-H3K9ac (Millipore, 07-352) or H3K9cr (PTM Biolabs, PTM-516) at 1:2000 in a 5% non-fat milk solution and detected with an HRP-conjugated anti-rabbit by enhanced chemiluminesence (ECL). Bromodomains pull-downs cDNAs of GST-fused bromodomains were obtained either from EpiCypher Inc. or as a kind gift from Katrin Chua (Stanford University). GST fusions were expressed as described above except that the preparation buffer contained 50 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol (v/v), and 1 mM DTT. Pull-down assays were preformed as described above except that the assay buffer contained 50 mM Tris (pH 8.0), 300 mM NaCl, and 0.1% NP-40. Supplementary Material Accession codes. Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5IOK. Author contributions F.H.A., S.A.S., E.K.S., J.B.B., A.G., I.K.T and K.K. performed experiments and together with X.S., B.D.S and T.G.K. analyzed the data. F.H.A., S.A.S., B.D.S. and T.G.K. wrote the manuscript with input from all authors. Competing Financial Interest The authors declare no competing financial interests. Additional information Any supplementary information is available in the online version of this paper. Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification Intracellular Crotonyl-CoA Stimulates Transcription through p300-Catalyzed Histone Crotonylation Protein lysine acylation and cysteine succination by intermediates of energy metabolism Identification of ‘erasers’ for lysine crotonylated histone marks using a chemical proteomics approach Perceiving the epigenetic landscape through histone readers Interpreting the language of histone and DNA modifications A Subset of Human Bromodomains Recognizes Butyryllysine and Crotonyllysine Histone Peptide Modifications Histone recognition and large-scale structural analysis of the human bromodomain family YEATS domain proteins: a diverse family with many links to chromatin modification and transcription AF9 YEATS domain links histone acetylation to DOT1L-mediated H3K79 methylation Association of Taf14 with acetylated histone H3 directs gene transcription and the DNA damage response Anc1 interacts with the catalytic subunits of the general transcription factors TFIID and TFIIF, the chromatin remodeling complexes RSC and INO80, and the histone acetyltransferase complex NuA3 Preparation and analysis of the INO80 complex TFG/TAF30/ANC1, a component of the yeast SWI/SNF complex that is similar to the leukemogenic proteins ENL and AF-9 The something about silencing protein, Sas3, is the catalytic subunit of NuA3, a yTAF(II)30-containing HAT complex that interacts with the Spt16 subunit of the yeast CP (Cdc68/Pob3)-FACT complex The essential role of acetyllysine binding by the YEATS domain in transcriptional regulation Phaser crystallographic software PHENIX: a comprehensive Python-based system for macromolecular structure solution Features and development of Coot Molecular basis for chromatin binding and regulation of MLL5 Association of UHRF1 with methylated H3K9 directs the maintenance of DNA methylation Association of Taf14 with acetylated histone H3 directs gene transcription and the DNA damage response The Saccharomyces cerevisiae histone H2A variant Htz1 is acetylated by NuA4 A phosphatase complex that dephosphorylates gammaH2AX regulates DNA damage checkpoint recovery The structural mechanism for the recognition of H3K9cr (a) Chemical structure of crotonyllysine. (b) The crystal structure of the Taf14 YEATS domain (wheat) in complex with the H3K9cr5-13 peptide (green). (c) H3K9cr is stabilized via an extensive network of intermolecular electrostatic and polar interactions with the Taf14 YEATS domain. (d) The π-π-π stacking mechanism involving the alkene moiety of crotonyllysine. H3K9cr is a selective target of the Taf14 YEATS domain (a, b) Western blot analysis comparing the levels of H3K9cr and H3K9ac in wild type (WT), HAT deletion, or HDAC deletion yeast strains. Total H3 was used as a loading control. (c) Superimposed 1H,15N HSQC spectra of Taf14 YEATS recorded as H3K9cr5-13 and H3K9ac5-13 peptides were titrated in. Spectra are color coded according to the protein:peptide molar ratio. (d) Western blot analyses of peptide pull-down assays using wild-type and mutated Taf14 YEATS domains and indicated 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+[{"sourceid":"4872110","sourcedb":"","project":"","target":"","text":"Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive. Here we identify the cytoplasmic ribosome biogenesis protein Tsr3 as the responsible enzyme in yeast and human cells. In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation. The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode. This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate. Structurally, Tsr3 therefore represents a novel class of acp transferase enzymes. INTRODUCTION Eukaryotic ribosome biogenesis is highly complex and requires a large number of non-ribosomal proteins and small non-coding RNAs in addition to ribosomal RNAs (rRNAs) and proteins. An increasing number of diseases—so called ribosomopathies—are associated with disturbed ribosome biogenesis. During eukaryotic ribosome biogenesis several dozens of rRNA nucleotides become chemically modified. The most abundant rRNA modifications are methylations at the 2′-OH ribose moieties and isomerizations of uridine residues to pseudouridine, catalyzed by small nucleolar ribonucleoprotein particles (snoRNPs). In addition, 18S and 25S (yeast)/ 28S (humans) rRNAs contain several base modifications catalyzed by site-specific and snoRNA-independent enzymes. In Saccharomyces cerevisiae 18S rRNA contains four base methylations, two acetylations and a single 3-amino-3-carboxypropyl (acp) modification, whereas six base methylations are present in the 25S rRNA. While in humans the 18S rRNA base modifications are highly conserved, only three of the yeast base modifications catalyzed by ScRrp8/HsNML, ScRcm1/HsNSUN5 and ScNop2/HsNSUN1 are preserved in the corresponding human 28S rRNA. Ribosomal RNA modifications have been suggested to optimize ribosome function, although in most cases this remains to be clearly established. They might contribute to increased RNA stability by providing additional hydrogen bonds (pseudouridines), improved base stacking (pseudouridines and base methylations) or an increased resistance against hydrolysis (ribose methylations). Most modified rRNA nucleotides cluster in the vicinity of the decoding or the peptidyl transferase center, suggesting an influence on ribosome functionality and stability. Defects of rRNA modification enzymes often lead to disturbed ribosome biogenesis or functionally impaired ribosomes, although the lack of individual rRNA modifications often has no or only a slight influence on the cell. Importantly, malfunctions of several base modifying enzymes are linked to developmental diseases, aging or tumorigenesis. The chemically most complex modification is located in the loop capping helix 31 of 18S rRNA (Supplementary Figure S1B). There a uridine (U1191 in yeast) is modified to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ, Figure 1A). This base modification was first described in 1968 for hamster cells and is conserved in eukaryotes. This hypermodified nucleotide, which is located at the P-site tRNA, is synthesized in three steps beginning with the snR35 H/ACA snoRNP guided conversion of uridine into pseudouridine. In a second step, the essential SPOUT-class methyltransferase Nep1/Emg1 modifies the pseudouridine to N1-methylpseudouridine. Methylation can only occur once pseudouridylation has taken place, as the latter reaction generates the substrate for the former. The final acp modification leading to N1-methyl-N3-aminocarboxypropyl-pseudouridine occurs late during 40S biogenesis in the cytoplasm, while the two former reactions are taking place in the nucleolus and nucleus, and is independent from pseudouridylation or methylation. Both the methyl and the acp group are derived from S-adenosylmethionine (SAM), but the enzyme responsible for acp modification remained elusive for more than 40 years. Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3Ψ is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3. The asterisk indicates the C1-atom labeled in the 14C-incorporation assay. (B) RP-HPLC elution profile of yeast 18S rRNA nucleosides. Hypermodified m1acp3Ψ elutes at 7.4 min (wild type, left profile) and is missing in Δtsr3 (middle profile) and Δnep1 Δnop6 mutants (right profile). (C) 14C-acp labeling of 18S rRNAs. Wild type (WT) and plasmid encoded 18S rRNA (U1191U) show the 14C-acp signal, whereas the 14C-acp signal is missing in the U1191A mutant plasmid encoded 18S rRNA (U1191A) and Δtsr3 mutants (Δtsr3). Upper lanes show the ethidium bromide staining of the 18S rRNAs for quantification. All samples were loaded on the gel with two different amounts of 5 and 10 μl. (D) Primer extension analysis of acp modification in yeast 18S rRNA (right gel) including a sequencing ladder (left gel). The primer extension stop at nucleotide 1191 is missing exclusively in Δtsr3 mutants and Δtsr3 Δsnr35 recombinants. (E) Primer extension analysis of human 18S rRNA after siRNA knockdown of HsNEP1/EMG1 (541, 542 and 543) and HsTSR3 (544 and 545) (right gel), including a sequencing ladder (left gel). The primer extension arrest is reduced in HTC116 cells transfected with siRNAs 544 and 545. The efficiency of siRNA mediated HsTSR3 repression correlates with the primer extension signals (see Supplementary Figure S2A). As a loading control, a structural stop is shown (asterisks). Only a few acp transferring enzymes have been characterized until now. During the biosynthesis of wybutosine, a tricyclic nucleoside present in eukaryotic and archaeal phenylalanine tRNA, Tyw2 (Trm12 in yeast) transfers an acp group from SAM to an acidic carbon atom. Archaeal Tyw2 has a structure very similar to Rossmann-fold (class I) RNA-methyltransferases, but its distinctive SAM-binding mode enables the transfer of the acp group instead of the methyl group of the cofactor. Another acp modification has been described in the diphtamide biosynthesis pathway, where an acp group is transferred from SAM to the carbon atom of a histidine residue of eukaryotic translation elongation factor 2 by use of a radical mechanism. In a recent bioinformatic study, the uncharacterized yeast gene YOR006c was predicted to be involved in ribosome biogenesis. It is highly conserved among eukaryotes and archaea (Supplementary Figure S1A) and its deletion leads to an accumulation of the 20S pre-rRNA precursor of 18S rRNA, suggesting an influence on D-site cleavage during the maturation of the small ribosomal subunit. On this basis, YOR006C was renamed ‘Twenty S rRNA accumulation 3′ (TSR3). However, its function remained unclear although recently a putative nuclease function during 18S rRNA maturation was predicted. Here, we identify Tsr3 as the long-sought acp transferase that catalyzes the last step in the biosynthesis of the hypermodified nucleotide m1acp3Ψ in yeast and human cells. Furthermore using catalytically defective mutants of yeast Tsr3 we demonstrated that the acp modification is required for 18S rRNA maturation. Surprisingly, the crystal structures of archaeal homologs revealed that Tsr3 is structurally similar to the SPOUT-class RNA methyltransferases. In contrast, the only other structurally characterized acp transferase enzyme Tyw2 belongs to the Rossmann-fold class of methyltransferase proteins. Interestingly, the two structurally very different enzymes use similar strategies in binding the SAM-cofactor in order to ensure that in contrast to methyltransferases the acp and not the methyl group of SAM is transferred to the substrate. MATERIALS AND METHODS Genetic constructions, growth conditions and yeast media Detailed descriptions are available in Supplementary Data. Cell culture HCT116(+/+) cells (CCL-247; ATCC) were grown at 37°C in a humidified incubator under 5% CO2 in the McCoy's 5a modified (Sigma-Aldrich)/10% FBS media. All media were supplemented with 50 U/ml penicillin and 50 μg/ml streptomycin (Life Technologies). DsiRNA inactivation and RT-qPCR Reverse transfection of HCT116 cells, DsiRNA inactivation and RT-qPCR using total human RNA are described in Supplementary Data. Sucrose gradient analysis Detailed descriptions for analytical or preparative separations of ribosomal subunits or polysome gradients are provided in Supplementary Data. HPLC analysis of 18S rRNA nucleosides 40S subunits from 200 ml yeast culture were isolated by sucrose gradient centrifugation in a SW28 rotor as described above, and precipitated with 2.5 vol of 100% ethanol (−20°C over night). Precipitated 40S subunits were dissolved in water and the 18S rRNA was purified via spin columns (Ambion PureLink RNA Mini Kit). RNA fragments were hydrolysed and dephosphorylated as described by Gehrke and Kuo. HPLC analysis of rRNA nucleoside composition was performed using a Supelcosil LC-18S column (Sigma; 250 × 4.6 mm, 5 μm) with a pre-column (4.6 × 20 mm) as previously described. 14C labeling of 18S rRNA nucleotide Ψ(U)1191 To enhance 14C-labeling, mutants of interest were recombined with a Δmet13 deletion. Resulting strains were cultivated with l-[1-14C]-methionine (Hartmann Analytic, 0.1 mCi/ml, 54 mCi/mmol) as described before. From isotope labeled cells total RNA was isolated with the PureLink RNA Mini Kit (Ambion) after enzymatic cell lysis with zymolyase. Ribosomal RNAs were separated on a 4% denaturing polyacrylamide gel. After ethidium bromide straining gels were dried and analyzed by autoradiography for 3–5 days using a storage phosphor screen. Signals were visualized with the Typhoon 9100 (GE Healthcare). Northern blot analysis 5 μg of total yeast RNAs extracted with phenol/chloroform were separated on 1.2% agarose gels in BPTE buffer for 16 h at 60V and afterwards transferred to a Biodyne B membrane by vacuum blotting. Oligonucleotides D/A2 or +1-A0 were radiolabeled using γ-[32P]-ATP and T4-polynucleotide kinase and hybridized to the membrane at 37°C. Signals were visualized by phosphoimaging with the Typhoon 9100 (GE Healthcare). RNA extraction from human cells, gel-electrophoresis and northern blotting were performed as described before. Primer extension 20 pmol of oligonucleotide PE-1191 complementary to yeast 18S rRNA nucleotides 1247–1228 were labeled with 50 μCi γ-[32P]-ATP using T4-polynucleotide kinase, purified via Sephadex G-25 and annealed to 500 ng of 18S rRNA. Primer annealing and reverse transcription were carried out as described by Sharma et al.. After precipitation with ethanol and 3 M NaAc pH 5.2 pellets were washed with 70% ethanol, dried and dissolved in 12 μl formamide loading dye. 2 μl of primer extension samples were separated on sequencing or mini gels which were dried after running and exposed on a storage phosphor screen. Signals were visualized with the Typhoon 9100 (GE Healthcare). Primer extension on human RNA was performed using 5 μg of total RNA with AMV Reverse Transcriptase (Promega) and oligonucleotide PE_1248. Following alkaline hydrolysis, cDNAs were precipitated with ethanol, resuspended in acrylamide loading buffer and separated on a 6% (v/v) denaturing acrylamide gel in 0.5× TBE at 80 W for 1.5 h. After migration, the gels were dried and exposed to Fuji Imaging plates (Fujifilm). The signal was acquired with a Phosphor imager (FLA-7000, Fujifilm). Protein detection and localization A description of the western blot detection of HA-fused Tsr3 in yeast crude extracts or sucrose gradients fractions is provided in Supplementary Data. For cellular localization Tsr3 was expressed as N-terminal fusion with yEGFP in a yeast strain encoding for ScNop56-mRFP. Protein localization in exponentially growing cells was visualized using a Leica TCS SP5. In vitro SAM binding Purified SsTsr3 protein in 25 mM Tris–HCl pH 7.8 250 mM NaCl was mixed with S-[methyl-14C]-adenosyl-l-methionine (PerkinElmer; 20 μCi/ml, 58 mCi/mmol) and 0–10 mM non-labeled SAM in a binding buffer (50 mM Tris–HCl pH 7.8, 250 mM NaCl) in a total volume of 50 μl and incubated at 30°C for 10 min. Samples were passed over HAWP02500 membrane filters (Millipore) and unbound 14C-SAM was removed by washing three times with 5 ml buffer using a vacuum filtering equipment. Filter bound 14C-SAM was measured by liquid scintillation spectrometry in a Wallac 1401 scintillation counter. Protein expression and purification Genes coding for archaeal Tsr3 homologs without any tags were obtained commercially (Genscript) in pET11a vectors and overexpressed in Escherichia coli BL21(DE3). Proteins were purified by a combination of heat shock and appropriate column chromatography steps as described in detail in the Supplementary Data. Crystallization, X-ray data collection, structure calculation and refinement Initial hits for VdTsr3 and SsTsr3 were obtained using the Morpheus Screen (Molecular dimensions) and further refined as described in the Supplementary Data. Diffraction data were collected at the Swiss Light Source (Paul Scherer Institut). The structure of VdTsr3 was determined at 1.6 Å by SAD using a selenomethionine derivative. The structure of SsTsr3 was determined at 2.25 Å by molecular replacement using VdTsr3 as the search model. A detailed description of the data collection, processing, structure calculation and refinement procedures can be found in the Supplementary Data and in Supplementary Table S1. Structures were deposited in the Protein Data Bank as entries 5APG (VdTsr3) and 5AP8 (SsTsr3). Analytical gel filtration For analytical gel filtration experiments a Sephadex S75 10/300 GL column (GE Healthcare) was used. 100 μl protein samples (25 mM Tris–HCl pH 7.8, 250 mM NaCl, 2 mM β-mercaptoethanol) with a protein concentration of 150 μM were used. The flow rate was 0.5 ml/min. The column was calibrated using the marker proteins of the LMW gel filtration calibration kit (GE Healthcare). Protein elution was followed by recording the adsorption at a wavelength of λ = 280 nm. Fluorescence quenching and fluorescence anisotropy measurements Fluorescence quenching and fluorescence anisotropy measurements were carried out in triplicates at 25°C on a Fluorolog 3 spectrometer (Horiba Jobin Yvon) equipped with polarizers. For fluorescence quenching with SAM, SAH and 5′-methylthioadenosine experiments the tryptophan fluorescence of SsTsr3 (200 nM in 25 mM Tris–HCl pH 7.8, 250 mM NaCl, 2 mM β-mercaptoethanol) was excited at 295 nm and emission spectra were recorded from 250 to 450 nm for each titration step. The fluorescence intensity at 351 nm for each titration step was normalized with regard to the fluorescence of the free protein and was used for deriving binding curves. KD's were derived by nonlinear regression with Origin 8.0 (Origin Labs) using Equation (1):  (F is the normalized fluorescence intensity, a is the change in fluorescence intensity, c is the ligand concentration and KD is the dissociation constant). 5′-Fluoresceine labeled RNAs for fluorescence anisotropy measurements were obtained commercially (Dharmacon), deprotected according to the manufacturer's protocol and the RNA concentration adjusted to 50 nM in 25 mM Tris–HCl pH 7.8, 250 mM NaCl. Fluoresceine fluorescence was excited at 492 nm and emission was recorded at 516 nm. The data were fitted to Equation (1) (F is the normalized fluorescence anisotropy, a is the change in fluorescence anisotropy). RESULTS Tsr3 is the enzyme responsible for 18S rRNA acp modification in yeast and humans The S. cerevisiae 18S rRNA acp transferase was identified in a systematic genetic screen where numerous deletion mutants from the EUROSCARF strain collection (www.euroscarf.de) were analyzed by HPLC for alterations in 18S rRNA base modifications. For the Δtsr3 deletion strain the HPLC elution profile of 18S rRNA nucleosides (Figure 1B) was very similar to that of the pseudouridine-N1 methyltransferase mutant Δnep1, where a shoulder at ∼ 7.4 min elution time was missing in the elution profile. As previously reported this shoulder was identified by ESI-MS as corresponding to m1acp3Ψ. In order to directly analyze the presence of the acp modification of nucleotide 1191 we used an in vivo14C incorporation assay with 1-14C-methionine. Whereas the acp labeling of 18S rRNA was clearly present in the wild type strain no radioactive labeling could be observed in a Δtsr3 strain (Figure 1C). No radioactive labeling was detected in the 18S U1191A mutant which served as a control for the specificity of the 14C-aminocarboxypropyl incorporation. As previously shown, only the acp but none of the other modifications at U1191 of yeast 18S rRNA blocks reverse transcriptase activity. Therefore the presence of the acp modification can be directly assessed by primer extension. Indeed, in wild-type yeast a strong primer extension stop signal occurred at position 1192. In contrast, in a Δtsr3 mutant no primer extension stop signal was present at this position. As expected, in a Δsnr35 deletion preventing pseudouridylation and N1-methylation (resulting in acp3U) as well as in a Δnep1 deletion strain where pseudouridine is not methylated (resulting in acp3Ψ) a primer extension stop signal of similar intensity as in the wild type was observed. In a Δtsr3 Δsnr35 double deletion strain the 18S rRNA contains an unmodified U and the primer extension stop signal was missing (Figure 1D). The Tsr3 protein is highly conserved in yeast and humans (50% identity). Human 18S rRNA has also been shown to contain m1acp3Ψ in the 18S rRNA at position 1248. After siRNA-mediated depletion of Tsr3 in human colon carcinoma HCT116(+/+) cells the acp primer extension arrest was reduced in comparison to cells transfected with a non-targeting scramble siRNA control (Figure 1E, compare lanes 544 and scramble). The efficiency of siRNA-mediated depletion was established by RT-qPCR and found to be very high with siRNA 544 (Supplementary Figure S2A, remaining TSR3 mRNA level of 2%). By comparison, treating cells with siRNA 545, which only reduced the TSR3 mRNA to 20%, did not markedly reduced the acp signal. This suggests that low residual levels of HsTsr3 are sufficient to modify the RNA. As a control for loading, a structural stop is shown (asterisk, Figure 1E). Thus, HsTsr3 is also responsible for the acp modification of 18S rRNA nucleotide Ψ1248 in helix 31. Similar to yeast, siRNA-mediated depletion of the Ψ1248 N1-methyltransferase Nep1/Emg1 had no influence on the primer extension arrest (Figure 1E). Phenotypic characterization of Δtsr3 mutants Although the acp modification of 18S rRNA is highly conserved in eukaryotes, yeast Δtsr3 mutants showed only a minor growth defect. However, the Δtsr3 deletion was synthetic sick with a Δsnr35 deletion preventing pseudouridylation and Nep1-catalyzed methylation of nucleotide 1191 (Figure 2A). Interestingly, no increased growth defect could be observed for Δtsr3 Δnep1 recombinants containing the nep1 suppressor mutation Δnop6 as well as for Δtsr3 Δsnr35 Δnep1 recombinants with unmodified U1191 (Supplementary Figure S2D and E). Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. The Δtsr3 deletion is synthetic sick with a Δsnr35 deletion preventing U1191 pseudouridylation. (B) In agar diffusion assays the yeast Δtsr3 deletion mutant shows a hypersensitivity against paromomycin and hygromycin B which is further increased by recombination with Δsnr35. (C) Northern blot analysis with an ITS1 hybridization probe after siRNA depletion of HsTSR3 (siRNAs 544 and 545) and a scrambled siRNA as control. The accumulation of 18SE and 47S and/or 45S pre-RNAs is enforced upon HsTSR3 depletion. Right gel: Ethidium bromide staining showing 18S and 28S rRNAs. (D) Cytoplasmic localization of yeast Tsr3 shown by fluorescence microscopy of GFP-fused Tsr3. From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). The TSR3 gene was genetically modified at its native locus, resulting in a C-terminal fusion of Tsr3 with a 3xHA epitope expressed by the native promotor in yeast strain CEN.BM258-5B. The influence of the acp modification of nucleotide 1191 on ribosome function was analyzed by treating Δtsr3 mutants with protein synthesis inhibitors. Similar to a temperature-sensitive nep1 mutant, the Δtsr3 deletion caused hypersensitivity to paromomycin and, to a lesser extent, to hygromycin B (Figure 2B), but not to G418 or cycloheximide (data not shown). In accordance with the synthetic sick growth phenotype the paromomycin and hygromycin B hypersensitivity further increased in a Δtsr3 Δsnr35 recombination strain (Figure 2B). In a yeast Δtsr3 strain as well as in the Δtsr3 Δsnr35 recombinant 20S pre-rRNA accumulated significantly and the level of mature 18S rRNA was reduced (Supplementary Figures S2C and S3D), as reported previously. A minor effect on 20S rRNA accumulation was also observed for Δsnr35, but - probably due to different strain backgrounds – to a weaker extent than described earlier. In human cells, the depletion of HsTsr3 in HCT116(+/+) cells caused an accumulation of the human 20S pre-rRNA equivalent 18S-E suggesting an evolutionary conserved role of Tsr3 in the late steps of 18S rRNA processing (Figure 2C and Supplementary Figure S2B). Surprisingly, early nucleolar processing reactions were also inhibited, and this was observed in both yeast Δtsr3 cells (see accumulation of 35S in Supplementary Figure S2C) and Tsr3 depleted human cells (see 47S/45S accumulation in Figure 2C and Northern blot quantification in Supplementary Figure S2B). Consistent with its role in late 18S rRNA processing, TSR3 deletion leads to a ribosomal subunit imbalance with a reduced 40S to 60S ratio of 0.81 (σ = 0.024) which was further increased in a Δtsr3 Δsnr35 recombinant to 0.73 (σ = 0.023) (Supplementary Figure S2F). In polysome profiles, a reduced level of 80S ribosomes and a strong signal for free 60S subunits was observed in line with the 40S subunit deficiency (Supplementary Figure S2G). Cellular localization of Tsr3 in S. cerevisiae Fluorescence microscopy of GFP-tagged Tsr3 localized the fusion protein in the cytoplasm of yeast cells and no co-localization with the nucleolar marker protein Nop56 could be observed (Figure 2D). This agrees with previous biochemical data suggesting that the acp modification of 18S rRNA occurs late during 40S subunit biogenesis in the cytoplasm, and makes an additional nuclear localization as reported in a previous large-scale analysis unlikely. After polysome gradient separation C-terminally epitope-labeled Tsr3-3xHA was exclusively detectable in the low-density fraction (Figure 2E). Such distribution on a density gradient suggests that Tsr3 only interacts transiently with pre-40S subunits, which presumably explains why it was not characterized in pre-ribosome affinity purifications. Structure of Tsr3 Searches for sequence homologs of S. cerevisiae Tsr3 (ScTsr3) by us and others revealed that the genomes of many archaea contain genes encoding Tsr3-like proteins. However, these archaeal homologs are significantly smaller than ScTsr3 (∼190 aa in archaea vs. 313 aa in yeast) due to shortened N- and C-termini (Supplementary Figure S1A). To locate the domains most important for Tsr3 activity, ScTsr3 fragments of different lengths containing the highly conserved central part were expressed in a Δtsr3 mutant (Figure 3A) and analyzed by primer extension (Figure 3B) and Northern blotting (Figure 3C). N-terminal truncations of up to 45 aa and C-terminal truncations of up to 76 aa mediated acp modification as efficiently as the full-length protein and no significant increased levels of 20S pre-RNA were detected. Even a Tsr3 fragment with a 90 aa C-terminal truncation showed a residual primer extension stop, whereas N-terminal truncations exceeding 46 aa almost completely abolished the primer extension arrest (Figure 3B). Domain characterization of yeast Tsr3 and correlation of acp modification with late 18S rRNA processing steps. (A) Scheme of the TSR3 gene with truncation positions in the open reading frame. TSR3 fragments of different length were expressed under the native promotor from multicopy plasmids in a Δtsr3 deletion strain. (B) Primer extension analysis of 18S rRNA acp modification in yeast cells expressing the indicated TSR3 fragments. N-terminal deletions of 36 or 45 amino acids and C-terminal deletions of 43 or 76 residues show a primer extension stop comparable to the wild type. Tsr3 fragments 37–223 or 46–223 cause a nearly complete loss of the arrest signal. The box highlights the shortest Tsr3 fragment (aa 46–270) with wild type activity (strong primer extension block). (C) Northern blot analysis of 20S pre-rRNA accumulation. A weak 20S rRNA signal, indicating normal processing, is observed for Tsr3 fragment 46–270 (highlighted in a box) showing its functionality. Strong 20S rRNA accumulation similar to that of the Δtsr3 deletion is observed for Tsr3 fragments 37–223 or 46–223. Thus, the archaeal homologs correspond to the functional core of Tsr3. In order to define the structural basis for Tsr3 function, homologs from thermophilic archaea were screened for crystallization. We focused on archaeal species containing a putative Nep1 homolog suggesting that these species are in principle capable of synthesizing N1-methyl-N3-acp-pseudouridine. Well diffracting crystals were obtained for Tsr3 homologs from the two crenarchaeal species Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) which share 36% (VdTsr3) and 38% (SsTsr3) identity with the ScTsr3 core region (ScTsr3 aa 46–223). While for S. solfataricus the existence of a modified nucleotide of unknown chemical composition in the loop capping helix 31 of its 16S rRNA has been demonstrated, no information regarding rRNA modifications is yet available for V. distributa. Crystals of VdTsr3 diffracted to a resolution of 1.6 Å whereas crystals of SsTsr3 diffracted to 2.25 Å. Serendipitously, VdTsr3 was purified and crystallized in complex with endogenous (E. coli) SAM (Supplementary Figure S4) while SsTsr3 crystals contained the protein in the apo state. The structure of VdTsr3 was solved ab initio, by single-wavelength anomalous diffraction phasing (Se-SAD) with Se containing derivatives (selenomethionine and seleno-substituted SAM). The structure of SsTsr3 was solved by molecular replacement using VdTsr3 as a search model (see Supplementary Table S1 for data collection and refinement statistics). The structure of VdTsr3 can be divided into two domains (Figure 4A). The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. The loop connecting β2 and β3 contains a single turn of a 310-helix. Helices α1 and α2 are located on one side of the five-stranded β-sheet while α3 packs against the opposite β-sheet surface. The C-terminal domain (aa 93–184) has a globular all α-helical structure comprising α-helices α4 to α9. Both domains are tightly packed against each other. Remarkably, the entire C-terminal domain (92 aa) of the protein is threaded through the loop which connects β-strand β3 and α-helix α2 of the N-terminal domain. Thus, the VdTsr3 structure contains a deep trefoil knot. The structure of SsTsr3 in the apo state is very similar to that of VdTsr3 (Figure 4C) with an RMSD for equivalent Cα atoms of 1.1 Å. The only significant difference in the global structure of the two proteins is the presence of an extended α-helix α8 and the absence of α-helix α9 in SsTsr3. Tsr3 has a fold similar to SPOUT-class RNA methyltransferases. (A) Cartoon representation of the X-ray structure of VdTsr3 in two orientations. β-strands are colored in crimson whereas α-helices in the N-terminal domain are colored light blue and α-helices in the C-terminal domain are colored dark blue. The bound S-adenosylmethionine is shown in a stick representation and colored by atom type. A red arrow marks the location of the topological knot in the structure. (B) Secondary structure representation of the VdTsr3 structure. The color coding is the same as in (A). (C) Structural superposition of the X-ray structures of VdTsr3 in the SAM-bound state (red) and SsTsr3 (blue) in the apo state. The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. Vertical lines indicate the elution volumes of molecular weight markers. Vd, Vulcanisaeta distributa; Ss, Sulfolobus solfataricus. Structure predictions suggested that Tsr3 might contain a so-called RLI domain which contains a ‘bacterial like’ ferredoxin fold and binds two iron-sulfur clusters through eight conserved cysteine residues. However, no structural similarity to an RLI-domain was detectable. This is in accordance with the functional analysis of alanine replacement mutations of cysteine residues in ScTsr3 (Supplementary Figure S3). The β-strand topology and the deep C-terminal trefoil knot of archaeal Tsr3 are the structural hallmarks of the SPOUT-class RNA-methyltransferase fold. The closest structural homolog identified in a DALI search is the tRNA methyltransferase Trm10 (DALI Z-score 6.8) which methylates the N1 nitrogen of G9/A9 in many archaeal and eukaryotic tRNAs by using SAM as the methyl group donor. In comparison to Tsr3 the central β-sheet element of Trm10 is extended by one additional β-strand pairing to β2. Furthermore, the trefoil knot of Trm10 is not as deep as that of Tsr3 (Figure 4D). Interestingly, Nep1—the enzyme preceding Tsr3 in the biosynthetic pathway for the synthesis of m1acp3Ψ—also belongs to the SPOUT-class of RNA methyltransferases. However, the structural similarities between Nep1 and Tsr3 (DALI Z-score 4.4) are less pronounced than between Tsr3 and Trm10. Most SPOUT-class RNA-methyltransferases are homodimers. A notable exception is Trm10. Gel filtration experiments with both VdTsr3 and SsTsr3 (Figure 4E) showed that both proteins are monomeric in solution thereby extending the structural similarities to Trm10. So far, structural information is only available for one other enzyme that transfers the acp group from SAM to an RNA nucleotide. This enzyme, Tyw2, is part of the biosynthesis pathway of wybutosine nucleotides in tRNAs. However, there are no structural similarities between Tsr3 and Tyw2, which contains an all-parallel β-sheet of a different topology and no knot structure. Instead, Tyw2 has a fold typical for the class-I-or Rossmann-fold class of methyltransferases (Supplementary Figure S5B). Cofactor binding of Tsr3 The SAM-binding site of Tsr3 is located in a deep crevice between the N- and C-terminal domains in the vicinity of the trefoil knot as typical for SPOUT-class RNA-methyltransferases (Figure 4A). The adenine base of the cofactor is recognized by hydrogen bonds between its N1 nitrogen and the backbone amide of L93 directly preceding β5 as well as between its N6-amino group and the backbone carbonyl group of Y108 located in the loop connecting β5 in the N-terminal and α4 in the C-terminal domain (Figure 5A). Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. The ribose 2′ and 3′ hydroxyl groups of SAM are hydrogen bonded to the backbone carbonyl group of I69. The acp side chain of SAM is fixed in position by hydrogen bonding of its carboxylate group to the backbone amide and the side chain hydroxyl group of T19 in α1 as well as the backbone amide group of T112 in α4 (C-terminal domain). Most importantly, the methyl group of SAM is buried in a hydrophobic pocket formed by the sidechains of W73 and A76 both located in α3 (Figure 5A and B). W73 is highly conserved in all known Tsr3 proteins, whereas A76 can be replaced by other hydrophobic amino acids. Consequently, the accessibility of this methyl group for a nucleophilic attack is strongly reduced in comparison with RNA-methyltransferases such as Trm10 (Figure 5B, C). In contrast, the acp side chain of SAM is accessible for reactions in the Tsr3-bound state (Figure 5B). SAM-binding by Tsr3. (A) Close-up view of the SAM-binding pocket of VdTsr3. Nitrogen atoms are dark blue, oxygen atoms red, sulfur atoms orange, carbon atoms of the protein light blue and carbon atoms of SAM yellow. Hydrogen bonds are indicated by dashed lines. (B) Solvent accessibility of the acp group of SAM bound to VdTsr3. The solvent accessible surface of the protein is shown in semitransparent gray whereas SAM is show in a stick representation. Atoms are colored as in (A). A red arrow indicates the reactive CH2-moiety of the acp group. (C) Solvent accessibility of the SAM methyl group for SAM bound to the RNA methyltransferase Trm10. Bound SAM was modelled based on the X-ray structure of the Trm10/SAH-complex (pdb4jwf). A red arrow indicates the SAM methyl group. (D) Binding of SAM analogs to SsTsr3. Tryptophan fluorescence quenching curves upon addition of SAM (blue), 5′-methyl-thioadenosine (red) and SAH (black). (E) Binding of 14C-labeled SAM to SsTsr3. Radioactively labeled SAM is retained on a filter in the presence of SsTsr3. Addition of unlabeled SAM competes with the binding of labeled SAM. A W66A-mutant of SsTsr3 (W73 in VdTsr3) does not bind SAM. (F) Primer extension (upper left) shows a strongly reduced acp modification of yeast 18S rRNA in Δtsr3 cells expressing Tsr3-S62D, -E111A or –W114A. This correlates with a 20S pre-rRNA accumulation comparable to the Δtsr3 deletion (right: northern blot). 3xHA tagged Tsr3 mutants are expressed comparable to the wild type as shown by western blot (lower left). Binding affinities for SAM and its analogs 5′-methylthioadenosin and SAH to SsTsr3 were measured using tryptophan fluorescence quenching. VdTsr3 could not be used in these experiments since we could not purify it in a stable SAM-free form. SsTsr3 bound SAM with a KD of 6.5 μM, which is similar to SAM-KD's reported for several SPOUT-class methyltransferases. 5′-methylthioadenosin—the reaction product after the acp-transfer—binds only ∼2.5-fold weaker (KD = 16.7 μM) compared to SAM. S-adenosylhomocysteine which lacks the methyl group of SAM binds with significantly lower affinity (KD = 55.5 μM) (Figure 5D). This suggests that the hydrophobic interaction between SAM's methyl group and the hydrophobic pocket of Tsr3 is thermodynamically important for the interaction. On the other hand, the loss of hydrogen bonds between the acp sidechain carboxylate group and the protein appears to be thermodynamically less important but these hydrogen bonds might play a crucial role for the proper orientation of the cofactor side chain in the substrate binding pocket. Accordingly, a W66A-mutation (W73 in VdTsr3) of SsTsr3 significantly diminished SAM-binding in a filter binding assay compared to the wild type (Figure 5E). Furthermore, a W to A mutation at the equivalent position W114 in ScTsr3 strongly reduced the in vivo acp transferase activity (Figure 5F). The side chain hydroxyl group of T19 seems of minor importance for SAM binding since mutations of T17 (T19 in VdTsr3) to either A or D did not significantly influence the SAM-binding affinity of SsTsr3 (KD's = 3.9 or 11.2 mM, respectively). Nevertheless, a mutation of the equivalent position S62 of ScTsr3 to D, but not to A, resulted in reduced acp modification in vivo, as shown by primer extension analysis (Figure 5F). The acp-transfer reaction catalyzed by Tsr3 most likely requires the presence of a catalytic base in order to abstract a proton from the N3 imino group of the modified pseudouridine. The side chain of D70 (VdTsr3) located in β4 is ∼5 Å away from the SAM sulfur atom. This residue is conserved as D or E both in archaeal and eukaryotic Tsr3 homologs. Mutations of the corresponding residue in SsTsr3 to A (D63) does not significantly alter the SAM-binding affinity of the protein (KD = 11.0 μM). However, the mutation of the corresponding residue of ScTsr3 (E111A) leads to a significant decrease of the acp transferase activity in vivo (Figure 5F). RNA-binding of Tsr3 Analysis of the electrostatic surface properties of VdTsr3 clearly identified positively charged surface patches in the vicinity of the SAM-binding site suggesting a putative RNA-binding site (Figure 6A). Furthermore, a negatively charged MES-ion is found in the crystal structure of VdTsr3 complexed to the side chain of K22 in helix α1. Its negatively charged sulfate group might mimic an RNA backbone phosphate. Helix α1 contains two more positively charged amino acids K17 and R25 as does the loop preceding it (R9). A second cluster of positively charged residues is found in or near helix α3 (K74, R75, K82, R85 and K87). Some of these amino acids are conserved between archaeal and eukaryotic Tsr3 (Supplementary Figure S1A). In the C-terminal domain, the surface exposed α-helices α5 and α7 carry a significant amount of positively charged amino acids. A triple mutation of the conserved positively charged residues R60, K65 and R131 to A in ScTsr3 resulted in a protein with a significantly impaired acp transferase activity in vivo (Figure 6D) in line with an important functional role for these positively charged residues. RNA-binding of Tsr3. (A) Electrostatic charge distribution on the surface of VdTsr3. Surface areas colored in blue are positively charged whereas red areas are negatively charged. SAM is shown in a stick representation. Also shown in stick representation is a negatively charged MES ion. Conserved basic amino acids are labeled. (B) Comparison of the secondary structures of helix 31 from the small ribosomal subunit rRNAs in S. cerevisiae and S. solfataricus with the location of the hypermodified nucleotide indicated in red. For S. solfataricus the chemical identity of the hypermodified nucleotide is not known but the existence of NEP1 and TSR3 homologs suggest that it is indeed N1-methyl-N3-acp-pseudouridine. (C) Binding of SsTsr3 to RNA. 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). Oligo-U9-RNA was used for comparison (black). The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. Combination of the three point mutations (R60A/K65A/R131A) leads to a strongly reduced acp modification of 18S rRNA. In order to explore the RNA-ligand specificity of Tsr3 we titrated SsTsr3 prepared in RNase-free form with 5′-fluoresceine-labeled RNA and determined the affinity by fluorescence anisotropy measurements. SsTsr3 in the apo state bound a 20mer RNA corresponding to helix 31 of S. solfataricus 16S rRNA (Figure 6B) with a KD of 1.9 μM and to a version of this hairpin stabilized by additional GC base pairs (20mer-GC) with a KD of 0.6 μM (Figure 6C). A single stranded oligoU-RNA bound with a 10-fold-reduced affinity (6.0 μM). The presence of saturating amounts of SAM (2 mM) did not have a significant influence on the RNA-affinity of SsTsr3 (KD of 1.7 μM for the 20mer-GC-RNA) suggesting no cooperativity in substrate binding. DISCUSSION U1191 is the only hypermodified base in the yeast 18S rRNA and is strongly conserved in eukaryotes. The formation of 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ) is very complex requiring three successive modification reactions involving one H/ACA snoRNP (snR35) and two protein enzymes (Nep1/Emg1 and Tsr3). This makes it unique in eukaryotic rRNA modification. The m1acp3Ψ base is located at the tip of helix 31 on the 18S rRNA (Supplementary Figure S1B) which, together with helices 18, 24, 34 and 44, contribute to building the decoding center of the small ribosomal subunit. A similar modification (acp3U) was identified in Haloferax volcanii and corresponding modified nucleotides were also shown to occur in other archaea. As shown here TSR3 encodes the transferase catalyzing the acp modification as the last step in the biosynthesis of m1acp3Ψ in yeast and human cells. Unexpectedly, archaeal Tsr3 has a structure similar to SPOUT-class RNA methyltransferases, and it is the first example for an enzyme of this class transferring an acp group, due to a modified SAM-binding pocket that exposes the acp instead of the methyl group of SAM to its RNA substrate. Similar to the structurally unrelated Rossmann-fold Tyw2 acp transferase, the SAM methyl group of Tsr3 is bound in an inaccessible hydrophobic pocket whereas the acp side chain becomes accessible for a nucleophilic attack by the N3 of pseudouridine. In contrast, in the structurally closely related RNA methyltransferase Trm10 the methyl group of the cofactor SAM is accessible whereas its acp side chain is buried inside the protein. This suggests that enzymes with a SAM-dependent acp transferase activity might have evolved from SAM-dependent methyltransferases by slight modifications of the SAM-binding pocket. Thus, additional examples for acp transferase enzymes might be found with similarities to other structural classes of methyltransferases. In contrast to Nep1, the enzyme preceding Tsr3 in the m1acp3Ψ biosynthesis pathway, Tsr3 binds rather weakly and with little specificity to its isolated substrate RNA. This suggests that Tsr3 is not stably incorporated into pre-ribosomal particles and that its binding to the nascent ribosomal subunit possibly requires additional interactions with other pre-ribosomal components. Consistently, in sucrose gradient analysis, Tsr3 was found in low-molecular weight fractions rather than with pre-ribosome containing high-molecular weight fractions. In contrast to several enzymes that catalyze base specific modifications in rRNAs Tsr3 is not an essential protein. Typically, other small subunit rRNA methyltransferases as Dim1, Bud23 and Nep1/Emg1 carry dual functions, in ribosome biogenesis and rRNA modification, and it is their involvement in pre-RNA processing that is essential rather than their RNA-methylating activity (, discussed in 7). In contrast, for several Tsr3 mutants (SAM-binding and cysteine mutations) we found a systematic correlation between the loss of acp modification and the efficiency of 18S rRNA maturation. This demonstrates that, unlike the other small subunit rRNA base modifications, the acp modification is required for efficient pre-rRNA processing. Recently, structural, functional, and CRAC (cross-linking and cDNA analysis) experiments of late assembly factors involved in cytoplasmic processing of 40S subunits, along with cryo-EM studies of the late pre-40S subunits have provided important insights into late pre-40S processing. Apart from most of the ribosomal proteins, cytoplasmic pre-40S particles contain 20S rRNA and at least seven non-ribosomal proteins including the D-site endonuclease Nob1 as well as Tsr1, a putative GTPase and Rio2 which block the mRNA channel and the initiator tRNA binding site, respectively, thus preventing translation initiation. After structural changes, possibly driven by GTP hydrolysis, which go together with the formation of the decoding site, the 20S pre-rRNA becomes accessible for Nob1 cleavage at site D. This also involves joining of pre-40S and 60S subunits to 80S-like particles in a translation-like cycle promoted by eIF5B. The cleavage step most likely acts as a quality control check that ensures the proper 40S subunit assembly with only completely processed precursors. Finally, termination factor Rli1, an ATPase, promotes the dissociation of assembly factors and the 80S-like complex dissociates and releases the mature 40S subunit. Interestingly, differences in the level of acp modification were demonstrated for different steps of the cytoplasmic pre-40S subunit maturation after analyzing purified 20S pre-rRNAs using different purification bait proteins. Early cytoplasmic pre-40S subunits still containing the ribosome assembly factors Tsr1, Ltv1, Enp1 and Rio2 were not or only partially acp modified. In contrast, late pre-40S subunits containing Nob1 and Rio1 or already associated with 60S subunits in 80S-like particles showed acp modification levels comparable to mature 40S subunits. Thus, the acp transfer to m1Ψ1191 occurs during the step at which Rio2 leaves the pre-40S particle. These data and the finding that a missing acp modification hinders pre-20S rRNA processing, suggest that the acp modification together with the release of Rio2 promotes the formation of the decoding site and thus D-site cleavage by Nob1. The interrelation between acp modification and Rio2 release is also supported by CRAC analysis showing that Rio2 binds to helix 31 next to the Ψ1191 residue that receives the acp modification. Therefore, Rio2 either blocks the access of Tsr3 to helix 31, and acp modification can only occur after Rio2 is released, or the acp modification of m1Ψ1191 and putative subsequent conformational changes of 20S rRNA weaken the binding of Rio2 to helix 31 and support its release from the pre-rRNA. In summary, by identifying Tsr3 as the enzyme responsible for introducing the acp group to the hypermodified m1acp3Ψ nucleotide at position 1191 (yeast)/ 1248 (humans) of 18S rRNA we added one of the last remaining pieces to the puzzle of eukaryotic small ribosomal subunit rRNA modifications. The current data together with the finding that acp modification takes place at the very last step in pre-40S subunit maturation indicate that the acp modification probably supports the formation of the decoding site and efficient 20S pre-rRNA D-site cleavage. Furthermore, our structural data unravelled how the regioselectivity of SAM-dependent group transfer reactions can be tuned by distinct small evolutionary adaptions of the ligand binding pocket of SAM-binding enzymes. ACCESSION NUMBERS Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes PDB 5APG (VdTsr3/SAM-complex) and PDB 5AP8 (SsTsr3). Supplementary Material SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. FUNDING DFG grant [En134/9-1]; SFB 902 (Molecular Principles of RNA-based Regulation); DFG SPP1784 (Chemical Biology of Native Nucleic Acid Modifications, DFG grants) [En134/13-1, Wo 901/5-1]; European Community's Seventh Framework Programme [FP7/2007-2013] under BioStruct-X [283570]; Goethe University and the State of Hesse; EMBO long-term fellowship [ALTF 644-2014 to S.S.]; Research in the Lab of DLJL is supported by the Université Libre de Bruxelles (ULB); Fonds National de la Recherche (F.R.S./FNRS); Walloon Region [DGO6]; Fédération Wallonie-Bruxelles; European Research Development Fund (ERDF). Funding for open access charge: DFG SPP1784 (Chemical Biology of Native Nucleic Acid Modifications, DFG grants) [En134/13-1, Wo 901/5-1]. Conflict of interest statement. None declared. REFERENCES Ribosome biogenesis in the yeast Saccharomyces cerevisiae Diverse diseases from a ubiquitous process: the ribosomopathy paradox Genetics. Mysterious ribosomopathies Human diseases of the SSU processome Site-specific ribose methylation of preribosomal RNA. a novel function for small nucleolar RNAs Site-specific pseudouridine formation in preribosomal RNA is guided by small nucleolar RNAs 'View from a bridge': a new perspective on eukaryotic rRNA base modification Yeast Rrp8p, a novel methyltransferase responsible for m1A 645 base modification of 25S rRNA Yeast Nop2 and Rcm1 methylate C2870 and C2278 of the 25S rRNA, respectively Methylation of ribosomal RNA by NSUN5 is a conserved mechanism modulating organismal lifespan Post-transcriptional nucleotide modification and alternative folding of RNA Expanding the nucleotide repertoire of the ribosome with post-transcriptional modifications Structure, dynamics, and function of RNA modification enzymes rRNA modifications and ribosome function Optimization of ribosome structure and function by rRNA base modification Loss of rRNA modifications in the decoding center of the ribosome impairs translation and strongly delays pre-rRNA processing Ribosome structure and activity are altered in cells lacking snoRNPs that form pseudouridines in the peptidyl transferase center Ribosomopathies: human disorders of ribosome dysfunction Noncoding RNAs in eukaryotic ribosome biogenesis and function Prognostic value of nucleolar protein p120 in patients with resected lung adenocarcinoma Incorporation of 14C from [2-14C]methionine into 18 s but not 28 s RNA of Chinese hamster cells Characterization of three new snRNAs from Saccharomyces cerevisiae. snR34, snR35 and snR36 The crystal structure of Nep1 reveals an extended SPOUT-class methyltransferase fold and a pre-organized SAM-binding site The ribosome assembly factor Nep1 responsible for Bowen-Conradi syndrome is a pseudouridine-N1-specific methyltransferase The Bowen-Conradi syndrome protein Nep1 (Emg1) has a dual role in eukaryotic ribosome biogenesis, as an essential assembly factor and in the methylation of Ψ1191 in yeast 18S rRNA Biosynthesis of a hypermodified nucleotide in Saccharomyces carlsbergensis 17S and HeLa-cell 18S ribosomal ribonucleic acid Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution S-Adenosylmethionine-dependent alkylation reactions: when are radical reactions used? Biosynthesis of wybutosine, a hyper-modified nucleoside in eukaryotic phenylalanine tRNA Structural basis of AdoMet-dependent aminocarboxypropyl transfer reaction catalyzed by tRNA-wybutosine synthesizing enzyme, TYW2 The diphthamide modification pathway from Saccharomyces cerevisiae - revisited Diphthamide synthesis in Saccharomyces cerevisiae: structure of the DPH2 gene Identification of the proteins required for biosynthesis of diphthamide, the target of bacterial ADP-ribosylating toxins on translation elongation factor 2 Rational extension of the ribosome biogenesis pathway using network-guided genetics Analysis of two domains with novel RNA-processing activities throws light on the complex evolution of ribosomal RNA biogenesis Ribonucleoside analysis by reversed-phase high-performance liquid chromatography  Yeast Kre33 and human NAT10 are conserved 18S rRNA cytosine acetyltransferases that modify tRNAs assisted by the adaptor Tan1/THUMPD1 Identification of novel methyltransferases, Bmt5 and Bmt6, responsible for the m3U methylations of 25S rRNA in Saccharomyces cerevisiae Global analysis of protein localization in budding yeast Presence of a hypermodified nucleotide in HeLa cell 18 S and Saccharomyces carlsbergensis 17 S ribosomal RNAs Mutations in the nucleolar proteins Tma23 and Nop6 suppress the malfunction of the Nep1 protein Nep1p (Emg1p), a novel protein conserved in eukaryotes and archaea, is involved in ribosome biogenesis Deciphering structure and topology of conserved COG2042 orphan proteins The phylogenetic relationships of three sulfur dependent archaebacteria X-ray structure of the complete ABC enzyme ABCE1 from Pyrococcus abyssi Identification of the yeast gene encoding the tRNA m1G methyltransferase responsible for modification at position 9 Crystal structure of tRNA m1G9 methyltransferase Trm10: insight into the catalytic mechanism and recognition of tRNA substrate Crystal structure of the eukaryotic ribosome Identities and phylogenetic comparisons of posttranscriptional modifications in 16 S ribosomal RNA from Haloferax volcanii Methanococcus jannaschii sp. nov., an extremely thermophilic methanogen from a submarine hydrothermal vent The DIM1 gene responsible for the conserved m6(2)Am6(2)A dimethylation in the 3'-terminal loop of 18 S rRNA is essential in yeast Bud23 methylates G1575 of 18S rRNA and is required for efficient nuclear export of pre-40S subunits The human 18S rRNA base methyltransferases DIMT1L and WBSCR22-TRMT112 but not rRNA modification are required for ribosome biogenesis The yeast ribosome synthesis factor Emg1 is a novel member of the superfamily of alpha/beta knot fold methyltransferases Cracking pre-40S ribosomal subunit structure by systematic analyses of RNA-protein cross-linking Ribosome assembly factors prevent premature translation initiation by 40S assembly intermediates Essential ribosome assembly factor Fap7 regulates a hierarchy of RNA-protein interactions during small ribosomal subunit biogenesis Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits A translation-like cycle is a quality control checkpoint for maturing 40S ribosome 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+[{"sourceid":"4880283","sourcedb":"","project":"","target":"","text":"Crystal Structures of Putative Sugar Kinases from Synechococcus Elongatus PCC 7942 and Arabidopsis Thaliana The genome of the Synechococcus elongatus strain PCC 7942 encodes a putative sugar kinase (SePSK), which shares 44.9% sequence identity with the xylulose kinase-1 (AtXK-1) from Arabidopsis thaliana. Sequence alignment suggests that both kinases belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. However, their exact physiological function and real substrates remain unknown. Here we solved the structures of SePSK and AtXK-1 in both their apo forms and in complex with nucleotide substrates. The two kinases exhibit nearly identical overall architecture, with both kinases possessing ATP hydrolysis activity in the absence of substrates. In addition, our enzymatic assays suggested that SePSK has the capability to phosphorylate D-ribulose. In order to understand the catalytic mechanism of SePSK, we solved the structure of SePSK in complex with D-ribulose and found two potential substrate binding pockets in SePSK. Using mutation and activity analysis, we further verified the key residues important for its catalytic activity. Moreover, our structural comparison with other family members suggests that there are major conformational changes in SePSK upon substrate binding, facilitating the catalytic process. Together, these results provide important information for a more detailed understanding of the cofactor and substrate binding mode as well as the catalytic mechanism of SePSK, and possible similarities with its plant homologue AtXK-1. Introduction Carbohydrates are essential cellular compounds involved in the metabolic processes present in all organisms. Phosphorylation is one of the various pivotal modifications of carbohydrates, and is catalyzed by specific sugar kinases. These kinases exhibit considerable differences in their folding pattern and substrate specificity. Based on sequence analysis, they can be divided into four families, namely HSP 70_NBD family, FGGY family, Mer_B like family and Parm_like family. The FGGY family carbohydrate kinases contain different types of sugar kinases, all of which possess different catalytic substrates with preferences for short-chained sugar substrates, ranging from triose to heptose. These sugar substrates include L-ribulose, erythritol, L-fuculose, D-glycerol, D-gluconate, L-xylulose, D-ribulose, L-rhamnulose and D-xylulose. Structures reported in the Protein Data Bank of the FGGY family carbohydrate kinases exhibit a similar overall architecture containing two protein domains, one of which is responsible for the binding of substrate, while the second is used for binding cofactor ATP. While the binding pockets for substrates are at the same position, each FGGY family carbohydrate kinases uses different substrate-binding residues, resulting in high substrate specificity. Synpcc7942_2462 from the cyanobacteria Synechococcus elongatus PCC 7942 encodes a putative sugar kinase (SePSK), and this kinase contains 426 amino acids. The At2g21370 gene product from Arabidopsis thaliana, xylulose kinase-1 (AtXK-1), whose mature form contains 436 amino acids, is located in the chloroplast (ChloroP 1.1 Server). SePSK and AtXK-1 display a sequence identity of 44.9%, and belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. Members of this sub-family are responsible for the phosphorylation of sugars similar to L-ribulose and D-ribulose. The sequence and the substrate specificity of ribulokinase-like carbohydrate kinases are different, but they share the common folding feature with two domains. Domain I exhibits a ribonuclease H-like folding pattern, and is responsible for the substrate binding, while domain II possesses an actin-like ATPase domain that binds cofactor ATP. Two possible xylulose kinases (xylulose kinase-1: XK-1 and xylulose kinase-2: XK-2) from Arabidopsis thaliana were previously proposed. It was shown that XK-2 (At5g49650) located in the cytosol is indeed xylulose kinase. However, the function of XK-1 (At2g21370) inside the chloroplast stroma has remained unknown. SePSK from Synechococcus elongatus strain PCC 7942 is the homolog of AtXK-1, though its physiological function and substrates remain unclear. In order to obtain functional and structural information about these two proteins, here we reported the crystal structures of SePSK and AtXK-1. Our findings provide new details of the catalytic mechanism of SePSK and lay the foundation for future studies into its homologs in eukaryotes. Results and Discussion Overall structures of apo-SePSK and apo-AtXK-1 The attempt to solve the SePSK structure by molecular replacement method failed with ribulokinase from Bacillus halodurans (PDB code: 3QDK, 15.7% sequence identity) as an initial model. We therefore used single isomorphous replacement anomalous scattering method (SIRAS) for successful solution of the apo-SePSK structure at a resolution of 2.3 Å. Subsequently, the apo-SePSK structure was used as molecular replacement model to solve all other structures identified in this study. Our structural analysis showed that apo-SePSK consists of one SePSK protein molecule in an asymmetric unit. The amino-acid residues were traced from Val2 to His419, except for the Met1 residue and the seven residues at the C-termini. Apo-SePSK contains two domains referred to further on as domain I and domain II (Fig 1A). Domain I consists of non-contiguous portions of the polypeptide chains (aa. 2–228 and aa. 402–419), exhibiting 11 α-helices and 11 β-sheets. Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. In addition, four β-sheets (β7, β10, β12 and β16) and five α-helices (α8, α9, α13, α14 and α15) flank the left side of the core region. Domain II is comprised of aa. 229–401 and classified into B2 (β31/β29/β22/β23/β25/β24) and A3 (α26/α27/α28/α30) (Fig 1A and S1 Fig). In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). The overall folding of SePSK resembles a clip, with A2 of domain I acting as a hinge region. As a consequence, a deep cleft is formed between the two domains. Overall structures of SePSK and AtXK-1. (A) Three-dimensional structure of apo-SePSK. The secondary structural elements are indicated (α-helix: cyan, β-sheet: yellow). (B) Three-dimensional structure of apo-AtXK-1. The secondary structural elements are indicated (α-helix: green, β-sheet: wheat). Apo-AtXK-1 exhibits a folding pattern similar to that of SePSK in line with their high sequence identity (Fig 1B and S1 Fig). However, superposition of structures of AtXK-1 and SePSK shows some differences, especially at the loop regions. A considerable difference is found in the loop3 linking β3 and α4, which is stretched out in the AtXK-1 structure, while in the SePSK structure, it is bent back towards the inner part. The corresponding residues between these two structures (SePSK-Lys35 and AtXK-1-Lys48) have a distance of 15.4 Å (S3 Fig). Activity assays of SePSK and AtXK-1 In order to understand the function of these two kinases, we performed structural comparison using Dali server. The structures most closely related to SePSK are xylulose kinase, glycerol kinase and ribulose kinase, implying that SePSK and AtXK-1 might function similarly to these kinases. We first tested whether both enzymes possessed ATP hydrolysis activity in the absence of substrates. As shown in Fig 2A, both SePSK and AtXK-1 exhibited ATP hydrolysis activity. This finding is in agreement with a previous result showing that xylulose kinase (PDB code: 2ITM) possessed ATP hydrolysis activity without adding substrate. To further identify the actual substrate of SePSK and AtXK-1, five different sugar molecules, including D-ribulose, L-ribulose, D-xylulose, L-xylulose and Glycerol, were used in enzymatic activity assays. As shown in Fig 2B, the ATP hydrolysis activity of SePSK greatly increased upon adding D-ribulose than adding other potential substrates, suggesting that it has D-ribulose kinase activity. In contrary, limited increasing of ATP hydrolysis activity was detected for AtXK-1 upon addition of D-ribulose (Fig 2C), despite its structural similarity with SePSK. The enzymatic activity assays of SePSK and AtXK-1. (A) The ATP hydrolysis activity of SePSK and AtXK-1. Both SePSK and AtXK-1 showed ATP hydrolysis activity in the absence of substrate. While the ATP hydrolysis activity of SePSK greatly increases upon addition of D-ribulose (DR). (B) The ATP hydrolysis activity of SePSK with addition of five different substrates. The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. Three single-site mutants of SePSK are D8A-SePSK, T11A-SePSK and D221A-SePSK. The ATP hydrolysis activity measured via luminescent ADP-Glo assay (Promega). To understand the catalytic mechanism of SePSK, we performed structural comparisons among xylulose kinase, glycerol kinase, ribulose kinase and SePSK. Our results suggested that three conserved residues (D8, T11 and D221 of SePSK) play an important role in SePSK function. Mutations of the corresponding residue in xylulose kinase and glycerol kinase from Escherichia coli greatly reduced their activity. To identify the function of these three residues of SePSK, we constructed D8A, T11A and D221A mutants. Using enzymatic activity assays, we found that all of these mutants exhibit much lower activity of ATP hydrolysis after adding D-ribulose than that of wild type, indicating the possibility that these three residues are involved in the catalytic process of phosphorylation D-ribulose and are vital for the function of SePSK (Fig 2D). SePSK and AtXK-1 possess a similar ATP binding site To obtain more detailed information of SePSK and AtXK-1 in complex with ATP, we soaked the apo-crystals in the reservoir adding cofactor ATP, and obtained the structures of SePSK and AtXK-1 bound with ATP at the resolution of 2.3 Å and 1.8 Å, respectively. In both structures, a strong electron density was found in the conserved ATP binding pocket, but can only be fitted with an ADP molecule (S4 Fig). Thus the two structures were named ADP-SePSK and ADP-AtXK-1, respectively. The extremely weak electron densities of ATP γ-phosphate in both structures suggest that the γ-phosphate group of ATP is either flexible or hydrolyzed by SePSK and AtXK-1. This result was consistent with our enzymatic activity assays where SePSK and AtXK-1 showed ATP hydrolysis activity without adding any substrates (Fig 2A and 2C). To avoid hydrolysis of ATP, we soaked the crystals of apo-SePSK and apo-AtXK-1 into the reservoir adding AMP-PNP. However, we found that the electron densities of γ-phosphate group of AMP-PNP (AMP-PNP γ-phosphate) are still weak in the AMP-PNP-SePSK and AMP-PNP-AtXK-1 structures, suggesting high flexibility of ATP-γ-phosphate. The γ-phosphate group of ATP is transferred to the sugar substrate during the reaction process, so this flexibility might be important for the ability of these kinases. The overall structures as well as the coordination modes of ADP and AMP-PNP in the AMP-PNP-AtXK-1, ADP-AtXK-1, ADP-SePSK and AMP-PNP-SePSK structures are nearly identical (S5 Fig), therefore the structure of AMP-PNP-SePSK is used here to describe the structural details and to compare with those of other family members. As shown in Fig 3A, one SePSK protein molecule is in an asymmetric unit with one AMP-PNP molecule. The AMP-PNP is bound at the domain II, where it fits well inside a positively charged groove. The AMP-PNP binding pocket consists of four α-helices (α26, α28, α27 and α30) and forms a shape resembling a half-fist (Fig 3A and 3B). The head group of the AMP-PNP is embedded in a pocket surrounded by Trp383, Asn380, Gly376 and Gly377. The purine ring of AMP-PNP is positioned in parallel to the indole ring of Trp383. In addition, it is hydrogen-bonded with the side chain amide of Asn380 (Fig 3B). The tail of AMP-PNP points to the hinge region of SePSK, and its α-phosphate and β-phosphate groups are stabilized by Gly376 and Ser243, respectively. Together, this structure clearly shows that the AMP-PNP-β-phosphate is sticking out of the ATP binding pocket, thus the γ-phosphate group is at the empty space between domain I and domain II and is unconstrained in its movement by the protein. Structure of SePSK in complex with AMP-PNP. (A) The electron density of AMP-PNP. The SePSK structure is shown in the electrostatic potential surface mode. The AMP-PNP is depicted as sticks with its ǀFoǀ-ǀFcǀ map contoured at 3 σ shown as cyan mesh. (B) The AMP-PNP binding pocket. The head of AMP-PNP is sandwiched by four residues (Leu293, Gly376, Gly377 and Trp383). The protein skeleton is shown as cartoon (cyan). The four α-helices (α26, α28, α27 and α30) are labeled in red. The AMP-PNP and coordinated residues are shown as sticks. The interactions between them are represented as black dashed lines. The numerical note near the black dashed line indicates the distance (Å). The potential substrate binding site in SePSK The results from our activity assays suggested that SePSK has D-ribulose kinase activity. To better understand the interaction pattern between SePSK and D-ribulose, the apo-SePSK crystals were soaked into the reservoir with 10 mM D-ribulose (RBL) and the RBL-SePSK structure was solved. As shown in S6 Fig, two residual electron densities are visible in domain I, which can be interpreted as two D-ribulose molecules with reasonable fit. As shown in Fig 4A, the nearest distance between the carbon skeleton of two D-ribulose molecules are approx. 7.1 Å (RBL1-C4 and RBL2-C1). RBL1 is located in the pocket consisting of α21 and the loop between β6 and β7. The O4 and O5 of RBL1 are coordinated with the side chain carboxyl group of Asp221. Furthermore, the O2 of RBL1 interacts with the main chain amide nitrogen of Ser72 (Fig 4B). This pocket is at a similar position of substrate binding site of other sugar kinase, such as L-ribulokinase (PDB code: 3QDK) (S7 Fig). However, structural comparison shows that the substrate ligating residues between the two structures are not strictly conserved. Based on the structures, the ligating residues of RBL1 in RBL-SePSK structure are Ser72, Asp221 and Ser222, and the interacting residues of L-ribulose with L-ribulokinase are Ala96, Lys208, Asp274 and Glu329 (S7 Fig). Glu329 in 3QDK has no counterpart in RBL-SePSK structure. In addition, although Lys208 of L-ribulokinase has the corresponding residue (Lys163) in RBL-SePSK structure, the hydrogen bond of Lys163 is broken because of the conformational change of two α-helices (α9 and α13) of SePSK. These differences might account for their different substrate specificity. The binding of D-ribulose (RBL) with SePSK. (A) The electrostatic potential surface map of RBL-SePSK and a zoom-in view of RBL binding site. The RBL1 and RBL2 are depicted as sticks. (B) Interaction of two D-ribulose molecules (RBL1 and RBL2) with SePSK. The RBL molecules (carbon atoms colored yellow) and amino acid residues of SePSK (carbon atoms colored green) involved in RBL interaction are shown as sticks. The hydrogen bonds are indicated by the black dashed lines and the numbers near the dashed lines are the distances (Å). (C) The binding affinity assays of SePSK with D-ribulose. Single-cycle kinetic data are reflecting the interaction of SePSK and D8A-SePSK with D-ribulose. It shows two experimental sensorgrams after minus the empty sensorgrams. The original data is shown as black curve, and the fitted data is shown as different color (wild type SePSK: red curve, D8A-SePSK: green curve). Dissociation rate constant of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively. The binding pocket of RBL2 with relatively weak electron density is near the N-terminal region of SePSK and is negatively charged. The side chain of Asp8 interacts strongly with O3 and O4 of RBL2. The hydroxyl group of Ser12 coordinates with O2 of RBL2. The backbone amide nitrogens of Gly13 and Arg15 also keep hydrogen bonds with RBL2 (Fig 4B). Structural comparison of SePSK and AtXK-1 showed that while the RBL1 binding pocket is conserved, the RBL2 pocket is disrupted in AtXK-1 structure, despite the fact that the residues interacting with RBL2 are highly conserved between the two proteins. In the RBL-SePSK structure, a 2.6 Å hydrogen bond is present between RBL2 and Ser12 (Fig 4B), while in the AtXK-1 structure this hydrogen bond with the corresponding residue (Ser22) is broken. This break is probably induced by the conformational change of the two β-sheets (β1 and β2), with the result that the linking loop (loop 1) is located further away from the RBL2 binding site. This change might be the reason that AtXK-1 only shows limited increasing in its ATP hydrolysis ability upon adding D-ribulose as a substrate after comparing with SePSK (Fig 2C). Our SePSK structure shows that the Asp8 residue forms strong hydrogen bond with RBL2 (Fig 4B). In addition, our enzymatic assays indicated that Asp8 is important for the activity of SePSK (Fig 2D). To further verified this result, we measured the binding affinity for D-ribulose of both wild type (WT) and D8A mutant of SePSK using a surface plasmon resonance method. The results showed that the affinity of D8A-SePSK with D-ribulose is weaker than that of WT with a reduction of approx. two third (Fig 4C). Dissociation rate constant (Kd) of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively. The results implied that the second RBL binding site plays a role in the D-ribulose kinase function of SePSK. However, considering the high concentration of D-ribulose used for crystal soaking, as well as the relatively weak electron density of RBL2, it is also possible that the second binding site of D-ribulose in SePSK is an artifact. Simulated conformational change of SePSK during the catalytic process It was reported earlier that the crossing angle between the domain I and domain II in FGGY family carbohydrate kinases is different. In addition, this difference may be caused by the binding of substrates and/or ATP. As reported previously, members of the sugar kinase family undergo a conformational change to narrow the crossing angle between two domains and reduce the distance between substrate and ATP in order to facilitate the catalytic reaction of phosphorylation of sugar substrates. After comparing structures of apo-SePSK, RBL-SePSK and AMP-PNP-SePSK, we noticed that these structures presented here are similar. Superposing the structures of RBL-SePSK and AMP-PNP-SePSK, the results show that the nearest distance between AMP-PNP γ-phosphate and RBL1/RBL2 is 7.5 Å (RBL1-O5)/6.7 Å (RBL2-O1) (S8 Fig). This distance is too long to transfer the γ-phosphate group from ATP to the substrate. Since the two domains of SePSK are widely separated in this structure, we hypothesize that our structures of SePSK represent its open form, and that a conformational rearrangement must occur to switch to the closed state in order to facilitate the catalytic process of phosphorylation of sugar substrates. For studying such potential conformational change, a simulation on the Hingeprot Server was performed to predict the movement of different SePSK domains. The results showed that domain I and domain II are closer to each other with Ala228 and Thr401 in A2 as Hinge-residues. Based on the above results, SePSK is divided into two rigid parts. The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). The results of superposition displayed different crossing angle between these two domains. After superposition, the distances of AMP-PNP γ-phosphate and the fifth hydroxyl group of RBL1 are 7.9 Å (superposed with AtXK-1), 7.4 Å (superposed with SePSK), 6.6 Å (superposed with 3LL3) and 6.1 Å (superposed with 1GLJ). Meanwhile, the distances of AMP-PNP γ-phosphate and the first hydroxyl group of RBL2 are 7.2 Å (superposed with AtXK-1), 6.7 Å (superposed with SePSK), 3.7 Å (superposed with 3LL3), until AMP-PNP γ-phosphate fully contacts RBL2 after superposition with 1GLJ (Fig 5). This distance between RBL2 and AMP-PNP-γ-phosphate is close enough to facilitate phosphate transferring. Together, our superposition results provided snapshots of the conformational changes at different catalytic stages of SePSK and potentially revealed the closed form of SePSK. Simulated conformational change of SePSK during the catalytic process. The structures are shown as cartoon and the ligands are shown as sticks. Domain I from D-ribulose-SePSK (green) and Domain II from AMP-PNP-SePSK (cyan) are superposed with apo-AtXK-1 (1st), apo-SePSK (2nd), 3LL3 (3rd) and 1GLJ (4th), respectively. The numbers near the black dashed lines show the distances (Å) between two nearest atoms of RBL and AMP-PNP. In summary, our structural and enzymatic analyses provide evidence that SePSK shows D-ribulose kinase activity, and exhibits the conserved features of FGGY family carbohydrate kinases. Three conserved residues in SePSK were identified to be essential for this function. Our results provide the detailed information about the interaction of SePSK with ATP and substrates. Moreover, structural superposition results enable us to visualize the conformational change of SePSK during the catalytic process. In conclusion, our results provide important information for a more detailed understanding of the mechanisms of SePSK and other members of FGGY family carbohydrate kinases. Materials and Methods Cloning, expression and purification of SePSK The gene encoding SePSK was amplified by polymerase chain reaction (PCR) with forward primer 5' CATGCCATGGGCATGGTCGTTGCACTTGGCCTCG 3' containing an internal Nco I restriction site (underlined) and reverse primer 5' CCGCTCGAGGGTTCTCTTTAACCCCGCCG 3' including an internal Xho I restriction site (underlined) from Synechococcus elongatus PCC 7942 genomic DNA. The amplified PCR product was digested with Nco I and Xho I (Takara) and ligated into linearized pET28-a vector (Novagen) between Nco I and Xho I restriction sites with a C-terminal his6 tag. The recombinant plasmids were transformed into competent Escherichia coli Trans10 cells for DNA production and purification, and the final constructs were verified by sequencing. The recombinant vectors were transformed into Escherichia coli BL21 (DE3) to express the protein. After induction with the 1 mM IPTG at 289 K in Luria-Bertani medium until the cell density reached an OD 600 nm of 0.6–0.8, the cells were harvested by centrifugation at 6,000 g at 4°C for 15 min, re-suspended in buffer A (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole) and disrupted by sonication. After centrifuge 40,000 g for 30 min, the protein was purified by passage through a Ni2+ affinity column in buffer A, and then washed the unbound protein with buffer B (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 60 mM imidazole), and eluted the fraction with the buffer C (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 500 mM imidazole). After that, the protein was further purified by size exclusion chromatography with Superdex 200 10/300 GL (GE Healthcare) equilibrated with the buffer D (20 mM Tris-HCl, pH 8.0, 300 mM NaCl). The eluted major peak fraction was concentrated to 20 mg/mL protein using 10,000 MCWO centrifugal filter units (Millipore) and stored at -80°C for crystallization trials. The purified product was analyzed by SDS-PAGE with a single band visible only. Cloning, expression and purification of AtXK-1 The gene encoding AtXK-1 was amplified by PCR using a forward primer 5' TACTTCCAATCCAATGCTGTTATGAGTGGCAATAAAGGAACGA 3' and reverse primer 5' TTATCCACTTCCAATGTTACAAACCACTGTTCTGTTTTGCGCCC 3' from cDNA library of Arabidopsis thaliana. The underlined nucleotides were used for the ligation-independent cloning. The PCR product was treated by T4 DNA polymerase (LIC-qualified, Novagen) and then cloned into linearized pMCSG7 vector treated by T4 DNA polymerase (LIC-qualified, Novagen) with an N-terminal his6 tag though ligation-independent cloning method. The final construct was confirmed by DNA sequencing after amplified in competent Escherichia coli Trans10 cells. The recombinant vectors were transformed into Escherichia coli BL21 (DE3) for protein expression. After induction with 1 mM IPTG at 289 K in Luria-Bertani medium, cells were grown until the cell density reached an OD 600 nm of 0.6–0.8. Subsequent purification was identical to that used for SePSK, except that there was one additional step, during which tobacco etch virus protease was used to digest the crude AtXK-1 protein for removal of the N-terminal his6 tag following Ni2+ affinity purification. Ni2+ affinity column buffer contained extra 20% glycerol. The protein was further purified by size exclusion chromatography with Superdex 200 10/300 GL (GE Healthcare) in elution buffer consisting of 20 mM HEPES, pH 7.5, 100 mM NaCl. Finally, AtXK-1 protein was concentrated to 40 mg/mL protein using 10,000 MCWO centrifugal filter units (Millipore) and stored at -80°C prior to crystallization trials. Purity was verified by SDS-PAGE with a single band visible only. Site-directed mutagenesis of SePSK The gene of D8A and T11A mutations were amplified by PCR with the forward primer 5' CATGCCATGGGCATGGTCGTTGCACTTGGCCTCGCCTTCGGCAC 3' and forward primer 5' CATGCCATGGGCATGGTCGTTGCACTTGGCCTCGACTTCGGCGCCTCTGGAGCCC 3' (mismatched base pairs are underlined). The reverse primers of D8A and T11A mutants, the further constructions and purification procedures were identical with those used for wild type SePSK. The N-terminal sequence of D221A was amplified with forward primer (T7 promoter primer) 5' TAATACGACTCACTATA 3' and reverse primer 5' AGCAGCAATGCTAGCCGTTGTACCG 3’, and the C-terminal sequence of D221A was amplified with forward primer 5' TGCCGGTACAACGGCTAGCATTGCT 3' and reverse primer (T7 terminator primer) CGATCAATAACGAGTCGCC (mismatched base pairs are underlined). The second cycle PCR used the above PCR products as templates, and the construction and purification procedures were identical to those used for wild type SePSK. Crystallization and data collection Crystallization trials of SePSK and AtXK-1 were carried out at 281 K by mixing equal volume of 20 mg/ml protein and reservoir solution with the sitting-drop vapor diffusion method. The reservoir solution was PEG Rx I-35 (0.1 M BIS-TRIS pH 6.5, 20% w/v Polyethylene glycol monomethyl ether 5,000) (Hampton research). After 2 or 3 days, the rod-like crystals could be observed. For phasing, the high-quality apo-SePSK crystals were soaked in mother liquor containing 1 mM ethylmercuricthiosalicylic acid, sodium salt (Hampton research, heavy atom kit) overnight at 281 K. In order to get the complexes with ATP and AMP-PNP, the crystals of apo-SePSK and apo-AtXK-1 were incubated with the reservoir including 10 mM ATP and 20 mM MgCl2 as well as 10 mM AMP-PNP and 20 mM MgCl2, respectively. The apo-SePSK crystals were incubated with the reservoir including 10 mM D-ribulose in order to obtain the complex D-ribulose-bound SePSK (RBL-SePSK). The crystals of three mutants (D8A, T11A and D221A) grew in the same condition as that of the wild type SePSK. The crystals were dipped into reservoir solution supplemented with 15% glycerol and then flash frozen in a nitrogen gas stream at 100 K. All data sets were collected at Shanghai Synchrotron Radiation Facility, Photo Factory in Japan and Institute of Biophysics, Chinese Academy of Sciences. Diffraction data were processed using the HKL2000 package. Structure determination and refinement The initial phases of SePSK were obtained from the Hg-derivative crystals by single isomorphous replacement anomalous scattering (SIRAS) using AutoSol from the PHENIX suite. AutoBuild from the PHENIX suite was used to build 75% of the main chain of apo-SePSK, and the remaining residues were built manually by Coot. All other structures were solved by molecular replacement method using apo-SePSK as an initial model. The model was refined using phenix.refine and REFMAC5. The final model was checked with PROCHECK. All structural figures were prepared by PyMOL. The summary of the data-collection and structure-refinement statistics is shown in Table 1 and S1 Table. Atomic coordinates and structure factors in this article have been deposited in the Protein Data Bank. The deposited codes of all structures listed in the Table 1 and S1 Table. Data collection and refinement statistics. Data set\tHg-SePSK\tapo-SePSK\tAMP-PNP-SePSK\tRBL-SePSK\tapo-AtXK-1\t \tData collection\t\t\t\t\t\t \tSpace group\tC 1 2 1\tC 1 2 1\tC 1 2 1\tC 1 2 1\tP21\t \tWavelength (Å)\t1.54178\t1.54178\t1.54178\t1.54178\t1.54178\t \tCell parameters\t\t\t\t\t\t \ta/b/c(Å)\t103.1, 46.6, 88.3\t110.2, 49.0, 86.9\t103.5, 46.6, 88.0\t102.6, 47.0, 88.7\t49.7, 87.9, 53.6\t \tα/β/γ(°)\t90.0, 91.9, 90.0\t90.0, 110.3, 90.0\t90.0, 91.0, 90.0\t90.0, 91.4, 90.0\t90.0, 97.0, 90.0\t \tResolution (Å)a\t50.00–2.20(2.28–2.20)\t50.00–2.30(2.38–2.30)\t50.00–2.30(2.38–2.30)\t50.00–2.35(2.43–2.35)\t50.00–2.00(2.07–2.00)\t \tR mergeb\t0.105(0.514)\t0.149(0.501)\t0.082(0.503)\t0.095(0.507)\t0.106(0.454)\t \t〈 I/σ(I)〉\t28.89(4.07)\t13.85(4.10)\t10.18(1.79)\t19.4(4.6)\t12.91(4.08)\t \tCompleteness (%)\t92.3(99.2)\t96.1(94.2)\t98.9(99.8)\t99.8(100.0)\t97.1(94.5)\t \tRedundancy\t6.7(5.1)\t7.4(7.5)\t2.4(2.4)\t6.9(6.7)\t7.2(6.9)\t \tRefinement statistics\t\t\t\t\t\t \tResolution (Å)\t\t32.501–2.301\t24.707–2.300\t24.475–2.344\t23.771–1.998\t \tRwork/ Rfreec\t\t0.1834/0.2276\t0.1975/0.2327\t0.2336/0.2687\t0.1893/0.2161\t \tNo. atoms\t\t\t\t\t\t \tProtein\t\t3503\t3196\t3209\t3256\t \tligand/ion\t\t-\t31\t20\t-\t \tWater\t\t313\t146\t143\t486\t \tRMSD Bond lengths (Å)d\t\t0.003\t0.005\t0.003\t0.003\t \tRMSD Bond angles (°)d\t\t0.674\t0.886\t0.649\t0.838\t \tRamachandran plot (%)\t\t\t\t\t\t \tfavoured\t\t98.1\t97.8\t96.7\t99.1\t \tallowed\t\t1.9\t2.2\t3.3\t0.9\t \tdisallowed\t\t0.0\t0.0\t0.0\t0.0\t \tPDB code\t\t5HTN\t5HTP\t5HV7\t5HTR\t \t a The values in parentheses correspond to the highest resolution shell. b Rmerge = ∑j∑h|Ij,h-\u003cIh\u003e|/∑j∑h\u003cIh\u003e where h are unique reflection indices and Ij,h are intensities of symmetry-related reflections and \u003cIh\u003e is the mean intensity. c R-work and R-free were calculated as follows: R = Σ (|Fobs-Fcalc|)/Σ |Fobs| ×100, where Fobs and Fcalc are the observed and calculated structure factor amplitudes, respectively. d Root mean square deviations (r.m.s.d.) from standard values. ADP-Glo kinase assay ADP-Glo kinase assay was used according to the manufacturer’s instructions (Promega). Each reaction mixture system consisted of 8 uM enzyme, 100 uM ATP, 1 mM MgCl2, 20 mM HEPES (pH 7.4), 5 mM substrate. The reaction was initiated by adding the purified enzyme into the reaction system. After incubation at 298 K for different time, equal volume ADP-Glo™ reagent was added to terminate the kinase reaction and to deplete any remaining ATP. Subsequently, kinase detection reagent with double volume of reaction system was added to convert ADP to ATP and allowed the newly synthesized ATP to be measured using a luciferase/luciferin reaction which produced luminescence signal and could be recorded. After incubation at room temperature for another 60 min, luminescence was detected by Varioskan Flash Multimode Reader (Thermo). The reference experiment was carried out in the same reaction system without the enzyme. For each assay, at least three repeats were performed for the calculation of mean values and standard deviations (SDs). The purity of five substrates in the activity assays was ≥98% (D-ribulose, Santa cruz), 99.7% (L-ribulose, Carbosynth), 99.3% (D-xylulose, Carbosynth), 99.5% (L-xylulose, Carbosynth) and 99.0% (Glycerol, AMRESCO). Surface plasmon resonance Surface plasmon resonance (SPR) was used to analyze the interaction of SePSK and D-ribulose. The SPR experiments were performed on a Biacore T100 system using series S CM5 sensor chips (GE Healthcare). All sensorgrams were recorded at 298 K. The proteins in buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCl, was diluted to 40 ug/ml by 10 mM sodium acetate buffer at pH 4.5. All flow cells on a CM5 sensor chip were activated with a freshly prepared solution of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.05 M N-hydroxysuccinimide (NHS) in a ratio of 1:1 at a constant flow rate of 10 ul/min for 420 s. Deactivation of the surface was performed with an injection of a 1 M solution of ethanolamine-HCl (pH 8.5) using the same flow rate and duration. Kinetic parameters were derived from data sets acquired in single-cycle mode. Each run consisted of five consecutive analytic injections at 125, 250, 500, 1000 and 2000 uM. Analytic injections lasted for 60 s, separated by 30 s dissociation periods. Each cycle was completed with an extended dissociation period of 300 s. The specific binding to a blank flow cell was subtracted to obtain corrected sensorgrams. Biacore data were analyzed using BiaEvaluation software (GE Healthcare) by fitting to a 1:1 Langmuir binding fitting model. Accession Codes Coordinates and structure factors for all the structures in this article have been deposited in the Protein Data Bank. These accession codes are 5HTN, 5HTP, 5HUX, 5HV7, 5HTJ, 5HU2, 5HTY, 5HTR, 5HTV and 5HTX. The corresponding-structures are apo-SePSK, AMP-PNP-SePSK, ADP-SePSK, RBL-SePSK, D8A-SePSK, T11A-SePSK, D221A-SePSK, apo-AtXK1, AMP-PNP-AtXK1 and ADP-AtXK1, respectively. Supporting Information References The FGGY carbohydrate kinase family: insights into the evolution of functional specificities Structure and function of human xylulokinase, an enzyme with important roles in carbohydrate metabolism Structural insight into mechanism and diverse substrate selection strategy of L-ribulokinase Structural and Kinetic Studies of Induced Fit in Xylulose Kinase from Escherichia coli ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites A cytosolic Arabidopsis D-xylulose kinase catalyzes the phosphorylation of 1-deoxy-D-xylulose into a precursor of the plastidial isoprenoid pathway Crystal structures of Escherichia coli glycerol kinase variant S58→ W in complex with nonhydrolyzable ATP analogues reveal a putative active conformation of the enzyme as a result of domain motion Glycerol kinase from Escherichia coli and an Ala65→ Thr mutant: the crystal structures reveal conformational changes with implications for allosteric regulation Structure and Reaction Mechanism of l-Rhamnulose Kinase from Escherichia coli Structural and Functional Analysis of Fucose-Processing Enzymes from Streptococcus pneumoniae Conserved active site aspartates and domain–domain interactions in regulatory properties of the sugar kinase superfamily Analyzing a kinetic titration series using affinity biosensors Structures of enterococcal glycerol kinase in the absence and presence of glycerol: correlation of conformation to substrate binding and a mechanism of activation by phosphorylation The sugar kinase/heat shock protein 70/actin superfamily: implications of conserved structure for mechanism HingeProt: automated prediction of hinges in protein structures A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site Processing of X-ray diffraction data collected in oscillation mode PHENIX: a comprehensive Python-based system for macromolecular structure solution Coot: model-building tools for molecular graphics On macromolecular refinement at subatomic resolution with interatomic scatterers PROCHECK: a program to check the stereochemical quality of protein structures DeLano. W. The PyMOL Molecular Graphics System. Available: http://www.pymol.org. Comparison of the luminescent ADP-Glo assay to a standard radiometric assay for measurement of protein kinase 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diff --git a/annotated_BioC_JSON/PMC4887326_ann.json b/annotated_BioC_JSON/PMC4887326_ann.json
new file mode 100644
index 0000000000000000000000000000000000000000..c092a6c6021ed4dd0c2afc21878a2b7610e7628d
--- /dev/null
+++ b/annotated_BioC_JSON/PMC4887326_ann.json
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+[{"sourceid":"4887326","sourcedb":"","project":"","target":"","text":"Structural insights into the regulatory mechanism of the Pseudomonas aeruginosa YfiBNR system YfiBNR is a recently identified bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) signaling system in opportunistic pathogens. It is a key regulator of biofilm formation, which is correlated with prolonged persistence of infection and antibiotic drug resistance. In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. However, the detailed regulatory mechanism remains elusive. Here, we report the crystal structures of YfiB alone and of an active mutant YfiBL43P complexed with YfiR with 2:2 stoichiometry. Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation. Based on the structural and biochemical data, we propose an updated regulatory model of the YfiBNR system. INTRODUCTION Bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) is a ubiquitous second messenger that bacteria use to facilitate behavioral adaptations to their ever-changing environment. An increase in c-di-GMP promotes biofilm formation, and a decrease results in biofilm degradation (Boehm et al.,; Duerig et al.,; Hickman et al.,; Jenal,; Romling et al.,). The c-di-GMP level is regulated by two reciprocal enzyme systems, namely, diguanylate cyclases (DGCs) that synthesize c-di-GMP and phosphodiesterases (PDEs) that hydrolyze c-di-GMP (Kulasakara et al.,; Ross et al.,; Ross et al.,). Many of these enzymes are multiple-domain proteins containing a variable N-terminal domain that commonly acts as a signal sensor or transduction module, followed by the relatively conserved GGDEF motif in DGCs or EAL/HD-GYP domains in PDEs (Hengge,; Navarro et al.,; Schirmer and Jenal,). Intriguingly, studies in diverse species have revealed that a single bacterium can have dozens of DGCs and PDEs (Hickman et al.,; Kirillina et al.,; Kulasakara et al.,; Tamayo et al.,). In Pseudomonas aeruginosa in particular, 42 genes containing putative DGCs and/or PDEs were identified (Kulasakara et al.,). The functional role of a number of downstream effectors of c-di-GMP has been characterized as affecting exopolysaccharide (EPS) production, transcription, motility, and surface attachment (Caly et al.,; Camilli and Bassler,; Ha and O’Toole,; Pesavento and Hengge,). However, due to the intricacy of c-di-GMP signaling networks and the diversity of experimental cues, the detailed mechanisms by which these signaling pathways specifically sense and integrate different inputs remain largely elusive. Biofilm formation protects pathogenic bacteria from antibiotic treatment, and c-di-GMP-regulated biofilm formation has been extensively studied in P. aeruginosa (Evans,; Kirisits et al.,; Malone,; Reinhardt et al.,). In the lungs of cystic fibrosis (CF) patients, adherent biofilm formation and the appearance of small colony variant (SCV) morphologies of P. aeruginosa correlate with prolonged persistence of infection and poor lung function (Govan and Deretic,; Haussler et al.,; Haussler et al.,; Parsek and Singh,; Smith et al.,). Recently, Malone and coworkers identified the tripartite c-di-GMP signaling module system YfiBNR (also known as AwsXRO (Beaumont et al.,; Giddens et al.,) or Tbp (Ueda and Wood,)) by genetic screening for mutants that displayed SCV phenotypes in P. aeruginosa PAO1 (Malone et al.,; Malone et al.,). The YfiBNR system contains three protein members and modulates intracellular c-di-GMP levels in response to signals received in the periplasm (Malone et al.,). More recently, this system was also reported in other Gram-negative bacteria, such as Escherichia coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), Klebsiella pneumonia (Huertas et al.,) and Yersinia pestis (Ren et al.,). YfiN is an integral inner-membrane protein with two potential transmembrane helices, a periplasmic Per-Arnt-Sim (PAS) domain, and cytosolic domains containing a HAMP domain (mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and a C-terminal GGDEF domain indicating a DGC’s function (Giardina et al.,; Malone et al.,). YfiN is repressed by specific interaction between its periplasmic PAS domain and the periplasmic protein YfiR (Malone et al.,). YfiB is an OmpA/Pal-like outer-membrane lipoprotein (Parsons et al.,) that can activate YfiN by sequestering YfiR (Malone et al.,) in an unknown manner. Whether YfiB directly recruits YfiR or recruits YfiR via a third partner is an open question. After the sequestration of YfiR by YfiB, the c-di-GMP produced by activated YfiN increases the biosynthesis of the Pel and Psl EPSs, resulting in the appearance of the SCV phenotype, which indicates enhanced biofilm formation (Malone et al.,). It has been reported that the activation of YfiN may be induced by redox-driven misfolding of YfiR (Giardina et al.,; Malone et al.,; Malone et al.,). It is also proposed that the sequestration of YfiR by YfiB can be induced by certain YfiB-mediated cell wall stress, and mutagenesis studies revealed a number of activation residues of YfiB that were located in close proximity to the predicted first helix of the periplasmic domain (Malone et al.,). In addition, quorum sensing-related dephosphorylation of the PAS domain of YfiN may also be involved in the regulation (Ueda and Wood,; Xu et al.,). Recently, we solved the crystal structure of YfiR in both the non-oxidized and the oxidized states, revealing breakage/formation of one disulfide bond (Cys71-Cys110) and local conformational change around the other one (Cys145-Cys152), indicating that Cys145-Cys152 plays an important role in maintaining the correct folding of YfiR (Yang et al.,). In the present study, we solved the crystal structures of an N-terminal truncated form of YfiB (34–168) and YfiR in complex with an active mutant YfiBL43P. Most recently, Li and coworkers reported the crystal structures of YfiB (27–168) alone and YfiRC71S in complex with YfiB (59–168) (Li et al.,). Compared with the reported complex structure, YfiBL43P in our YfiB-YfiR complex structure has additional visible N-terminal residues 44–58 that are shown to play essential roles in YfiB activation and biofilm formation. Therefore, we are able to visualize the detailed allosteric arrangement of the N-terminal structure of YfiB and its important role in YfiB-YfiR interaction. In addition, we found that the YfiBL43P shows a much higher PG-binding affinity than wild-type YfiB, most likely due to its more compact PG-binding pocket. Moreover, we found that Vitamin B6 (VB6) or L-Trp can bind YfiR with an affinity in the ten millimolar range. Together with functional data, these results provide new mechanistic insights into how activated YfiB sequesters YfiR and releases the suppression of YfiN. These findings may facilitate the development and optimization of anti-biofilm drugs for the treatment of chronic infections. RESULTS Overall structure of YfiB We obtained two crystal forms of YfiB (residues 34–168, lacking the signal peptide from residues 1–26 and periplasmic residues 27–33), crystal forms I and II, belonging to space groups P21 and P41, respectively. Overall structure of YfiB. (A) The overall structure of the YfiB monomer. (B) A topology diagram of the YfiB monomer. (C and D) The analytical ultracentrifugation experiment results for the wild-type YfiB and YfiBL43P\n Two dimeric types of YfiB dimer. (A–C) The “head to head” dimer. (D–F) The “back to back” dimer. (A) and (E) indicate the front views of the two dimers, (B) and (F) indicate the top views of the two dimers, and (C) and (D) indicate the details of the two dimeric interfaces The crystal structure of YfiB monomer consists of a five-stranded β-sheet (β1-2-5-3-4) flanked by five α-helices (α1–5) on one side. In addition, there is a short helix turn connecting the β4 strand and α4 helix (Fig. 1A and 1B). Each crystal form contains three different dimeric types of YfiB, two of which are present in both, suggesting that the rest of the dimeric types may result from crystal packing. Here, we refer to the two dimeric types as “head to head” and “back to back” according to the interacting mode (Fig. 2A and 2E), with the total buried surface areas being 316.8 Å2 and 554.3 Å2, respectively. The “head to head” dimer exhibits a clamp shape. The dimerization occurs mainly via hydrophobic interactions formed by A37 and I40 on the α1 helices, L50 on the β1 strands, and W55 on the β2 strands of both molecules, making a hydrophobic interacting core (Fig. 2A–C). The “back to back” dimer presents a Y shape. The dimeric interaction is mainly hydrophilic, occurring among the main-chain and side-chain atoms of N68, L69, D70 and R71 on the α2-α3 loops and R116 and S120 on the α4 helices of both molecules, resulting in a complex hydrogen bond network (Fig. 2D–F). The YfiB-YfiR interaction Overall structure of the YfiB-YfiR complex and the conserved surface in YfiR. (A) The overall structure of the YfiB-YfiR complex. The YfiBL43P molecules are shown in cyan and yellow. The YfiR molecules are shown in green and magenta. Two interacting regions are highlighted by red rectangles. (B) Structural superposition of apo YfiB and YfiR-bound YfiBL43P. To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34–70 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44–70 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P. The residues proposed to contribute to YfiB activation are illustrated in sticks. The key residues in apo YfiB are shown in red and those in YfiBL43P are shown in blue. (D) Close-up views showing interactions in regions I and II. YfiBL43P and YfiR are shown in cyan and green, respectively. (E and F) The conserved surface in YfiR contributes to the interaction with YfiB. (G) The residues of YfiR responsible for interacting with YfiB are shown in green sticks, and the proposed YfiN-interacting residues are shown in yellow sticks. The red sticks, which represent the YfiB-interacting residues, are also responsible for the proposed interactions with YfiN To gain structural insights into the YfiB-YfiR interaction, we co-expressed YfiB (residues 34–168) and YfiR (residues 35–190, lacking the signal peptide), but failed to obtain the complex, in accordance with a previous report in which no stable complex of YfiB-YfiR was observed (Malone et al.,). It has been reported that single mutants of Q39, L43, F48 and W55 contribute to YfiB activation leading to the induction of the SCV phenotype in P. aeruginosa PAO1 (Malone et al.,). It is likely that these residues may be involved in the conformational changes of YfiB that are related to YfiR sequestration (Fig. 3C). Therefore, we constructed two such single mutants of YfiB (YfiBL43P and YfiBF48S). As expected, both mutants form a stable complex with YfiR. Finally, we crystalized YfiR in complex with the YfiBL43P mutant and solved the structure at 1.78 Å resolution by molecular replacement using YfiR and YfiB as models. The YfiB-YfiR complex is a 2:2 heterotetramer (Fig. 3A) in which the YfiR dimer is clamped by two separated YfiBL43P molecules with a total buried surface area of 3161.2 Å2. The YfiR dimer in the complex is identical to the non-oxidized YfiR dimer alone (Yang et al.,), with only Cys145-Cys152 of the two disulfide bonds well formed, suggesting Cys71-Cys110 disulfide bond formation is not essential for forming YfiB-YfiR complex. The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the α1 helix (residues 34–43) is invisible on the electron density map, and the α2 helix and β1 and β2 strands are rearranged to form a long loop containing two short α-helix turns (Fig. 3B and 3C), thus embracing the YfiR dimer. The observed changes in conformation of YfiB and the results of mutagenesis suggest a mechanism by which YfiB sequesters YfiR. The YfiB-YfiR interface can be divided into two regions (Fig. 3A and 3D). Region I is formed by numerous main-chain and side-chain hydrophilic interactions between residues E45, G47 and E53 from the N-terminal extended loop of YfiB and residues S57, R60, A89 and H177 from YfiR (Fig. 3D-I(i)). Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). In region II, the side chains of R96, E98 and E157 from YfiB interact with the side chains of E163, S146 and R171 from YfiR, respectively. Additionally, the main chains of I163 and V165 from YfiB form hydrogen bonds with the main chains of L166 and A164 from YfiR, respectively, and the main chain of P166 from YfiB interacts with the side chain of R185 from YfiR (Fig. 3D-II). These two regions contribute a robust hydrogen-bonding network to the YfiB-YfiR interface, resulting in a tightly bound complex. Based on the observations that two separated YfiBL43P molecules form a 2:2 complex structure with YfiR dimer, we performed an analytical ultracentrifugation experiment to check the oligomeric states of wild-type YfiB and YfiBL43P. The results showed that wild-type YfiB exists in both monomeric and dimeric states in solution, while YfiBL43P primarily adopts the monomer state in solution (Fig. 1C–D). This suggests that the N-terminus of YfiB plays an important role in forming the dimeric YfiB in solution and that the conformational change of residue L43 is associated with the stretch of the N-terminus and opening of the dimer. Therefore, it is possible that both dimeric types might exist in solution. For simplicity, we only discuss the “head to head” dimer in the following text. The PG-binding site of YfiB The PG-binding site in YfiB. (A) Structural superposition of the PG-binding sites of the H. influenzae Pal/PG-P complex and YfiR-bound YfiBL43P complexed with sulfate ions. (B) Close-up view showing the key residues of Pal interacting with the m-Dap5 ε-carboxylate group of PG-P. Pal is shown in wheat and PG-P is in magenta. (C) Close-up view showing the key residues of YfiR-bound YfiBL43P interacting with a sulfate ion. YfiR-bound YfiBL43P is shown in cyan; the sulfate ion, in green; and the water molecule, in yellow. (D) Structural superposition of the PG-binding sites of apo YfiB and YfiR-bound YfiBL43P, the key residues are shown in stick. Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA PG-associated lipoprotein (Pal) is highly conserved in Gram-negative bacteria and anchors to the outer membrane through an N-terminal lipid attachment and to PG layer through its periplasmic domain, which is implicated in maintaining outer membrane integrity. Previous homology modeling studies suggested that YfiB contains a Pal-like PG-binding site (Parsons et al.,), and the mutation of two residues at this site, D102 and G105, reduces the ability for biofilm formation and surface attachment (Malone et al.,). In the YfiB-YfiR complex, one sulfate ion is found at the bottom of each YfiBL43P molecule (Fig. 3A) and forms a strong hydrogen bond with D102 of YfiBL43P (Fig. 4A and 4C). Structural superposition between YfiBL43P and Haemophilus influenzae Pal complexed with biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala (m-Dap is meso-diaminopimelate) (PDB code: 2aiz) (Parsons et al.,), revealed that the sulfate ion is located at the position of the m-Dap5 ϵ-carboxylate group in the Pal/PG-P complex (Fig. 4A). In the Pal/PG-P complex structure, the m-Dap5 ϵ-carboxylate group interacts with the side-chain atoms of D71 and the main-chain amide of D37 (Fig. 4B). Similarly, in the YfiR-bound YfiBL43P structure, the sulfate ion interacts with the side-chain atoms of D102 (corresponding to D71 in Pal) and R117 (corresponding to R86 in Pal) and the main-chain amide of N68 (corresponding to D37 in Pal). Moreover, a water molecule was found to bridge the sulfate ion and the side chains of N67 and D102, strengthening the hydrogen bond network (Fig. 4C). In addition, sequence alignment of YfiB with Pal and the periplasmic domain of OmpA (proteins containing PG-binding site) showed that N68 and D102 are highly conserved (Fig. 4G, blue stars), suggesting that these residues contribute to the PG-binding ability of YfiB. Interestingly, superposition of apo YfiB with YfiR-bound YfiBL43P revealed that the PG-binding region is largely altered mainly due to different conformation of the N68 containing loop. Compared to YfiBL43P, the N68-containing loop of the apo YfiB flips away about 7 Å, and D102 and R117 swing slightly outward; thus, the PG-binding pocket is enlarged with no sulfate ion or water bound (Fig. 4D). Therefore, we proposed that the PG-binding ability of inactive YfiB might be weaker than that of active YfiB. To validate this, we performed a microscale thermophoresis (MST) assay to measure the binding affinities of PG to wild-type YfiB and YfiBL43P, respectively. The results indicated that the PG-binding affinity of YfiBL43P is 65.5 μmol/L, which is about 16-fold stronger than that of wild-type YfiB (Kd = 1.1 mmol/L) (Fig. 4E–F). As the experiment is performed in the absence of YfiR, it suggests that an increase in the PG-binding affinity of YfiB is not a result of YfiB-YfiR interaction and is highly coupled to the activation of YfiB characterized by a stretched N-terminal conformation. The conserved surface in YfiR is functional for binding YfiB and YfiN Calculation using the ConSurf Server (http://consurf.tau.ac.il/), which estimates the evolutionary conservation of amino acid positions and visualizes information on the structure surface, revealed a conserved surface on YfiR that contributes to the interaction with YfiB (Fig. 3E and 3F). Interestingly, the majority of this conserved surface contributes to the interaction with YfiB (Fig. 3E and 3F). Malone JG et al. have reported that F151, E163, I169 and Q187, located near the C-terminus of YfiR, comprise a putative YfiN binding site (Malone et al.,). Interestingly, these residues are part of the conserved surface of YfiR (Fig. 3G). F151, E163 and I169 form a hydrophobic core while, Q187 is located at the end of the α6 helix. E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). Collectively, a part of the YfiB-YfiR interface overlaps with the proposed YfiR-YfiN interface, suggesting alteration in the association-disassociation equilibrium of YfiR-YfiN and hence the ability of YfiB to sequester YfiR. YfiR binds small molecules Previous studies indicated that YfiR constitutes a YfiB-independent sensing device that can activate YfiN in response to the redox status of the periplasm, and we have reported YfiR structures in both the non-oxidized and the oxidized states earlier, revealing that the Cys145-Cys152 disulfide bond plays an essential role in maintaining the correct folding of YfiR (Yang et al.,). However, whether YfiR is involved in other regulatory mechanisms is still an open question. Overall Structures of VB6-bound and Trp-bound YfiR. (A) Superposition of the overall structures of VB6-bound and Trp-bound YfiR. (B) Close-up views showing the key residues of YfiR that bind VB6 and L-Trp. The electron densities of VB6 and Trp are countered at 3.0σ and 2.3σ, respectively, in |Fo|-|Fc| maps. (C) Superposition of the hydrophobic pocket of YfiR with VB6, L-Trp and F57 of YfiB Intriguingly, a Dali search (Holm and Rosenstrom,) indicated that the closest homologs of YfiR shared the characteristic of being able to bind several structurally similar small molecules, such as L-Trp, L-Phe, B-group vitamins and their analogs, encouraging us to test whether YfiR can recognize these molecules. For this purpose, we co-crystallized YfiR or soaked YfiR crystals with different small molecules, including L-Trp and B-group vitamins. Fortunately, we found obvious small-molecule density in the VB6-bound and Trp-bound YfiR crystal structures (Fig. 5A and 5B), and in both structures, the YfiR dimers resemble the oxidized YfiR structure in which both two disulfide bonds are well formed (Yang et al.,). Functional analysis of VB6 and L-Trp. (A and B) The effect of increasing concentrations of VB6 or L-Trp on YfiBL43P-induced attachment (bars). The relative optical density is represented as curves. Wild-type YfiB is used as negative control. (C and D) BIAcore data and analysis for binding affinities of (C) VB6 and (D) L-Trp with YfiR. (E–G) ITC data and analysis for titration of (E) YfiB wild-type, (F) YfiBL43P, and (G) YfiBL43P/F57A into YfiR Structural analyses revealed that the VB6 and L-Trp molecules are bound at the periphery of the YfiR dimer, but not at the dimer interface. Interestingly, VB6 and L-Trp were found to occupy the same hydrophobic pocket, formed by L166/I169/V176/P178/L181 of YfiR, which is also a binding pocket for F57 of YfiB, as observed in the YfiB-YfiR complex (Fig. 5C). To evaluate the importance of F57 in YfiBL43P-YfiR interaction, the binding affinities of YfiBL43P and YfiBL43P/F57A for YfiR were measured by isothermal titration calorimetry (ITC). The results showed Kd values of 1.4 × 10−7 mol/L and 5.3 × 10−7 mol/L for YfiBL43P and YfiBL43P/F57A, respectively, revealing that the YfiBL43P/F57A mutant caused a 3.8-fold reduction in the binding affinity compared with the YfiBL43P mutant (Fig. 6F and 6G). In parallel, to better understand the putative functional role of VB6 and L-Trp, yfiB was deleted in a PAO1 wild-type strain, and a construct expressing the YfiBL43P mutant was transformed into the PAO1 ΔyfiB strain to trigger YfiBL43P-induced biofilm formation. Growth and surface attachment assays were carried out for the yfiB-L43P strain in the presence of increasing concentrations of VB6 or L-Trp. As shown in Fig. 6A and 6B, the over-expression of YfiBL43P induced strong surface attachment and much slower growth of the yfiB-L43P strain, and as expected, a certain amount of VB6 or L-Trp (4–6 mmol/L for VB6 and 6–10 mmol/L for L-Trp) could reduce the surface attachment. Interestingly, at a concentration higher than 8 mmol/L, VB6 lost its ability to inhibit biofilm formation, implying that the VB6-involving regulatory mechanism is highly complicated and remains to be further investigated. Of note, both VB6 and L-Trp have been reported to correlate with biofilm formation in certain Gram-negative bacteria (Grubman et al.,; Shimazaki et al.,). In Helicobacter pylori in particular, VB6 biosynthetic enzymes act as novel virulence factors, and VB6 is required for full motility and virulence (Grubman et al.,). In E. coli, mutants with decreased tryptophan synthesis show greater biofilm formation, and matured biofilm is degraded by L-tryptophan addition (Shimazaki et al.,). However, the detailed mechanism remains elusive. To answer the question whether competition of VB6 or L-Trp for the YfiB F57-binding pocket of YfiR plays an essential role in inhibiting biofilm formation, we measured the binding affinities of VB6 and L-Trp for YfiR via BIAcore experiments. The results showed relatively weak Kd values of 35.2 mmol/L and 76.9 mmol/L for VB6 and L-Trp, respectively (Fig. 6C and 6D). Based on our results, we concluded that VB6 or L-Trp can bind to YfiR, however, VB6 or L-Trp alone may have little effects in interrupting the YfiB-YfiR interaction, the mechanism by which VB6 or L-Trp inhibits biofilm formation remains unclear and requires further investigation. DISCUSSION Previous studies suggested that in response to cell stress, YfiB in the outer membrane sequesters the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus inducing the diguanylate cyclase activity of YfiN to allow c-di-GMP production (Giardina et al.,; Malone et al.,; Malone et al.,). However, the pattern of interaction between these proteins and the detailed regulatory mechanism remain unknown due to a lack of structural information. Here, we report the crystal structures of YfiB alone and an active mutant YfiBL43P in complex with YfiR, indicating that YfiR forms a 2:2 complex with YfiB via a region composed of conserved residues. Our structural data analysis shows that the activated YfiB has an N-terminal portion that is largely altered, adopting a stretched conformation compared with the compact conformation of the apo YfiB. The apo YfiB structure constructed beginning at residue 34 has a compact conformation of approximately 45 Å in length. In addition to the preceding 8 aa loop (from the lipid acceptor Cys26 to Gly34), the full length of the periplasmic portion of apo YfiB can reach approximately 60 Å. It was reported that the distance between the outer membrane and the cell wall is approximately 50 Å and that the thickness of the PG layer is approximately 70 Å (Matias et al.,). Thus, YfiB alone represents an inactive form that may only partially insert into the PG matrix. By contrast, YfiR-bound YfiBL43P (residues 44–168) has a stretched conformation of approximately 55 Å in length. In addition to the 17 preceding intracellular residues (from the lipid acceptor Cys26 to Leu43), the length of the intracellular portion of active YfiB may extend over 100 Å, assuming a fully stretched conformation. Provided that the diameter of the widest part of the YfiB dimer is approximately 64 Å, which is slightly smaller than the smallest diameter of the PG pore (70 Å) (Meroueh et al.,), the YfiB dimer should be able to penetrate the PG layer. Regulatory model of the YfiBNR tripartite system. The periplasmic domain of YfiB and the YfiB-YfiR complex are depicted according to the crystal structures. The lipid acceptor Cys26 is indicated as blue ball. The loop connecting Cys26 and Gly34 of YfiB is modeled. The PAS domain of YfiN is shown as pink oval. Once activated by certain cell stress, the dimeric YfiB transforms from a compact conformation to a stretched conformation, allowing the periplasmic domain of the membrane-anchored YfiB to penetrate the cell wall and sequester the YfiR dimer, thus relieving the repression of YfiN These results, together with our observation that activated YfiB has a much higher cell wall binding affinity, and previous mutagenesis data showing that (1) both PG binding and membrane anchoring are required for YfiB activity and (2) activating mutations possessing an altered N-terminal loop length are dominant over the loss of PG binding (Malone et al.,), suggest an updated regulatory model of the YfiBNR system (Fig. 7). In this model, in response to a particular cell stress that is yet to be identified, the dimeric YfiB is activated from a compact, inactive conformation to a stretched conformation, which possesses increased PG binding affinity. This allows the C-terminal portion of the membrane-anchored YfiB to reach, bind and penetrate the cell wall and sequester the YfiR dimer. The YfiBNR system provides a good example of a delicate homeostatic system that integrates multiple signals to regulate the c-di-GMP level. Homologs of the YfiBNR system are functionally conserved in P. aeruginosa (Malone et al.,; Malone et al.,), E. coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), K. pneumonia (Huertas et al.,) and Y. pestis (Ren et al.,), where they affect c-di-GMP production and biofilm formation. The mechanism by which activated YfiB relieves the repression of YfiN may be applicable to the YfiBNR system in other bacteria and to analogous outside-in signaling for c-di-GMP production, which in turn may be relevant to the development of drugs that can circumvent complicated antibiotic resistance. MATERIALS AND METHODS Protein expression and purification P. aeruginosa YfiR (residues 35–190, lacking the predicted N-terminal periplasmic localization signaling peptide) and YfiB (residues 34–168, lacking the signal peptide from residues 1–26 and periplasmic residues 27–33) were cloned into ORF1 of the pETDuet-1 (Merck Millipore, Darmstadt, Germany) vector via the BamHI and HindIII restriction sites, with a constructed N-terminal His6 and a TEV cleavage site, respectively. In addition, YfiB (residues 34–168) was ligated into the NdeI and XhoI restriction sites of ORF2 in the previously constructed YfiR expression vector. Site-directed mutagenesis was carried out using a QuikChange kit (Agilent Technologies, Santa Clara, CA), following the manufacturer’s instructions. The proteins were over-expressed in the E. coli BL21-CodonPlus(DE3)-RIPL strain. Protein expression was induced by adding 0.5–1 mmol/L IPTG at an OD600 of approximately 0.8. The cell cultures were then incubated for an additional 4.5 h at 37°C. The cells were subsequently harvested by centrifugation and stored at −80°C. Cell suspensions were thawed and homogenized using a high-pressure homogenizer (JNBIO, Beijing, China). YfiR was first purified by Ni affinity chromatography and then incubated with His6-tagged TEV protease overnight. The His6-TEV cleavage site was subsequently removed by incubation with Ni-NTA resin. Finally, YfiR was purified with a HiTrap STM column (GE Healthcare), followed by a Superdex 200 (GE Healthcare) column. YfiB was purified with Ni affinity chromatography, followed by a Superdex 200 (GE Healthcare) column. The YfiB-YfiR complex was first purified by Ni affinity chromatography, then by a Superdex 200 (GE Healthcare) column, and finally by a HiTrap STM column (GE Healthcare). All of the purified fractions were collected and concentrated to ~40 mg/mL in 20 mmol/L Tris-HCl (pH 8.0) and 200 mmol/L NaCl, frozen in liquid nitrogen and stored at −80°C. Crystallization and data collection Crystal screening was performed with commercial screening kits (Hampton Research, CA, USA) using the sitting-drop vapor diffusion method, and positive hits were optimized using the hanging-drop vapor diffusion method at 293 K. Crystals of the YfiB protein were obtained and optimized in buffer containing 0.2 mol/L lithium sulfate monohydrate, 0.1 mol/L Tris-HCl (pH 8.0) and 30% w/v polyethylene glycol 4000. After being soaked for a few seconds in cryoprotection solution (well solution complemented with 25% xylitol), the crystals were cooled by plunging them into liquid nitrogen. Diffraction-quality crystals of the YfiB-YfiR complex were grown in buffer containing 0.2 mol/L ammonium sulfate, 0.1 mol/L Tris-HCl (pH 8.0) and 12% w/v polyethylene glycol 8000. The crystals were cryoprotected with 8% (w/v) polyethylene glycol 8000 and 0.1 mol/L Tris-HCl (pH 7.5) supplemented with saturated sucrose prior to being flash frozen. Crystals of the native YfiR were obtained and optimized in 0.1 mol/L HEPES (pH 7.5) and 1.8 mol/L ammonium sulfate. VB6-bound YfiR crystals were obtained by soaking the native YfiR crystals in 2 mmol/L VB6 molecules. Trp-bound YfiR crystals were obtained by co-crystalizing the YfiR protein and 4 mmol/L L-Trp molecules in 0.2 mol/L NaCl, 0.1 mol/L BIS-TRIS (pH 5.5), and 25% w/v polyethylene glycol 3350. For cryoprotection, both the VB6-bound and the L-Trp-bound YfiR crystals were soaked in 2.5 mol/L lithium sulfate monohydrate for a few seconds before data collection. Diffraction data for the YfiB crystal belonging to space group P21 was collected in house, the data for the YfiB crystal belonging to space group P41 and for the Trp-bound YfiR crystal were collected on beamline BL17U at the Shanghai Synchrotron Radiation Facility (SSRF), and the data for the VB6-bound YfiR crystal were collected on beamline BL18U at SSRF. Finally, the data for the YfiB-YfiR complex crystal were collected on beamline BL-1A at the Photon Factory in Japan. The diffraction data were processed with the HKL2000 software program (Otwinowski and Minor,). Structure determination and refinement Data collection, phasing and refinement statistics Data collection\tYfiB (crystal form I)\tYfiB (crystal form II)\tVB6-bound YfiR\tTrp-bound YfiR\tYfiBL43P-YfiR\t \tSpace group\tP21\tP41\tP43212\tP43212\tP1\t \tWavelength (Å)\t1.54187\t0.9791\t0.97861\t0.9791\t1.10000\t \tResolution (Å)a\t50.0–2.15 (2.19–2.15)\t50.0–2.80 (2.85–2.8)\t50.0–2.4 (2.44–2.4)\t50.0–2.5 (2.54–2.5)\t50–1.78 (1.86–1.78)\t \tCell dimensions\t \t a, b, c (Å)\t65.85, 90.45, 66.30\t46.95, 46.95, 154.24\t120.24, 120.24, 84.99\t120.88, 120.88, 88.46\t49.50, 58.57, 69.86\t \t α, β, γ (°)\t90, 113.87, 90\t90, 90, 90\t90, 90, 90\t90, 90, 90\t72.93, 96.98, 90.19\t \t Unique reflections\t37,625 (1866)\t8,105 (412)\t24,776 (1202)\t23170 (1132)\t67,774 (6615)\t \t I/σI\t19.59 (2.62)\t12.36 (4.15)\t20.17 (2.4)\t39.5 (4.68)\t17.75 (1.89)\t \t Completeness (%)\t97.1 (95.4)\t97.8 (100)\t99.1 (98.8)\t99.9 (100)\t96.5 (94.6)\t \t Rmerge (%)\t6.5 (44.5)\t14.6 (49.7)\t8.9 (56.8)\t9.4 (89.2)\t5.6 (46.3)\t \t Rmeas (%)\t7.4 (51.6)\t15.4 (52.0)\t9.6 (61.7)\t9.6 (90.8)\t6.6 (55.1)\t \t CC1/2b\t0.747\t0.952\t0.899\t0.974\t0.849\t \tRefinement\t \t Rwork (%)\t20.14\t19.17\t17.82\t18.66\t17.90\t \t Rfree(%)\t26.29\t26.49\t19.81\t23.05\t20.61\t \tAverage B factors (Å2)\t \t Protein\t25.54\t42.70\t38.68\t35.03\t32.54\t \t VB6\t-\t-\t44.08\t-\t-\t \t Trp\t-\t-\t-\t87.51\t-\t \t SO42−\t37.16\t66.52\t51.55\t41.93\t45.51\t \t H2O\t32.91\t36.09\t40.58\t34.75\t43.52\t \tRoot mean square deviations\t \t Bond lengths (Å)\t0.009\t0.009\t0.007\t0.007\t0.007\t \t Bond angles (°)\t1.085\t1.132\t1.021\t0.977\t1.110\t \tRamachandran plot\t \t Most favored (%)\t92.6\t87.7\t96.5\t98.1\t94.2\t \t Additionally allowed (%)\t7.4\t12.3\t3.5\t1.9\t5.8\t \t Generously allowed (%)\t0\t0\t0\t0\t0\t \t Disallowed\t0\t0\t0\t0\t0\t \t \na Numbers in parentheses are for the highest resolution shell \nb The values of CC1/2 are for the highest resolution shell The two YfiB crystal structures respectively belonging to space groups P21 and P41 were both solved by molecular replacement (Lebedev et al.,) using the putative MotB-like protein DVU_2228 from D. vulgaris as a model (PDB code: 3khn) at 2.15 Å and 2.8 Å resolution, respectively. Both the VB6-bound and the Trp-bound YfiR crystals belonging to space group P43212, with a dimer in the asymmetric unit, were solved by molecular replacement (Lebedev et al.,) using native YfiR as a model (PDB code: 4YN7) at 2.4 Å and 2.5 Å resolution, respectively. The YfiB-YfiR crystal belonging to space group P1, with a 2:2 heterotetramer in the asymmetric unit, was solved by molecular replacement using YfiR and YfiB as models. Electron density maps were calculated using PHENIX (Adams et al.,). Model building was performed using COOT (Emsley et al.,) and refined with PHENIX (Adams et al.,; Afonine et al.,). The final structures were analyzed with PROCHECK (Laskowski et al.,). Data collection and refinement statistics are presented in Table 1. The figures depicting structures were prepared using PyMOL (http://www.pymol.org). Atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank (http://www.pdb.org) under accession codes 5EAZ, 5EB0, 5EB1, 5EB2 and 5EB3. Analytical ultracentrifugation Sedimentation velocity measurements were performed on a Beckman ProteomeLab XL-I at 25°C. All protein samples were diluted to an OD280 of 0.7 in 20 mmol/L Tris (pH 8.0) and 200 mmol/L NaCl. Data were collected at 60,000 rpm. (262,000 ×g) every 3 min at a wavelength of 280 nm. Interference sedimentation coefficient distributions, or c(M), were calculated from the sedimentation velocity data using SEDFIT (Schuck,). PG preparation PG was extracted from the E. coli DH5α strain by following a method described previously (Desmarais et al.,). Briefly, cells were cultured until they reached an OD600 of 0.7–0.8 and then collected at 5,000 ×g, 4°C. The collected bacteria were dripped into the boiling 6% (w/v) SDS and stirred at 500 rpm in a boiling water bath for 3 h before incubating overnight at room temperature. The large PG polymers were collected by ultracentrifugation at 130,000 ×g for 1 h at room temperature and washed repeatedly to remove SDS. The pellet was treated with Pronase E (200 μg/mL final concentration) for 3 h at 60°C followed by SDS to remove contaminating proteins and washed three times to remove the SDS by ultracentrifugation. Next, the samples were treated with lysozyme (200 μg/mL final concentration) for 16 h at 37°C. Finally, the purified PG is obtained by treating the samples in a boiling water bath for 10 min and centrifuging it at 13,000 ×g to remove the contaminating lysozyme. Microscale thermophoresis (MST) Purified YfiB wild-type and it mutant YfiBL43P were fluorescently labeled using the NanoTemper blue protein-labeling kit according to the manufacturer’s protocol. This resulted in coupling of the fluorescent dye NT-495. PG was titrated in 1:1 dilutions starting at 1 mmol/L. To determine of the Kd values, 10 μL labeled protein was mixed with 10 μL PG at various concentrations in Hepes buffer (20 mmol/L Hepes, 200 mmol/L NaCl, 0.005% Tween-20, pH 7.5). After 10 min of incubation, all binding reaction mixtures were loaded into the MST-grade glass capillaries (NanoTemper Technologies), and thermophoresis was measured with a NanoTemper Monolith-NT115 system (20% light-emitting diode, 20% IR laser power). Deletion of the yfiB genes The yfiB deletion construct was produced by SOE-PCR (Hmelo et al.,) and contained homologous flanking regions to the target gene. This construct was ligated into the pEX18Gm vector between the HindIII and the KpnI sites. The resulting vector was then used to delete yfiB by two-step allelic exchange (Hmelo et al.,). After being introduced into PAO1 via biparental mating with E. coli SM10 (λpir), single crossovers were selected on Vogel-Bonner Minimal Medium (VBMM), which was used for counter-selection against E. coli (P. aeruginosa can utilize citrate as a sole carbon source and energy source, whereas E. coli cannot), containing 50 μg/mL gentamycin. Restreaking was then performed on no-salt Luria-Bertani (NSLB) agar that contained 15% sucrose to force the resolution of double crossovers. Deletion of yfiB in the strains was confirmed by colony PCR. For complementation experiments, yfiB wild-type and L43P mutant genes were cloned into the pJN105 vector via the EcoRI and XbaI restriction sites, respectively. The plasmids were then individually transformed into the PAO1 ΔyfiB strain using the rapid electroporation method described in Choi KH et al. (Choi et al.,). Transformants were selected on LB plates containing 50 μg/mL gentamycin. For induction, arabinose was added to a final concentration of 0.2%. Attachment assays The attachment assays were carried out using the MBECTM (Minimum Biofilm Eradication Concentration, Innovotech, Inc.) biofilm inoculator, which consists of a plastic lid with 96 pegs and 96 individual wells. The MBEC plates containing 150 μL LB medium/well were inoculated with 1% overnight cultures of the yfiB-L43P strain and incubated overnight at 37°C without shaking. VB6, L-Trp and arabinose were added as appropriate. The peg lids were washed with distilled water, and the attached cell material was then stained with 0.1% crystal violet solution (5% methanol, 5% isopropanol) before further washing to remove excess dye. The crystal violet was re-dissolved in 20% acetic acid solution, and the absorbance was measured at 600 nm. Assays were performed with 12 wells/strain and repeated independently for each experiment. BIAcore analysis The interaction kinetics of YfiR with VB6 and L-Trp were examined on a SPR machine Biacore 3000 (GE Healthcare) at 25°C. The running buffer (20 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 0.005% (v/v) Tween-20) was vacuum filtered, and degassed immediately prior to use. YfiR at 10 μg/mL in 10 mmol/L sodium acetate (pH 5.5) was immobilized to 3000 response units on the carboxymethylated dextran surface-modified chip (CM5 chip). The binding affinities were evaluated over a range of 2.5–40 mmol/L concentrations. Meanwhile, for both binding assays, the concentration of 10 mmol/L was repeated as an internal control. All of the data collected were analyzed using BIAevaluation software version 4.1. ITC assays ITC experiments were performed in a buffer composed of 20 mmol/L Tris (pH 8.0) and 150 mmol/L NaCl at 25°C using an iTC200 calorimeter (GE Healthcare). YfiB wild-type or its mutants (YfiBL43P, YfiBL43P/F57A) (0.4 mmol/L, in the syringe) was titrated into YfiR (0.04 mmol/L, in the cell), respectively. The titration sequence included a single 0.5 µL injection, followed by 19 injections of 2 µL each, with a 2-min interval between injections and a stirring rate of 1000 rpm. The calorimetric data were then analyzed with OriginLab software (GE Healthcare). Min Xu, Xuan Yang and Xiu-An Yang have contributed equally to this work. References PHENIX: a comprehensive Python-based system for macromolecular structure solution Towards automated crystallographic structure refinement with phenix.refine Experimental evolution of bet hedging Second messenger-mediated adjustment of bacterial swimming velocity Targeting cyclic di-GMP signalling: a strategy to control biofilm formation? Bacterial small-molecule signaling pathways A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation Desmarais SM, Cava F, de Pedro MA, Huang KC (2014) Isolation and preparation of bacterial cell walls for compositional analysis by ultra performance liquid chromatography. J Vis Exp 83:e51183 Second messenger-mediated spatiotemporal control of protein degradation regulates bacterial cell cycle progression Features and development of Coot Small colony variants of Pseudomonas aeruginosa in chronic bacterial infection of the lung in cystic fibrosis Investigating the allosteric regulation of YfiN from Pseudomonas aeruginosa: clues from the structure of the catalytic domain Mutational activation of niche-specific genes provides insight into regulatory networks and bacterial function in a complex environment Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia Grubman A, Phillips A, Thibonnier M, Kaparakis-Liaskos M, Johnson C, Thiberge JM, Radcliff FJ, Ecobichon C, Labigne A, de Reuse H. et al (2010) Vitamin B6 is required for full motility and virulence in Helicobacter pylori. MBio 1 Ha DG, O’Toole GA (2015) c-di-GMP and its effects on biofilm formation and dispersion: a Pseudomonas aeruginosa review. Microbiol Spectr 3, MB-0003-2014 Small-colony variants of Pseudomonas aeruginosa in cystic fibrosis Highly adherent small-colony variants of Pseudomonas aeruginosa in cystic fibrosis lung infection Principles of c-di-GMP signalling in bacteria A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange Dali server: conservation mapping in 3D Klebsiella pneumoniae yfiRNB operon affects biofilm formation, polysaccharide production and drug susceptibility The disulfide bonding system suppresses CsgD-independent cellulose production in Escherichia coli Cyclic di-guanosine-monophosphate comes of age: a novel secondary messenger involved in modulating cell surface structures in bacteria? HmsP, a putative phosphodiesterase, and HmsT, a putative diguanylate cyclase, control Hms-dependent biofilm formation in Yersinia pestis Characterization of colony morphology variants isolated from Pseudomonas aeruginosa biofilms Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3′-5′)-cyclic-GMP in virulence PROCHECK: a program to check the stereochemical quality of protein structures Model preparation in MOLREP and examples of model improvement using X-ray data Structural insights into YfiR sequestering by YfiB in Pseudomonas aeruginosa PAO1 Role of small colony variants in persistence of Pseudomonas aeruginosa infections in cystic fibrosis lungs YfiBNR mediates cyclic di-GMP dependent small colony variant formation and persistence in Pseudomonas aeruginosa The YfiBNR signal transduction mechanism reveals novel targets for the evolution of persistent Pseudomonas aeruginosa in cystic fibrosis airways Cryo-transmission electron microscopy of frozen-hydrated sections of Escherichia coli and Pseudomonas aeruginosa Three-dimensional structure of the bacterial cell wall peptidoglycan Structural basis for c-di-GMP-mediated inside-out signaling controlling periplasmic proteolysis Processing of X-ray diffraction data collected in oscillation mode Bacterial biofilms: an emerging link to disease pathogenesis Peptidoglycan recognition by Pal, an outer membrane lipoprotein Bacterial nucleotide-based second messengers Genetic analysis of the role of yfiR in the ability of Escherichia coli CFT073 to control cellular cyclic dimeric GMP levels and to persist in the urinary tract Development and persistence of antimicrobial resistance in Pseudomonas aeruginosa: a longitudinal observation in mechanically ventilated patients HmsC, a periplasmic protein, controls biofilm formation via repression of HmsD, a diguanylate cyclase in Yersinia pestis Cyclic di-GMP: the first 25 years of a universal bacterial second messenger Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid Cellulose biosynthesis and function in bacteria GGDEF proteins YeaI, YedQ, and YfiN reduce early biofilm formation and swimming motility in Escherichia coli Structural and mechanistic determinants of c-di-GMP signalling Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling L-Tryptophan prevents Escherichia coli biofilm formation and triggers biofilm degradation Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients The EAL domain protein VieA is a cyclic diguanylate phosphodiesterase Connecting quorum sensing, c-di-GMP, pel polysaccharide, and biofilm formation in Pseudomonas aeruginosa through tyrosine phosphatase TpbA (PA3885) Structural and biochemical analysis of tyrosine phosphatase related to biofilm formation A (TpbA) from the opportunistic pathogen Pseudomonas aeruginosa PAO1 Crystal structures of 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diff --git a/annotated_BioC_JSON/PMC4919469_ann.json b/annotated_BioC_JSON/PMC4919469_ann.json
new file mode 100644
index 0000000000000000000000000000000000000000..fe489b63c69da31ce00d3a78071b985d29abbda6
--- /dev/null
+++ b/annotated_BioC_JSON/PMC4919469_ann.json
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+[{"sourceid":"4919469","sourcedb":"","project":"","target":"","text":"Investigation of the Interaction between Cdc42 and Its Effector TOCA1 Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42. It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain. TOCA1 binding to Cdc42 leads to actin rearrangements, which are thought to be involved in processes such as endocytosis, filopodia formation, and cell migration. We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein. We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains. We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42. NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery. Introduction The Ras superfamily of small GTPases comprises over 150 members that regulate a multitude of cellular processes in eukaryotes. The superfamily can be divided into five families based on structural and functional similarities: Ras, Rho, Rab, Arf, and Ran. All members share a well defined core structure of ∼20 kDa known as the G domain, which is responsible for guanine nucleotide binding. It is this guanine nucleotide binding that underlies their function as molecular switches, controlling a vast array of signaling pathways. These molecular switches cycle between active, GTP-bound, and inactive, GDP-bound, states with the help of auxiliary proteins. The guanine nucleotide exchange factors mediate formation of the active state by promoting the dissociation of GDP, allowing GTP to bind. The GTPase-activating proteins stimulate the rate of intrinsic GTP hydrolysis, mediating the return to the inactive state (reviewed in Ref.). The overall conformation of small G proteins in the active and inactive states is similar, but they differ significantly in two main regions known as switch I and switch II. These regions are responsible for “sensing” the nucleotide state, with the GTP-bound state showing greater rigidity and the GDP-bound state adopting a more relaxed conformation (reviewed in Ref.). In the active state, G proteins bind to an array of downstream effectors, through which they exert their extensive roles within the cell. The structures of more than 60 small G protein·effector complexes have been solved, and, not surprisingly, the switch regions have been implicated in a large proportion of the G protein-effector interactions (reviewed in Ref.). However, because each of the 150 members of the superfamily interacts with multiple effectors, there are still a huge number of known G protein-effector interactions that have not yet been studied structurally. The Rho family comprises 20 members, of which three, RhoA, Rac1, and Cdc42, have been relatively well studied. The role of these three proteins in the coordination of the actin cytoskeleton has been examined extensively. RhoA acts to rearrange existing actin structures to form stress fibers, whereas Rac1 and Cdc42 promote de novo actin polymerization to form lamellipodia and filopodia, respectively. A number of RhoA and Rac1 effector proteins, including the formins and members of the protein kinase C-related kinase (PRK)6 family, along with Cdc42 effectors, including the Wiskott-Aldrich syndrome (WASP) family and the transducer of Cdc42-dependent actin assembly (TOCA) family, have also been linked to the pathways that govern cytoskeletal dynamics. Cdc42 effectors, TOCA1 and the ubiquitously expressed member of the WASP family, N-WASP, have been implicated in the regulation of actin polymerization downstream of Cdc42 and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). N-WASP exists in an autoinhibited conformation, which is released upon PI(4,5)P2 and Cdc42 binding or by other factors, such as phosphorylation. Following their release, the C-terminal regions of N-WASP are free to interact with G-actin and a known nucleator of actin assembly, the Arp2/3 complex. The importance of TOCA1 in actin polymerization has been demonstrated in a range of in vitro and in vivo studies, but the exact role of TOCA1 in the many pathways involving actin assembly remains unclear. The most widely studied role of TOCA1 is in membrane invagination and endocytosis, although it has also been implicated in filopodia formation, neurite elongation, transcriptional reprogramming via nuclear actin, and interaction with ZO-1 at tight junctions. A role in cell motility and invasion has also been established. TOCA1 comprises an N-terminal F-BAR domain, a central homology region 1 (HR1) domain, and a C-terminal SH3 domain. The F-BAR domain is a known dimerization, membrane-binding, and membrane-deforming module found in a number of cell signaling proteins. The TOCA1 SH3 domain has many known binding partners, including N-WASP and dynamin. The HR1 domain has been directly implicated in the interaction between TOCA1 and Cdc42, representing the first Cdc42-HR1 domain interaction to be identified. Other HR1 domains studied so far, including those from the PRK family, have been found to bind their cognate Rho family G protein-binding partner with high specificity and affinities in the nanomolar range. The structures of the PRK1 HR1a domain in complex with RhoA and the HR1b domain in complex with Rac1 show that the HR1 domain comprises an anti-parallel coiled-coil that interacts with its G protein binding partner via both helices. Both of the G protein switch regions are involved in the interaction. The coiled-coil fold is shared by the HR1 domain of the TOCA family protein, CIP4, and, based on sequence homology, by TOCA1 itself. These HR1 domains, however, show specificity for Cdc42, rather than RhoA or Rac1. How different HR1 domain proteins distinguish their specific G protein partners remains only partially understood, and structural characterization of a novel G protein-HR1 domain interaction would add to the growing body of information pertaining to these protein complexes. Furthermore, the biological function of the interaction between TOCA1 and Cdc42 remains poorly understood, and so far there has been no biophysical or structural insight. The interactions of TOCA1 and N-WASP with Cdc42 as well as with each other have raised questions as to whether the two Cdc42 effectors can interact with a single molecule of Cdc42 simultaneously. There is some evidence for a ternary complex between Cdc42, N-WASP, and TOCA1, but there was no direct demonstration of simultaneous contacts between the two effectors and a single molecule of Cdc42. Nonetheless, the substantial difference between the structures of the G protein-binding regions of the two effectors is intriguing and implies that they bind to Cdc42 quite differently, providing motivation for investigating the possibility that Cdc42 can bind both effectors concurrently. WASP interacts with Cdc42 via a conserved, unstructured binding motif known as the Cdc42- and Rac-interactive binding region (CRIB), which forms an intermolecular β-sheet, expanding the anti-parallel β2 and β3 strands of Cdc42. In contrast, the TOCA family proteins are thought to interact via the HR1 domain, which may form a triple coiled-coil with switch II of Rac1, like the HR1b domain of PRK1. Here, we present the solution NMR structure of the HR1 domain of TOCA1, providing the first structural data for this protein. We also present data pertaining to binding of the TOCA HR1 domain to Cdc42, which is the first biophysical description of an HR1 domain binding this particular Rho family small G protein. Finally, we investigate the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and N-WASP, contributing to our understanding of G protein-effector interactions as well as the roles of Cdc42, N-WASP, and TOCA1 in the pathways that govern actin dynamics. Experimental Procedures Expression Constructs The Xenopus tropicalis TOCA1 HR1 domain (residues 330–426 and N-terminally extended constructs as indicated) were amplified from cDNA (TOCA1 accession number NM_001005148) and cloned into pGEX-6P-1 (GE Healthcare) or pGEX-HisP. The HR1 domain of human CIP4 (residues 388–481) was amplified from IMAGE clone 3532036, the Xenopus laevis FBP17 HR1 domain (residues 385–486) from IMAGE clone 5514481, and the X. tropicalis N-WASP G protein-binding domain (GBD) (residues 197–255) from IMAGE clone 5379332, and all were cloned into pGEX-6P-1. The resulting constructs express the proteins as N-terminal GST fusions with a 3C protease-cleavable tag, with pGEX-HisP expressing an additional C-terminal His6 tag. Human Cdc42Δ7Q61L and full-length Cdc42 were cloned into pGEX-2T (GE Healthcare) and pGEX-6P-1, respectively. A C-terminally extended construct of TOCA1 comprising residues 330–545 was cloned into pMAT10-P.7 The resulting construct expresses TOCA1 330–545 as an N-terminal His-MBP fusion protein with a 3C protease-cleavable tag. Full-length X. tropicalis TOCA1, TOCA1 F-BAR (residues 1–287), and TOCA1 ΔSH3 (residues 1–480) were PCR-amplified from a cDNA clone (IMAGE 5157175) and cloned into pET-His6-SNAP using FseI and AscI sites that had been incorporated into the primers to create His-SNAP-TOCA1 proteins. Protein Expression GST fusion proteins (HR1 domains and Cdc42) were expressed in E. coli BL21 cells (Invitrogen). Stationary cultures were diluted 1:10 and grown at 37 °C until an A600 of ∼0.8 was reached and then induced with 0.1 mm isopropyl-β-d-thiogalactopyranoside for 20 h at 20 °C. The GST-N-WASP GBD construct was expressed in E. coli BL21-CodonPlus®-RIL (Agilent Technologies). The proteins were purified using glutathione-agarose beads (Sigma) and eluted from the beads by cleavage of the GST tag with 3C protease (HR1 domains, N-WASP GBD, and full-length Cdc42Q61L) or thrombin (Novagen, Cdc42Δ7Q61L) prior to gel filtration on a 16/60 S75 column (GE Healthcare). His-MBP-HR1-SH3 was purified using nickel-nitrilotriacetic acid-agarose beads (Life Technologies) prior to cleavage with 3C protease and gel filtration. Full-length TOCA1, TOCA1 F-BAR, and TOCA1 ΔSH3 were expressed from pET-His6-SNAP in BL21 pLysS, grown at 37 °C until an A600 of ∼0.6 was reached, and induced with 0.3 mm isopropyl-β-d-thiogalactopyranoside overnight at 19 °C. Proteins were coupled to nickel-nitrilotriacetic acid-agarose (Qiagen), eluted using increasing concentrations of imidazole, and further purified by gel filtration using a 16/60 S200 column (GE Healthcare). All protein concentrations were determined by amino acid analysis (Protein and Nucleic Acid Chemistry Facility, Department of Biochemistry, University of Cambridge). Nucleotide Exchange For NMR experiments, Cdc42 was nucleotide-exchanged for the non-hydrolyzable GTP analogue GMPPNP (Sigma) as described previously. For scintillation proximity assays (SPAs), Cdc42 was loaded with [3H]GTP using [8-3H]GTP (PerkinElmer Life Sciences), as described previously. The protein was confirmed as full-length using mass spectrometry (PNAC facility, Department of Biochemistry, University of Cambridge). SPAs For direct assays, GST-PAK, GST-ACK, or His-tagged TOCA1 constructs were attached to a fluoromicrosphere via an anti-GST or anti-His antibody in the presence of Cdc42Δ7Q61L·[3H]GTP. Binding curves were fitted using a direct binding isotherm to obtain Kd values and their curve-fitting errors for the G protein-effector interactions. For competition assays, free ACK GBD, TOCA1 HR1, TOCA1 HR1SH3, or N-WASP GBD was titrated into a mixture of 30 nm Cdc42Δ7Q61L·[3H]GTP and 30 nm GST-ACK immobilized on a fluoromicrosphere as above. Data were fitted to competition binding isotherms to obtain Kd values and curve-fitting errors, as described previously. NMR Spectroscopy The NMR experiments and resonance assignments of the HR1 domain are described. The NMR experiments were carried out with 0.9 mm 13C/15N-labeled HR1 domain in 20 mm sodium phosphate, pH 7.5, 150 mm NaCl, 5 mm MgCl2, 5 mm DTT, 10% D2O. Distance restraints were derived from a 15N-separated NOESY (100-ms mixing time) recorded on a Bruker DRX500 and a 13C-separated NOESY (100-ms mixing time) recorded on an Avance AV600. NMR data were processed using AZARA (W. Boucher, University of Cambridge) and analyzed using ANALYSIS. Structure Calculation Structures were calculated iteratively using CNS version 1.0 interfaced to Aria version 2.3.1. The PROSLQ force field was used for non-bonded parameters. Backbone torsion angles were estimated from CA, CO, CB, N, and HA chemical shifts using TALOS-N. The “strong” φ and ψ restraints were included with an error of ±2 S.D. values of the averaged TALOS-N predictions. Dihedral angle predictions for residues 323–340 were weak, so no restraints were included for this region. NMR Titrations All of the 15N and 13C HSQCs were recorded at 25 °C in 50 mm sodium phosphate, pH 5.5, 25 mm NaCl, 5 mm MgCl2, 5 mm DTT, 10% D2O on a Bruker DRX500. 15N-HR1 HSQC experiments were recorded on 0.2 mm 15N-HR1 domain with HR1/Cdc42·GMPPNP ratios of 1:0, 1:0.25, 1:0.5, 1:1, and 1:4. Experiments were recorded on 0.27 mm 15N-Cdc42·GMPPNP at Cdc42/HR1 ratios of 1:0, 1:0.25, 1:0.5, and 1:2.2. The 15N HSQC titrations with N-WASP were recorded on 0.6 mm 15N-HR1 domain or 0.15 mm 15N-Cdc42 at the ratios indicated in the figures. Chemical Shift Mapping The chemical shift changes, δ, were calculated using the equation,  where δ(1H) and δ(15N) are the chemical shift changes for the 1H and 15N dimensions, respectively. Residues that had disappeared were assigned a δ value larger than the maximum calculated δ for the data set, and residues that were too overlapped to be reliably assigned in the complex spectra were assigned δ = 0. The residues that had shifted more than the mean chemical shift change across the spectrum were classed as significant and were filtered for solvent accessibility using NACCESS. Residues with \u003c50% solvent accessibility were considered to be buried and unavailable for binding. Pyrene Actin Assays Pyrene actin assays were carried out as described previously. Xenopus high speed supernatant was used at 5 mg/ml and supplemented with 0.12 mg/ml pyrene actin as described previously. TOCA1 HR1 domain or N-WASP CRIB domain was added at the concentrations indicated. Liposomes were made, using methods described previously, from 60% phosphatidylcholine, 30% phosphatidylserine, and 10% PI(4,5)P2 to 2 mm final lipid concentration. All of the lipids used were natural brain or liver lipids from Avanti Polar Lipids. The assays were initiated by the addition of 5 μl of liposomes per 200 μl of reaction mix. Results Cdc42-TOCA1 Binding TOCA1 was identified in Xenopus extracts as a protein necessary for Cdc42-dependent actin assembly and was shown to bind to Cdc42·GTPγS but not to Cdc42·GDP or to Rac1 and RhoA. Given its homology to other Rho family binding modules, it is likely that the HR1 domain of TOCA1 is sufficient to bind Cdc42. The C. elegans TOCA1 orthologues also bind to Cdc42 via their consensus HR1 domain. The HR1 domains from the PRK family bind their G protein partners with a high affinity, exhibiting a range of submicromolar dissociation constants (Kd) as low as 26 nm. A Kd in the nanomolar range was therefore expected for the interaction of the TOCA1 HR1 domain with Cdc42. We generated an X. tropicalis TOCA1 HR1 domain construct encompassing residues 330–426. This region comprises the complete HR1 domain based on secondary structure predictions and sequence alignments with another TOCA family member, CIP4, whose structure has been determined. The interaction between [3H]GTP·Cdc42 and a C-terminally His-tagged TOCA1 HR1 domain construct was investigated using SPA. The binding isotherm for the interaction is shown in Fig. 1A, together with the Cdc42-PAK interaction as a positive control. The binding of TOCA1 HR1 to Cdc42 was unexpectedly weak, with a Kd of \u003e1 μm. It was not possible to estimate the Kd more accurately using direct SPA experiments, because saturation could not be reached due to nonspecific signal at higher protein concentrations. The TOCA1 HR1-Cdc42 interaction is low affinity.\nA, curves derived from direct binding assays in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-PAK or HR1-His6 in SPAs. The SPA signal was corrected by subtraction of control data with no GST-PAK or HR1-His6. The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal; B and C, competition SPA experiments were carried out with the indicated concentrations of ACK GBD (B) or HR1 domain (C) titrated into 30 nm GST-ACK and either 30 nm Cdc42Δ7Q61L·[3H]GTP or full-length Cdc42Q61L·[3H]GTP. The Kd values derived for the ACK GBD with Cdc42Δ7 and full-length Cdc42 were 0.032 ± 0.01 and 0.011 ± 0.01 μm, respectively. The Kd values derived for the TOCA1 HR1 with Cdc42Δ7 and full-length Cdc42 were 6.05 ± 1.96 and 5.39 ± 1.69 μm, respectively. It was possible that the low affinity observed was due to negative effects of immobilization of the HR1 domain, so other methods were employed, which utilized untagged proteins. Isothermal titration calorimetry was carried out, but no heat changes were observed at a range of concentrations and temperatures (data not shown), suggesting that the interaction is predominantly entropically driven. Other G protein-HR1 domain interactions have also failed to show heat changes in our hands.7 Infrared interferometry with immobilized Cdc42 was also attempted but was unsuccessful for both TOCA1 HR1 and for the positive control, ACK. The affinity was therefore determined using competition SPAs. A complex of a GST fusion of the GBD of ACK, which binds with a high affinity to Cdc42, with radiolabeled [3H]GTP·Cdc42 was preformed, and the effect of increasing concentrations of untagged TOCA1 HR1 domain was examined. Competition of GST-ACK GBD bound to [3H]GTP·Cdc42 by free ACK GBD was used as a control and to establish the value of background counts when Cdc42 is fully displaced. The data were fitted to a binding isotherm describing competition. Free ACK competed with itself with an affinity of 32 nm, similar to the value obtained by direct binding of 23 nm. The TOCA1 HR1 domain also fully competed with the GST-ACK but bound with an affinity of 6 μm (Fig. 1, B and C), in agreement with the low affinity observed in the direct binding experiments. The Cdc42 construct used in the binding assays has seven residues deleted from the C terminus to facilitate purification. These residues are not generally required for G protein-effector interactions, including the interaction between RhoA and the PRK1 HR1a domain. In contrast, the C terminus of Rac1 contains a polybasic sequence, which is crucial for Rac1 binding to the HR1b domain from PRK1. As the observed affinity between TOCA1 HR1 and Cdc42 was much lower than expected, we reasoned that the C terminus of Cdc42 might be necessary for a high affinity interaction. The binding experiments were repeated with full-length [3H]GTP·Cdc42, but the affinity of the HR1 domain for full-length Cdc42 was similar to its affinity for truncated Cdc42 (Kd ≈ 5 μm; Fig. 1C). Thus, the C-terminal region of Cdc42 is not required for maximal binding of TOCA1 HR1. Another possible explanation for the low affinities observed was that the HR1 domain alone is not sufficient for maximal binding of the TOCA proteins to Cdc42 and that the other domains are required. Indeed, GST pull-downs performed with in vitro translated human TOCA1 fragments had suggested that residues N-terminal to the HR1 domain may be required to stabilize the HR1 domain structure. Furthermore, both BAR and SH3 domains have been implicated in interactions with small G proteins (e.g. the BAR domain of Arfaptin2 binds to Rac1 and Arl1), while an SH3 domain mediates the interaction between Rac1 and the guanine nucleotide exchange factor, β-PIX. TOCA1 dimerizes via its F-BAR domain, which could also affect Cdc42 binding, for example by presenting two HR1 domains for Cdc42 interactions. Various TOCA1 fragments (Fig. 2A) were therefore assessed for binding to full-length Cdc42 by direct SPA. The isolated F-BAR domain showed no binding to full-length Cdc42 (Fig. 2B). Full-length TOCA1 and ΔSH3 TOCA1 bound with micromolar affinity (Fig. 2B), in a similar manner to the isolated HR1 domain (Fig. 1A). The HR1-SH3 protein could not be purified to homogeneity as a fusion protein, so it was assayed in competition assays after cleavage of the His tag. This construct competed with GST-ACK GBD to give a similar affinity to the HR1 domain alone (Kd = 4.6 ± 4 μm; Fig. 2C). Taken together, these data suggest that the TOCA1 HR1 domain is sufficient for maximal binding and that this binding is of a relatively low affinity compared with many other Cdc42·effector complexes. The Cdc42-HR1 interaction is of low affinity in the context of full-length protein and in TOCA1 paralogues.\nA, diagram illustrating the TOCA1 constructs assayed for Cdc42 binding. Domain boundaries are derived from secondary structure predictions; B, binding curves derived from direct binding assays, in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-ACK or His-tagged TOCA1 constructs, as indicated, in SPAs. The SPA signal was corrected by subtraction of control data with no fusion protein. The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal. C–E, representative examples of competition SPA experiments carried out with the indicated concentrations of the TOCA1 HR1-SH3 construct titrated into 30 nm GST-ACK and 30 nm Cdc42Δ7Q61L·[3H]GTP (C) or HR1CIP4 (D) or HR1FBP17 (E) titrated into 30 nm GST-ACK and 30 nm Cdc42FLQ61L·[3H]GTP. The low affinity of the TOCA1 HR1-Cdc42 interaction raised the question of whether the other known Cdc42-binding TOCA family proteins, FBP17 and CIP4, also bind weakly. The HR1 domains from FBP17 and CIP4 were purified and assayed for Cdc42 binding in competition SPAs, analogous to those carried out with the TOCA1 HR1 domain. The affinities of both the FBP17 and CIP4 HR1 domains were also in the low micromolar range (10 and 5 μm, respectively) (Fig. 2, D and E), suggesting that low affinity interactions with Cdc42 are a common feature within the TOCA family. Structure of the TOCA1 HR1 Domain Because the TOCA1 HR1 domain was sufficient for maximal Cdc42-binding, we used this construct for structural studies. Initial experiments were performed with TOCA1 residues 324–426, but we observed that the N terminus was cleaved during purification to yield a new N terminus at residue 330 (data not shown). We therefore engineered a construct comprising residues 330–426 to produce the minimal, stable HR1 domain. Backbone and side chain resonances were assigned as described. 2,778 non-degenerate NOE restraints were used in initial structure calculations (1,791 unambiguous and 987 ambiguous), derived from three-dimensional 15N-separated NOESY and 13C-separated NOESY experiments. There were 1,845 unambiguous NOEs and 757 ambiguous NOEs after eight iterations. 100 structures were calculated in the final iteration; the 50 lowest energy structures were water-refined; and of these, the 35 lowest energy structures were analyzed. Table 1 indicates that the HR1 domain structure is well defined by the NMR data. Experimental restraints and structural statistics a \u003cSA\u003e, the average root mean square deviations for the ensemble ± S.D. b \u003cSA\u003ec, values for the structure that is closest to the mean. The structure closest to the mean is shown in Fig. 3A. The two α-helices of the HR1 domain interact to form an anti-parallel coiled-coil with a slight left-handed twist, reminiscent of the HR1 domains of CIP4 (PDB code 2KE4) and PRK1 (PDB codes 1CXZ and 1URF). A sequence alignment illustrating the secondary structure elements of the TOCA1 and CIP4 HR1 domains and the HR1a and HR1b domains from PRK1 is shown in Fig. 3B. The structure of the TOCA1 HR1 domain.\nA, the backbone trace of the 35 lowest energy structures of the HR1 domain overlaid with the structure closest to the mean is shown alongside a schematic representation of the structure closest to the mean. Flexible regions at the N and C termini (residues 330–333 and 421–426) are omitted for clarity. B, a sequence alignment of the HR1 domains from TOCA1, CIP4, and PRK1. The secondary structure was deduced using Stride, based on the Ramachandran angles, and is indicated as follows: gray, turn; yellow, α-helix; blue, 310 helix; white, coil. C, a close-up of the N-terminal region of TOCA1 HR1, indicating some of the NOEs defining its position with respect to the two α-helices. Dotted lines, NOE restraints. D, a close-up of the interhelix loop region showing some of the contacts between the loop and helix 1. NOEs are indicated with dotted lines. All structural figures were generated using PyMOL. In the HR1a domain of PRK1, a region N-terminal to helix 1 forms a short α-helix, which packs against both helices of the HR1 domain. This region of TOCA1 HR1 (residues 334–340) is well defined in the family of structures (Fig. 3A) but does not form an α-helix. It instead forms a series of turns, defined by NOE restraints observed between residues separated by one (residues 332–334, 333–335, etc.) or two (residues 337–340) residues in the sequence and the φ and ψ angles, assessed using Stride. These turns cause the chain to reverse direction, allowing the N-terminal segment (residues 334–340) to contact both helices of the HR1 domain. Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1. These contacts are summarized in Fig. 3C. The two α-helices of TOCA1 HR1 are separated by a long loop of 10 residues (residues 380–389) that contains two short 310 helices (residues 381–383 and 386–389). Interestingly, side chains of residues within the loop region point back toward helix 1; for example, there are numerous distinct NOEs between the side chains of Asn-380 and Met-383 of the loop region and Tyr-377 and Val-376 of helix 1 (Fig. 3D). The backbone NH and CHα groups of Gly-384 and Asp-385 also show NOEs with the side chain of Tyr-377. Mapping the TOCA1 and Cdc42 Binding Interfaces The HR1TOCA1-Cdc42 interface was investigated using NMR spectroscopy. A series of 15N HSQC experiments was recorded on 15N-labeled TOCA1 HR1 domain in the presence of increasing concentrations of unlabeled Cdc42Δ7Q61L·GMPPNP to map the Cdc42-binding surface. A comparison of the 15N HSQC spectra of free HR1 and HR1 in the presence of excess Cdc42 shows that although some peaks were shifted, several were much broader in the complex, and a considerable subset had disappeared (Fig. 4A). This behavior cannot be explained by the increase in molecular mass (from 12 to 33 kDa) when Cdc42 binds and is more likely to be due to conformational exchange. This leads to broadening of the peaks so that they are not detectable. Overall chemical shift perturbations (CSPs) were calculated for each residue, whereas those that had disappeared were assigned a shift change of 0.2 (Fig. 4B). A peak that disappeared or had a CSP above the mean CSP for the spectrum was considered to be significantly affected. Mapping the binding surface of Cdc42 onto the TOCA1 HR1 domain.\nA, the 15N HSQC of 200 μm TOCA1 HR1 domain is shown in the free form (black) and in the presence of a 4-fold molar excess of Cdc42Δ7Q61L·GMPPNP (red). Expansions of two regions are shown with peak assignments, showing backbone amides in fast or intermediate exchange. B, CSPs were calculated as described under “Experimental Procedures” and are shown for backbone and side chain NH groups. The mean CSP is marked with a red line. Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.2 but were excluded when calculating the mean CSP and are indicated with open bars. Those that were not traceable due to spectral overlap were assigned a CSP of zero and are marked with an asterisk below the bar. Residues with affected side chain CSPs derived from 13C HSQCs are marked with green asterisks above the bars. Secondary structure elements are shown below the graph. C, a schematic representation of the HR1 domain. Residues with significantly affected backbone or side chain chemical shifts when Cdc42 bound and that are buried are colored dark blue, whereas those that are solvent-accessible are colored yellow. Residues with significantly affected backbone and side chain groups that are solvent-accessible are colored red. A close-up of the binding region is shown, with affected side chain heavy atoms shown as sticks. D, the G protein-binding region is marked in red onto structures of the HR1 domains as indicated. 15N HSQC shift mapping experiments report on changes to amide groups, which are mainly inaccessible because they are buried inside the helices and are involved in hydrogen bonds. Therefore, 13C HSQC and methyl-selective SOFAST-HMQC experiments were also recorded on 15N,13C-labeled TOCA1 HR1 to yield more information on side chain involvement. The affected CH groups underwent significant line broadening, similarly to the NH peaks. Side chains whose CH groups disappeared in the presence of Cdc42 are marked on the graph in Fig. 4B with green asterisks. TOCA1 residues whose signals were affected by Cdc42 binding were mapped onto the structure of TOCA1 HR1 (Fig. 4C). The changes were localized to one end of the coiled-coil, and the binding site appeared to include residues from both α-helices and the loop region that joins them. Residues outside of this region were not significantly affected, indicating that there was no widespread conformational change. The residues in the interhelical loop and helix 1 that contact each other (Fig. 3D) show shift changes in their backbone NH and side chains in the presence of Cdc42. For example, the side chain of Asn-380 and the backbones of Val-376 and Tyr-377 were significantly affected but are all buried in the free TOCA1 HR1 structure, indicating that local conformational changes in the loop may facilitate complex formation. The chemical shift mapping data indicate that the G protein-binding region of the TOCA1 HR1 domain is broadly similar to that of the CIP4 and PRK1 HR1 domains (Figs. 3B and 4D). The corresponding 15N and 13C NMR experiments were also recorded on 15N-Cdc42Δ7Q61L·GMPPNP or 15N/13C -Cdc42Δ7Q61L·GMPPNP in the presence of unlabeled HR1 domain. The overall CSP was calculated for each residue. As was the case when labeled HR1 was observed, several peaks were shifted in the complex, but many disappeared, indicating exchange on an unfavorable, millisecond time scale (Fig. 5A). Detailed side chain data could not be obtained for all residues due to spectral overlap, but constant time 13C HSQC and methyl-selective SOFAST-HMQC experiments provided further information on certain well resolved side chains (marked with green asterisks in Fig. 5B). Mapping the binding surface of the HR1 domain onto Cdc42.\nA, the 15N HSQC of Cdc42Δ7Q61L·GMPPNP is shown in its free form (black) and in the presence of excess TOCA1 HR1 domain (1:2.2, red). Expansions of two regions are shown, with most peaks in fast or intermediate exchange. B, CSPs are shown for backbone NH groups. The red line indicates the mean CSP, plus one S.D. Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.1 and are indicated with open bars. Those that were not traceable due to overlap are marked with an asterisk. Residues with disappeared peaks in 13C HSQC experiments are marked on the chart with green asterisks. Secondary structure elements are indicated below the graph. C, the residues with significantly affected backbone and side chain groups are highlighted on an NMR structure of free Cdc42Δ7Q61L·GMPPNP; those that are buried are colored dark blue, whereas those that are solvent-accessible are colored red. Residues with either side chain or backbone groups affected are colored blue if buried and yellow if solvent-accessible. Residues without information from shift mapping are colored gray. The flexible switch regions are circled. As many of the peaks disappeared, the mean chemical shift change was relatively low, so a threshold of the mean plus one S.D. value was used to define a significant CSP. Residues that disappeared were also classed as significantly affected. Parts of the switch regions (Fig. 5, B and C) are invisible in NMR spectra recorded on free Cdc42 due to conformational exchange. These switch regions become visible in Cdc42 and other small G protein·effector complexes due to a decrease in conformational freedom upon complex formation. The switch regions of Cdc42 did not, however, become visible in the presence of the TOCA1 HR1 domain. Indeed, Ser-30 of switch I and Arg-66, Arg-68, Leu-70, and Ser-71 of switch II are visible in free Cdc42 but disappear in the presence of the HR1 domain. This suggests that the switch regions are not rigidified in the HR1 complex and are still in conformational exchange. Nevertheless, mapping of the affected residues onto the NMR structure of free Cdc42Δ7Q61L·GMPPNP (Fig. 5C)8 shows that, although they are relatively widespread compared with changes in the HR1 domain, in general, they are on the face of the protein that includes the switches. Although the binding interface may be overestimated, this suggests that the switch regions are involved in binding to TOCA1. Modeling the Cdc42·TOCA1 HR1 Complex The Cdc42·HR1TOCA1 complex was not amenable to full structural analysis due to the weak interaction and the extensive exchange broadening seen in the NMR experiments. HADDOCK was therefore used to perform rigid body docking based on the structures of free HR1 domain and Cdc42 and ambiguous interaction restraints derived from the titration experiments described above. Residues with significantly affected resonances and more than 50% solvent accessibility were defined as active. Passive residues were defined automatically as those neighboring active residues. The orientation of the HR1 domain with respect to Cdc42 cannot be definitively concluded in the absence of unambiguous distance restraints; hence, HADDOCK produced a set of models in which the HR1 domain contacts the same surface on Cdc42 but is in various orientations with respect to Cdc42. The cluster with the lowest root mean square deviation from the lowest energy structure is assumed to be the best model. By these criteria, in the best model, the HR1 domain is in a similar orientation to the HR1a domain of PRK1 bound to RhoA and the HR1b domain bound to Rac1. A representative model from this cluster is shown in Fig. 6A alongside the Rac1-HR1b structure (PDB code 2RMK) in Fig. 6B. Model of Cdc42·HR1 complex.\nA, a representative model of the Cdc42·HR1 complex from the cluster closest to the lowest energy model produced using HADDOCK. Residues of Cdc42 that are affected in the presence of the HR1 domain but are not in close proximity to it are colored in red and labeled. B, structure of Rac1 in complex with the HR1b domain of PRK1 (PDB code 2RMK). C, sequence alignment of RhoA, Cdc42 and Rac1. Contact residues of RhoA and Rac1 to PRK1 HR1a and HR1b, respectively, are colored cyan. Residues of Cdc42 that disappear or show chemical shift changes in the presence of TOCA1 are colored cyan if also identified as contacts in RhoA and Rac1 and yellow if they are not. Residues equivalent to Rac1 and RhoA contact sites but that are invisible in free Cdc42 are gray. D, regions of interest of the Cdc42·HR1 domain model. The four lowest energy structures in the chosen HADDOCK cluster are shown overlaid, with the residues of interest shown as sticks and labeled. Cdc42 is shown in cyan, and TOCA1 is shown in purple. A sequence alignment of RhoA, Cdc42, and Rac1 is shown in Fig. 6C. The RhoA and Rac1 contact residues in the switch regions are invisible in the spectra of Cdc42, but they are generally conserved between all three G proteins. Several Cdc42 residues identified by chemical shift mapping are not in close contact in the Cdc42·TOCA1 model (Fig. 6A). Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II. Other residues that are affected in the Cdc42·TOCA1 complex but that do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) include Gln-2Cdc42, Lys-16Cdc42, Thr-52Cdc42, and Arg-68Cdc42. Lys-16Cdc42 is unlikely to be a contact residue because it is involved in nucleotide binding, but the others may represent specific Cdc42-TOCA1 contacts. In the model, these side chains are involved in direct contacts (Fig. 6D). Competition between N-WASP and TOCA1 From the known interactions and effects of the proteins in biological systems, it has been suggested that TOCA1 and N-WASP could bind Cdc42 simultaneously. Studies in CHO cells indicated that a Cdc42·N-WASP·TOCA1 complex existed because FRET was observed between RFP-TOCA1 and GFP-N-WASP, and the efficiency was decreased when an N-WASP mutant was used that no longer binds Cdc42. An overlay of the HADDOCK model of the Cdc42·HR1TOCA1 complex and the structure of Cdc42 in complex with the GBD of the N-WASP homologue, WASP (PDB code 1CEE), shows that the HR1 and GBD binding sites only partly overlap, and, therefore, a ternary complex remained possible (Fig. 7A). Interestingly, the presence of the TOCA1 HR1 would not prevent the core CRIB of WASP from binding to Cdc42, although the regions C-terminal to the CRIB that are required for high affinity binding of WASP would interfere sterically with the TOCA1 HR1. A basic region in WASP including three lysines (residues 230–232), N-terminal to the core CRIB, has been implicated in an electrostatic steering mechanism, and these residues would be free to bind in the presence of TOCA1 HR1 (Fig. 7A). The N-WASP GBD displaces the TOCA1 HR1 domain.\nA, the model of the Cdc42·TOCA1 HR1 domain complex overlaid with the Cdc42-WASP structure. Cdc42 is shown in green, and TOCA1 is shown in purple. The core CRIB region of WASP is shown in red, whereas its basic region is shown in orange and the C-terminal region required for maximal affinity is shown in cyan. A semitransparent surface representation of Cdc42 and WASP is shown overlaid with the schematic. B, competition SPA experiments carried out with indicated concentrations of the N-WASP GBD construct titrated into 30 nm GST-ACK or GST-WASP GBD and 30 nm Cdc42Δ7Q61L·[3H]GTP. C, Selected regions of the 15N HSQC of 145 μm Cdc42Δ7Q61L·GMPPNP with the indicated ratios of the TOCA1 HR1 domain, the N-WASP GBD, or both, showing that the TOCA HR1 domain does not displace the N-WASP GBD. D, selected regions of the 15N HSQC of 600 μm TOCA1 HR1 domain in complex with Cdc42 in the absence and presence of the N-WASP GBD, showing displacement of Cdc42 from the HR1 domain by N-WASP. An N-WASP GBD construct was produced, and its affinity for Cdc42 was measured by competition SPA (Fig. 7B). The Kd that was determined (37 nm) is consistent with the previously reported affinity. Unlabeled N-WASP GBD was titrated into 15N-Cdc42Δ7Q61L·GMPPNP, and the backbone NH groups were monitored using HSQCs (Fig. 7C). Unlabeled HR1TOCA1 was then added to the Cdc42·N-WASP complex, and no changes were seen, suggesting that the N-WASP GBD was not displaced even in the presence of a 5-fold excess of HR1TOCA1. These experiments were recorded at sufficiently high protein concentrations (145 μm Cdc42, 145 μm N-WASP GBD, 725 μm TOCA1 HR1 domain) to be far in excess of the Kd values of the individual interactions (TOCA1 Kd ≈ 5 μm, N-WASP Kd = 37 nm). A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C). Furthermore, 15N-TOCA1 HR1 was monitored in the presence of unlabeled Cdc42Δ7Q61L·GMPPNP (1:1) before and after the addition of 0.25 and 1.0 eq of unlabeled N-WASP GBD. The spectrum when N-WASP and TOCA1 were equimolar was identical to that of the free HR1 domain, whereas the spectrum in the presence of 0.25 eq of N-WASP was intermediate between the TOCA1 HR1 free and complex spectra (Fig. 7D). When in fast exchange, the NMR signal represents a population-weighted average between free and bound states, so the intermediate spectrum indicates that the population comprises a mixture of free and bound HR1 domain. Hence, a third, intermediate state that includes all three proteins is unlikely. Again, the experiments were recorded on protein samples far in excess of the individual Kd values (600 μm each protein). These data indicate that the HR1 domain is displaced from Cdc42 by N-WASP and that a ternary complex comprising TOCA1 HR1, N-WASP GBD, and Cdc42 is not formed. Taken together, the data in Fig. 7, C and D, indicate unidirectional competition for Cdc42 binding in which the N-WASP GBD displaces TOCA1 HR1 but not vice versa. To extend these studies to a more complex system and to assess the ability of TOCA1 HR1 to compete with full-length N-WASP, pyrene actin assays were employed. These assays, described in detail elsewhere, were carried out using pyrene actin-supplemented Xenopus extracts into which exogenous TOCA1 HR1 domain or N-WASP GBD was added, to assess their effects on actin polymerization. Actin polymerization in all cases was initiated by the addition of PI(4,5)P2-containing liposomes. Actin polymerization triggered by the addition of PI(4,5)P2-containing liposomes has previously been shown to depend on TOCA1 and N-WASP. Endogenous N-WASP is present at ∼100 nm in Xenopus extracts, whereas TOCA1 is present at a 10-fold lower concentration than N-WASP. The addition of the isolated N-WASP GBD significantly inhibited the polymerization of actin at concentrations as low as 100 nm and completely abolished polymerization at higher concentrations (Fig. 8). The GBD presumably acts as a dominant negative, sequestering endogenous Cdc42 and preventing endogenous full-length N-WASP from binding and becoming activated. The addition of the TOCA1 HR1 domain to 100 μm had no significant effect on the rate of actin polymerization or maximum fluorescence. This is consistent with endogenous N-WASP, activated by other components of the assay, outcompeting the TOCA1 HR1 domain for Cdc42 binding. Actin polymerization downstream of Cdc42·N-WASP·TOCA1 is inhibited by excess N-WASP GBD but not by the TOCA1 HR1 domain. Fluorescence curves show actin polymerization in the presence of increasing concentrations of N-WASP GBD or TOCA1 HR1 domain as indicated. Maximal rates of actin polymerization derived from the linear region of the curves are represented in bar charts below. Error bars, S.E. Discussion The Cdc42-TOCA1 Interaction The TOCA1 HR1 domain alone is sufficient for Cdc42 binding in vitro, yet the affinity of the TOCA1 HR1 domain for Cdc42 is remarkably low (Kd ≈ 5 μm). This is over 100 times lower than that of the N-WASP GBD (Kd = 37 nm) and considerably lower than other known G protein-HR1 domain interactions. The polybasic tract within the C-terminal region of Cdc42 does not appear to be required for binding to TOCA1, which is in contrast to the interaction between Rac1 and the HR1b domain of PRK1 but more similar to the PRK1 HR1a-RhoA interaction. A single binding interface on both the HR1 domain and Cdc42 can be concluded from the data presented here. Furthermore, the interfaces are comparable with those of other G protein-HR1 interactions (Fig. 4), and the lowest energy model produced in rigid body docking resembles previously studied G protein·HR1 complexes (Fig. 6). It seems, therefore, that the interaction, despite its relatively low affinity, is specific and sterically similar to other HR1 domain-G protein interactions. The TOCA1 HR1 domain is a left-handed coiled-coil comparable with other known HR1 domains. A short region N-terminal to the coiled-coil exhibits a series of turns and contacts residues of both helices of the coiled-coil (Fig. 3). The corresponding sequence in CIP4 also includes a series of turns but is flexible, whereas in the HR1a domain of PRK1, the equivalent region adopts an α-helical structure that packs against the coiled-coil. The contacts between the N-terminal region and the coiled-coil are predominantly hydrophobic in both cases, but sequence-specific contacts do not appear to be conserved. This region is distant from the G protein-binding interface of the HR1 domains, so the structural differences may relate to the structure and regulation of these domains rather than their G protein interactions. The interhelical loops of TOCA1 and CIP4 differ from the same region in the HR1 domains of PRK1 in that they are longer and contain two short stretches of 310-helix. This region lies within the G protein-binding surface of all of the HR1 domains (Fig. 4D). TOCA1 and CIP4 both bind weakly to Cdc42, whereas the HR1a domain of PRK1 binds tightly to RhoA and Rac1, and the HR1b domain binds to Rac1. The structural features shared by TOCA1 and CIP4 may therefore be related to Cdc42 binding specificity and the low affinities. In free TOCA1, the side chains of the interhelical region make extensive contacts with residues in helix 1. Many of these residues are significantly affected in the presence of Cdc42, so it is likely that the conformation of this loop is altered in the Cdc42 complex. These observations therefore provide a molecular mechanism whereby mutation of Met383-Gly384-Asp385 to Ile383-Ser384-Thr385 abolishes TOCA1 binding to Cdc42. The lowest energy model produced by HADDOCK using ambiguous interaction restraints from the titration data resembled the NMR structures of RhoA and Rac1 in complex with their HR1 domain partners. Some speculative conclusions can be made based on this model. For example, Phe-56Cdc42, which is not visible in free Cdc42 or Cdc42·HR1TOCA1, is close to the TOCA1 HR1 (Fig. 6A). Phe-56Cdc42, which is a Trp in both Rac1 and RhoA (Fig. 6C), is thought to pack behind switch I when Cdc42 interacts with ACK, maintaining the switch in a binding-competent orientation. This residue has also been identified as important for Cdc42-WASP binding. Phe-56Cdc42 is therefore likely to be involved in the Cdc42-TOCA1 interaction, probably by stabilizing the position of switch I. Some residues that are affected in the Cdc42·HR1TOCA1 complex but do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) may contact HR1TOCA1 directly (Fig. 6D). Gln-2Cdc42, which has also been identified as a contact residue in the Cdc42·ACK complex, contacts Val-376TOCA1 and Asn-380TOCA1 in the model and disrupts the contacts between the interhelical loop and the first helix of the TOCA1 coiled-coil. Thr-52Cdc42, which has also been identified as making minor contacts with ACK, falls near the side chains of HR1TOCA1 helix 1, particularly Lys-372TOCA1, whereas the equivalent position in Rac1 is Asn-52Rac1. N52T is one of a combination of seven residues found to confer ACK binding on Rac1 and so may represent a specific Cdc42-effector contact residue. The position equivalent to Lys-372TOCA1 in PRK1 is Glu-58HR1a or Gln-151HR1b. Thr-52Cdc42-Lys-372TOCA1 may therefore represent a specific Cdc42-HR1TOCA1 contact. Arg-68Cdc42 of switch II is positioned close to Glu-395TOCA1 (Fig. 6D), suggesting a direct electrostatic contact between switch II of Cdc42 and helix 2 of the HR1 domain. The equivalent Arg in Rac1 and RhoA is pointing away from the HR1 domains of PRK1. The importance of this residue in the Cdc42-TOCA1 interaction remains unclear, although its mutation reduces binding to RhoGAP, suggesting that it can be involved in Cdc42 interactions. The solution structure of the TOCA1 HR1 domain presented here, along with the model of the HR1TOCA1·Cdc42 complex is consistent with a conserved mode of binding across the known HR1 domain-Rho family interactions, despite their differing affinities. The weak binding prevented detailed structural and thermodynamic studies of the complex. Nonetheless, structural studies of the TOCA1 HR1 domain, combined with chemical shift mapping, have highlighted some potentially interesting differences between Cdc42-HR1TOCA1 and RhoA/Rac1-HR1PRK1 binding. We have previously postulated that the inherent flexibility of HR1 domains contributes to their ability to bind to different Rho family G proteins, with Rho-binding HR1 domains displaying increased flexibility, reflected in their lower melting temperatures (Tm) and Rac binders being more rigid. The Tm of the TOCA1 HR1 domain is 61.9 °C (data not shown), which is the highest Tm that we have measured for an HR1 domain thus far. As such, the ability of the TOCA1 HR1 domain to bind to Cdc42 (a close relative of Rac1 rather than RhoA) fits this trend. An investigation into the local motions, particularly in the G protein-binding regions, may offer further insight into the differential specificities and affinities of G protein-HR1 domain interactions. Significance of a Weak, Transient Interaction The low affinity of the Cdc42-HR1TOCA1 interaction is consistent with a tightly spatially and temporally regulated pathway, requiring combinatorial signals leading to a series of coincident weak interactions that elicit full activation. The HR1 domains from other TOCA family members, CIP4 and FBP17, also bind at low micromolar affinities to Cdc42, so the low affinity interaction appears to be commonplace among this family of HR1 domain proteins, in contrast to the PRK family. Weak, transient protein-protein interactions are functionally significant in several systems; for example, the binding of adaptor proteins to protein cargo during the formation of clathrin-coated vesicles in endocytosis involves multiple interactions of micromolar affinity. The low affinity of the HR1TOCA1-Cdc42 interaction in the context of the physiological concentration of TOCA1 in Xenopus extracts (∼10 nm) suggests that binding between TOCA1 and Cdc42 is likely to occur in vivo only when TOCA1 is at high local concentrations and membrane-localized and therefore in close proximity to activated Cdc42. Evidence suggests that the TOCA family of proteins are recruited to the membrane via an interaction between their F-BAR domain and specific signaling lipids. For example, electrostatic interactions between the F-BAR domain and the membrane are required for TOCA1 recruitment to membrane vesicles and tubules, and TOCA1-dependent actin polymerization is known to depend specifically on PI(4,5)P2. Furthermore, the isolated F-BAR domain of FBP17 has been shown to induce membrane tubulation of brain liposomes and BAR domain proteins that promote tubulation cluster on membranes at high densities. Once at the membrane, high local concentrations of TOCA1 could exceed the Kd of F-BAR dimerization (likely to be comparable with that of the FCHo2 F-BAR domain (2.5 μm)) and that of the Cdc42-HR1TOCA1 interaction. Cdc42-HR1TOCA1 binding would then be favorable, as long as coincident activation of Cdc42 had occurred, leading to stabilization of TOCA1 at the membrane and downstream activation of N-WASP. It has been postulated that WASP and N-WASP exist in equilibrium between folded (inactive) and unfolded (active) forms, and the affinity of Cdc42 for the unfolded WASP proteins is significantly enhanced. The unfolded, high affinity state of WASP is represented by a short peptide, the GBD, which binds with a low nanomolar affinity to Cdc42. In contrast, the best estimate of the affinity of full-length WASP for Cdc42 is low micromolar. In the inactive state of WASP, the actin- and Arp2/3-binding VCA domain contacts the GBD, competing for Cdc42 binding. The high affinity of Cdc42 for the unfolded, active form pushes the equilibrium in favor of (N-)WASP activation. Binding of PI(4,5)P2 to the basic region just N-terminal to the GBD further favors the active conformation. A substantial body of data has illuminated the complex regulation of WASP/N-WASP proteins, and current evidence suggests that these allosteric activation mechanisms and oligomerization combine to regulate WASP activity, allowing the synchronization and integration of multiple potential activation signals (reviewed in Ref.). Our data are easily reconciled with this model. We envisage that TOCA1 is first recruited to the appropriate membrane in response to PI(4,5)P2 via its F-BAR domain, where the local increase in concentration favors F-BAR-mediated dimerization of TOCA1. Cdc42 is activated in response to co-incident signals and can then bind to TOCA1, further stabilizing TOCA1 at the membrane. TOCA1 can then recruit N-WASP via an interaction between its SH3 domain and the N-WASP proline-rich region. The recruitment of N-WASP alone and of the N-WASP·WIP complex by TOCA1 and FBP17 has been demonstrated. WIP inhibits the activation of N-WASP by Cdc42, an effect that is reversed by TOCA1. It may therefore be envisaged that WIP and TOCA1 exert opposing allosteric effects on N-WASP, with TOCA1 favoring the unfolded, active conformation of N-WASP and increasing its affinity for Cdc42. TOCA1 may also activate N-WASP by effective oligomerization because clustering of TOCA1 at the membrane following coincident interactions with PI(4,5)P2 and Cdc42 would in turn lead to clustering of N-WASP, in addition to pushing the equilibrium toward the unfolded, active state. In a cellular context, full-length TOCA1 and N-WASP are likely to have similar affinities for active Cdc42, but in the unfolded, active conformation, the affinity of N-WASP for Cdc42 dramatically increases. Our binding data suggest that TOCA1 HR1 binding is not allosterically regulated, and our NMR data, along with the high stability of TOCA1 HR1, suggest that there is no widespread conformational change in the presence of Cdc42. As full-length TOCA1 and the isolated HR1 domain bind Cdc42 with similar affinities, the N-WASP-Cdc42 interaction will be favored because the N-WASP GBD can easily outcompete the TOCA1 HR1 for Cdc42. A combination of allosteric activation by PI(4,5)P2, activated Cdc42 and TOCA1, and oligomeric activation implemented by TOCA1 would lead to full activation of N-WASP and downstream actin polymerization. In such an array of molecules localized to a discrete region of the membrane, it is plausible that WASP could bind to a second Cdc42 molecule rather than displacing TOCA1 from its cognate Cdc42. Our NMR and affinity data, however, are consistent with displacement of the TOCA1 HR1 by the N-WASP GBD. Furthermore, TOCA1 is required for Cdc42-mediated activation of N-WASP·WIP, implying that it may not be possible for Cdc42 to bind and activate N-WASP prior to TOCA1-Cdc42 binding. The commonly used MGD → IST (Cdc42-binding deficient) mutant of TOCA1 has a reduced ability to activate the N-WASP·WIP complex, further indicating the importance of the Cdc42-HR1TOCA1 interaction prior to downstream activation of N-WASP. In light of this, we favor an “effector handover” scheme whereby TOCA1 interacts with Cdc42 prior to N-WASP activation, after which N-WASP displaces TOCA1 from its bound Cdc42 in order to be fully activated rather than binding a second Cdc42 molecule. Potentially, the TOCA1-Cdc42 interaction functions to position N-WASP and Cdc42 such that they are poised to interact with high affinity. The concomitant release of TOCA1 from Cdc42 while still bound to N-WASP presumably enhances the ability of TOCA1 to further activate N-WASP·WIP-induced actin polymerization. There is an advantage to such an effector handover, in that N-WASP would only be robustly recruited when F-BAR domains are already present. Hence, actin polymerization cannot occur until F-BAR domains are poised for membrane distortion. Our model of the Cdc42·HR1TOCA1 complex indicates a mechanism by which such a handover could take place (Fig. 9) because it shows that the effector binding sites only partially overlap on Cdc42. The lysine residues thought to be involved in an electrostatic steering mechanism in WASP-Cdc42 binding are conserved in N-WASP and would be able to interact with Cdc42 even when the TOCA1 HR1 domain is already bound. It has been postulated that the initial interactions between this basic region and Cdc42 could stabilize the active conformation of WASP, leading to high affinity binding between the core CRIB and Cdc42. The region C-terminal to the core CRIB, required for maximal affinity binding, would then fully displace the TOCA1 HR1. A simplified model of the early stages of Cdc42·N-WASP·TOCA1-dependent actin polymerization.\nStep 1, TOCA1 is recruited to the membrane via its F-BAR domain and/or Cdc42 interactions. F-BAR oligomerization is expected to occur following membrane binding, but a single monomer is shown for clarity. Step 2, N-WASP exists in an inactive, folded conformation. The TOCA1 SH3 domain interacts with N-WASP, causing an activatory allosteric effect. The HR1TOCA1-Cdc42 and SH3TOCA1-N-WASP interactions position Cdc42 and N-WASP for binding. Step 3, electrostatic interactions between Cdc42 and the basic region upstream of the CRIB initiate Cdc42·N-WASP binding. Step 4, the core CRIB binds with high affinity while the region C-terminal to the CRIB displaces the TOCA1 HR1 domain and increases the affinity of the N-WASP-Cdc42 interaction further. The VCA domain is released for downstream interactions, and actin polymerization proceeds. WH1, WASP homology 1 domain; PP, proline-rich region; VCA, verprolin homology, cofilin homology, acidic region. In conclusion, the data presented here show that the TOCA1 HR1 domain is sufficient for Cdc42 binding in vitro and that the interaction is of micromolar affinity, lower than that of other G protein-HR1 domain interactions. The analogous HR1 domains from other TOCA1 family members, FBP17 and CIP4, also exhibit micromolar affinity for Cdc42. A role for the TOCA1-, FBP17-, and CIP4-Cdc42 interactions in the recruitment of these proteins to the membrane therefore appears unlikely. Instead, our findings agree with earlier suggestions that the F-BAR domain is responsible for membrane recruitment. The role of the Cdc42-TOCA1 interaction remains somewhat elusive, but it may serve to position activated Cdc42 and N-WASP to allow full activation of N-WASP and as such serve to couple F-BAR-mediated membrane deformation with N-WASP activation. We envisage a complex interplay of equilibria between free and bound, active and inactive Cdc42, TOCA family, and WASP family proteins, facilitating a tightly spatially and temporally regulated pathway requiring numerous simultaneous events in order to achieve appropriate and robust activation of the downstream pathway. Our data are therefore easily reconciled with the dynamic instability models described in relation to the formation of endocytic vesicles and with the current data pertaining to the complex activation of WASP/N-WASP pathways by allosteric and oligomeric effects. It is clear from the data presented here that TOCA1 and N-WASP do not bind Cdc42 simultaneously and that N-WASP is likely to outcompete TOCA1 for Cdc42 binding. We therefore postulate an effector handover mechanism based on current evidence surrounding WASP/N-WASP activation and our model of the Cdc42·HR1TOCA1 complex. The displacement of the TOCA1 HR1 domain from Cdc42 by N-WASP may represent a unidirectional step in the pathway of Cdc42·N-WASP·TOCA1-dependent actin assembly. Author Contributions J. R. W. generated constructs and proteins, set up NMR experiments, analyzed NMR data, and performed binding experiments; D. N. set up NMR experiments; H. M. F. generated longer TOCA clones and proteins; J. L. G. supervised the pyrene actin assays; D. O. supervised the protein binding assays; and H. R. M. performed NMR experiments and analyzed NMR data. J. R. W., D. O., and H. R. M. wrote the paper with input from all authors. The authors declare that they have no conflicts of interest with the contents of this article. The atomic coordinates and structure factors (code 5FRG) have been deposited in the Protein Data Bank (http://wwpdb.org/). D. Owen, unpublished data. H. R. Mott and D. Owen, unpublished data. PRK protein kinase C related kinase WASP Wiskott-Aldrich syndrome protein TOCA transducer of Cdc42-dependent actin assembly protein N-WASP neural Wiskott-Aldrich syndrome protein PI(4,5)P2 phosphatidylinositol 4,5-bisphosphate HR1 homology region 1 F-BAR Fes/CIP4 homology BAR SH3 Src homology 3 CRIB Cdc42- and Rac-interactive binding CIP4 Cdc42-interacting protein 4 MBP maltose-binding protein GBD G protein binding domain SPA scintillation proximity assay PAK p21-activated kinase ACK activated Cdc42-associated kinase HSQC heteronuclear single quantum correlation GMPPNP guanosine 5′-[β,γ-imido] triphosphate GTPγS guanosine 5′-3-O-(thio)triphosphate GBD G protein-binding domain CSP chemical shift perturbation PDB Protein Data Bank. The abbreviations used are:  References The GTPase superfamily: conserved structure and molecular mechanism Regulation of small GTPases by GEFs, GAPs, and GDIs The guanine nucleotide-binding switch in three dimensions Structures of Ras superfamily effector complexes: what have we learnt in two decades? Rho: a connection between membrane receptor signalling and the cytoskeleton The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors The small GTP-binding protein rac regulates growth factor-induced membrane ruffling Rho: theme and variations Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia Rho GTPases and the actin cytoskeleton Signaling to actin dynamics The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts Formins as effector proteins of Rho GTPases Protein kinase N (PKN) and PKN-related protein rhophilin as targets of small GTPase Rho Wiskott-Aldrich syndrome protein, a novel effector for the GTPase CDC42Hs, is implicated in actin polymerization Toca-1 mediates Cdc42-dependent actin nucleation by activating the N-WASP-WIP complex A Cdc42 target protein with homology to the non-kinase domain of FER has a potential role in regulating the actin cytoskeleton Calcium oscillations-coupled conversion of actin travelling waves to standing oscillations WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4,5-bisphosphate Corequirement of specific phosphoinositides and small GTP-binding protein Cdc42 in inducing actin assembly in Xenopus egg extracts Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein Physical mechanisms of signal integration by WASP family proteins The Arp2/3 complex mediates actin polymerization induced by the small GTP-binding protein Cdc42 EFC/F-BAR proteins and the N-WASP-WIP complex induce membrane curvature-dependent actin polymerization The Toca-1-N-WASP complex links filopodial formation to endocytosis Drosophila Cip4/Toca-1 integrates membrane trafficking and actin dynamics through WASP and SCAR/WAVE Requirements for F-BAR proteins TOCA-1 and TOCA-2 in actin dynamics and membrane trafficking during Caenorhabditis elegans oocyte growth and embryonic epidermal morphogenesis Cdc42 interaction with N-WASP and Toca-1 regulates membrane tubulation, vesicle formation and vesicle motility: implications for endocytosis Self-assembly of filopodia-like structures on supported lipid bilayers Phosphoinositides and membrane curvature switch the mode of actin polymerization via selective recruitment of toca-1 and Snx9 Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling Regulation of neuronal morphology by Toca-1, an F-BAR/EFC protein that induces plasma membrane invagination Nuclear actin polymerization is required for transcriptional reprogramming of Oct4 by oocytes A complex of ZO-1 and the BAR-domain protein TOCA-1 regulates actin assembly at the tight junction Transducer of Cdc42-dependent actin assembly promotes epidermal growth factor-induced cell motility and invasiveness Transducer of Cdc42-dependent actin assembly promotes breast cancer invasion and metastasis Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins Structure and analysis of FCHo2 F-BAR domain: a dimerizing and membrane recruitment module that effects membrane curvature The structural basis of Rho effector recognition revealed by the crystal structure of human RhoA complexed with the effector domain of PKN/PRK1 Molecular dissection of the interaction between the small G proteins Rac1 and RhoA and protein kinase C-related kinase 1 (PRK1) Mutational analysis reveals a single binding interface between RhoA and its effector, PRK1 Differential binding of RhoA, RhoB, and RhoC to protein kinase C-related kinase (PRK) isoforms PRK1, PRK2, and PRK3: PRKs have the highest affinity for RhoB The Rac1 polybasic region is required for interaction with its effector PRK1 The NMR structure of the TC10- and Cdc42-interacting domain of CIP4 A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases Structure of Cdc42 in complex with the GTPase-binding domain of the “Wiskott-Aldrich syndrome” protein Residues in Cdc42 that specify binding to individual CRIB effector proteins The structure of binder of Arl2 (BART) reveals a novel G protein binding domain: implications for function Delineation of the Cdc42/Rac-binding domain of p21-activated kinase The IQGAP1-Rac1 and IQGAP1-Cdc42 interactions: interfaces differ between the complexes 1H, 13C and 15N resonance assignments of the Cdc42-binding domain of TOCA1 The CCPN data model for NMR spectroscopy: development of a software pipeline ARIA2: automated NOE assignment and data integration in NMR structure calculation Protein backbone and side chain torsion angles predicted from NMR chemical shifts using artificial neural networks  Triggering actin polymerization in Xenopus egg extracts from phosphoinositide-containing lipid bilayers Activation of the WAVE complex by coincident signals controls actin assembly Structure of the small G protein Cdc42 bound to the GTPase-binding domain of ACK Structural basis for membrane binding specificity of the Bin/Amphiphysin/Rvs (BAR) domain of Arfaptin-2 determined by Arl1 GTPase Targeting and activation of Rac1 are mediated by the exchange factor β-Pix STRIDE: a web server for secondary structure assignment from known atomic coordinates of proteins SOFAST-HMQC experiments for recording two-dimensional heteronuclear correlation spectra of proteins within a few seconds The RalB-RLIP76 complex reveals a novel mode of Ral-effector interaction The HADDOCK web server for data-driven biomolecular docking The Cdc42/Rac interactive binding region motif of the Wiskott Aldrich syndrome protein (WASP) is necessary but not sufficient for tight binding to Cdc42 and structure formation An electrostatic steering mechanism of Cdc42 recognition by Wiskott-Aldrich syndrome proteins Double mutant cycle thermodynamic analysis of the hydrophobic Cdc42-ACK protein-protein interaction Single-molecule dynamics reveals cooperative binding-folding in protein recognition Specificity determinants on Cdc42 for binding its effector protein ACK Transient protein-protein interactions: structural, functional, and network properties Transient protein-protein interactions Integrating molecular and network biology to decode endocytosis Evolving nature of the AP2 α-appendage hub during clathrin-coated vesicle endocytosis Phosphatidylinositol-(4,5)-bisphosphate regulates sorting signal recognition by the clathrin-associated adaptor complex AP2 The nucleotide switch in Cdc42 modulates coupling between the GTPase-binding and allosteric equilibria of Wiskott-Aldrich syndrome protein A two-state allosteric model for autoinhibition rationalizes WASP signal integration and targeting Direct binding of the verprolin-homology domain in N-WASP to actin is essential for cytoskeletal 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+[{"sourceid":"4937829","sourcedb":"","project":"","target":"","text":"Visualizing chaperone-assisted protein folding Challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding, where obtaining structural ensembles of chaperone:substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a novel structural biology approach based on X-ray crystallography, termed Residual Electron and Anomalous Density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy. This study resulted in a series of snapshots depicting the various folding states of Im7 while bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone. High-resolution structural models of protein-protein interactions are critical for obtaining mechanistic insights into biological processes. However, many protein-protein interactions are highly dynamic, making it difficult to obtain high-resolution data. Particularly challenging are interactions of intrinsically or conditionally disordered sections of proteins with their partner proteins. Recent advances in X-ray crystallography and NMR spectroscopy continue to improve our ability to analyze biomolecules that exist in multiple conformations. X-ray crystallography has historically provided valuable information on small-scale conformational changes, but observing large-amplitude heterogeneous conformational changes often falls beyond the reach of current crystallographic techniques. NMR can theoretically be used to determine heterogeneous ensembles, but in practice, this proves to be very challenging. Despite the importance of understanding how proteins fold into their native state within the cell, our knowledge about this critical process remains limited. It is clear that molecular chaperones aid in protein folding. However, exactly how they facilitate the folding process is still being debated. Structural characterization of chaperone-assisted protein folding likely would help bring clarity to this question. Structural models of chaperone-substrate complexes have recently begun to provide information as to how a chaperone can recognize its substrate. However, the impact that chaperones have on their substrates, and how these interactions affect the folding process remain largely unknown. For most chaperones, it is still unclear whether the chaperone actively participates in and affects the folding of the substrate proteins, or merely provides a suitable microenvironment enabling the substrate to fold on its own. This is a truly fundamental question in the chaperone field, and one that has eluded the community largely because of the highly dynamic nature of the chaperone-substrate complexes. To address this question, we investigated the ATP-independent Escherichia coli periplasmic chaperone Spy. Spy prevents protein aggregation and aids in protein folding under various stress conditions, including treatment with tannin and butanol. We originally discovered Spy by its ability to stabilize the protein-folding model Im7 in vivo and recently demonstrated that Im7 folds while associated with Spy. The crystal structure of Spy revealed that it forms a thin α-helical homodimeric cradle. Crosslinking and genetic experiments suggested that Spy interacts with substrates somewhere on its concave side. By using a novel X-ray crystallography-based approach to model disorder in crystal structures, we have now determined the high-resolution ensemble of the dynamic Spy:Im7 complex. This work provides a detailed view of chaperone-mediated protein folding and shows how substrates like Im7 find their native fold while bound to their chaperones. RESULTS Crystallizing the Spy:Im7 complex We reasoned that to obtain crystals of complexes between Spy (domain boundaries in Supplementary Fig. 1) and its substrate proteins, our best approach was to identify crystallization conditions that yielded Spy crystals in the presence of protein substrates but not in their absence. We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. We found conditions in which all four substrates co-crystallized with Spy, but in which Spy alone did not yield crystals. Subsequent crystal washing and dissolution experiments confirmed the presence of the substrates in the co-crystals (Supplementary Fig. 2). The crystals diffracted to ~1.8 Å resolution. We used Spy:Im76-45 selenomethionine crystals for phasing with single-wavelength anomalous diffraction (SAD) experiments, and used this solution to build the well-ordered Spy portions of all four complexes. However, modeling of the substrate in the complex proved to be a substantial challenge, as the electron density of the substrate was discontinuous and fragmented. Even the minimal binding portion of Im7 (Im76-45) showed highly dispersed electron density (Fig. 1a). We hypothesized that the fragmented density was due to multiple, partially occupied conformations of the substrate bound within the crystal. Such residual density is typically not considered usable by traditional X-ray crystallography methods. Thus, we developed a new approach to interpret the chaperone-bound substrate in multiple conformations. READ: a strategy to visualize heterogeneous and dynamic biomolecules To determine the structure of the substrate portion of these Spy:substrate complexes, we conceived of an approach that we term READ, for Residual Electron and Anomalous Density. We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate. (2) We then labeled individual residues in the flexible regions of the substrate with the strong anomalous scatterer iodine, which serves to locate these residues in three-dimensional space using their anomalous density. (3) We performed molecular dynamics (MD) simulations to generate a pool of energetically reasonable conformations of the dynamic complex and (4) applied a sample-and-select algorithm to determine the minimal set of substrate conformations that fit both the residual and anomalous density. (5) Finally, we validated the ensemble using multiple statistical tests. Importantly, even though we only labeled a subset of the residues in the flexible regions of the substrate with iodine, the residual electron density can provide spatial information on many of the other flexible residues. These two forms of data are therefore complementary: by labeling individual residues, one can locate them to specific points in space. The electron density then allowed us to connect the labeled residues of the substrate by confining the protein chain within regions of detectable density. In this way, the two forms of data together were able to describe multiple conformations of the substrate within the crystal. As described in detail below, we developed the READ method to uncover the ensemble of conformations that the Spy-binding domain of Im7 (i.e., Im76-45) adopts while bound to Spy. However, we believe that READ will prove generally applicable to visualizing heterogeneous and dynamic complexes that have previously escaped detailed structural analysis. Collecting READ data for the Spy:Im76-45 complex To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3). Moreover, binding experiments indicated that Im76-45 comprises the entire Spy-binding region. To introduce the anomalous scatterer iodine, we replaced eight Im76-45 residues with the non-canonical amino acid 4-iodophenylalanine (pI-Phe). Its strong anomalous scattering allowed us to track the positions of these individual Im76-45 residues one at a time, potentially even if the residue was found in several locations in the same crystal. We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2). Consistent with our electron density map, we found that the majority of anomalous signals emerged in the cradle of Spy, implying that this is the likely Im7 substrate binding site. Consistent with the fragmented density, however, we observed multiple iodine positions for seven of the eight substituted residues. Together, these results indicated that the Im7 substrate binds Spy in multiple conformations. READ sample-and-select procedure To determine the structural ensemble that Im76-45 adopts while bound to Spy, we combined the residual electron density and the anomalous signals from our pI-Phe substituted Spy:Im76-45 complexes. To generate an accurate depiction of the chaperone-substrate interactions, we devised a selection protocol based on a sample-and-select procedure employed in NMR spectroscopy. This procedure iteratively constructs structural ensembles and then compares them to the experimental data. During each round of the selection, a genetic algorithm alters the ensemble and its agreement to the experimental data is re-evaluated. If successful, the selection identifies the smallest group of specific conformations that best fits the residual electron density and anomalous signals. The READ sample-and-select algorithm is diagrammed in Fig. 2. Prior to performing the selection, we generated a large and diverse pool of chaperone-substrate complexes using coarse-grained MD simulations in a pseudo-crystal environment (Fig. 2 and Supplementary Fig. 4). The coarse-grained simulations are based on a single-residue resolution model for protein folding and were extended here to describe Spy-Im76-45 binding events (Online Methods). The initial conditions of the binding simulations are not biased toward a particular conformation of the substrate or any specific chaperone-substrate interaction (Online Methods). Im76-45 binds and unbinds to Spy throughout the simulations. This strategy allows a wide range of substrate conformations to interact with the chaperone. From the MD simulations, we extracted ~10,000 diverse Spy:Im76-45 complexes to be used by the ensuing selection. Each complex within this pool comprises one Spy dimer bound to a single Im76-45 substrate. This pool was then used by the selection algorithm to identify the minimal ensemble that best satisfies both the residual electron and anomalous crystallographic data. The anomalous scattering portion of the selection uses our basic knowledge of pI-Phe geometry: the iodine is separated from its respective Cα atom in each coarse-grained conformer by 6.5 Å. The selection then picks ensembles that best reproduce the collection of iodine anomalous signals. Simultaneously, it uses the residual electron density to help choose ensembles. To make the electron density selection practical, we needed to develop a method to rapidly evaluate the agreement between the selected sub-ensembles and the experimental electron density on-the-fly during the selection procedure. To accomplish this task, we generated a compressed version of the experimental 2mFo−DFc electron density map for use in the selection. This process provided us with a target map that the ensuing selection tried to recapitulate. To reduce the extent of 3D space to be explored, this compressed map was created by only using density from regions of space significantly sampled by Im76-45 in the Spy:Im76-45 MD simulations. For each of the ~10,000 complexes in the coarse-grained MD pool, the electron density at the Cα positions of Im76-45 was extracted and used to construct an electron density map (Online Methods). These individual electron density maps from the separate conformers could then be combined (Fig. 2) and compared to the averaged experimental electron density map as part of the selection algorithm. This approach allowed us to simultaneously use both the iodine anomalous signals and the residual electron density in the selection procedure. The selection resulted in small ensembles from the MD pool that best fit the READ data (Fig. 1c,d). Before analyzing the details of the Spy:Im76-45 complex, we first engaged in a series of validation tests to verify the ensemble and selection procedure (Supplementary Note 1, Figures 1c,d, Supplemental Figures 5-7). Combined, these validation tests confirmed that the selection procedure and selected six-member ensemble recapitulate the experimental data. Of note, the final six-membered ensemble was the largest ensemble that could simultaneously decrease the RFree and pass the 10-fold cross-validation test. This ensemble depicts the conformations that the substrate Im76-45 adopts while bound to the chaperone Spy (Fig. 3 Supplementary Movie 1, and Table 1). Folding and interactions of Im7 while bound to Spy Our results showed that by using this novel READ approach, we were able to obtain structural information about the dynamic interaction of a chaperone with its substrate protein. We were particularly interested in finding answers to one of the most fundamental questions in chaperone biology—how does chaperone binding affect substrate structure and vice versa. By analyzing the individual structures of the six-member ensemble of Im76-45 bound to Spy, we observed that Im76-45 takes on several different conformations while bound. We found these conformations to be highly heterogeneous and to include unfolded, partially folded, and native-like states (Fig. 3). The ensemble primarily encompasses Im76-45 laying diagonally within the Spy cradle in several different orientations, but some conformations traverse as far as the tips or even extend over the side of the cradle (Figs. 3,4a). We constructed a contact map of the complex, which shows the frequency of interactions for chaperone-substrate residue pairs (Fig. 4). We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46). The Spy-contacting residues comprise a mixture of charged, polar, and hydrophobic residues. Surprisingly, we noted that in the ensemble, Im76-45 interacts with only 38% of the hydrophobic residues in the Spy cradle, but interacts with 61% of the hydrophilic residues in the cradle. This mixture suggests the importance of both electrostatic and hydrophobic components in binding the Im76-45 ensemble. With respect to the substrate, we observed that nearly every residue in Im76-45 is in contact with Spy (Fig. 4a). However, we did notice that despite this uniformity, regions of Im76-45 preferentially interact with different regions in Spy (Fig. 4b). For example, the N-terminal half of Im76-45 binds more consistently in the Spy cradle, whereas the C-terminal half predominantly binds to the outer edges of Spy’s concave surface. Not unexpectedly, we found that as Im76-45 progresses from the unfolded to the native state, its interactions with Spy shift accordingly. Whereas the least-folded Im76-45 pose in the ensemble forms the most hydrophobic contacts with Spy (Fig. 3), the two most-folded conformations form the fewest hydrophobic contacts (Fig. 3). This shift in contacts is likely due to hydrophobic residues of Im76-45 preferentially forming intra-molecular contacts upon folding (i.e., hydrophobic collapse), effectively removing themselves from the interaction sites. The diversity of conformations and binding sites observed here emphasizes the dynamic and heterogeneous nature of the chaperone-substrate ensemble. Although we do not yet have time resolution data of these various snapshots of Im76-45, this ensemble illustrates how a substrate samples its folding landscape while bound to a chaperone. Spy changes conformation upon substrate binding Comparing the structure of Spy in its substrate-bound and apo states revealed that the Spy dimer also undergoes significant conformational changes upon substrate binding (Fig. 5a and Supplementary Movie 2). Upon substrate binding, the Spy dimer twists 9° about its center relative to its apo form. This twist yields asymmetry and results in substantially different interaction patterns in the two Spy monomers (Fig. 4b). It is possible that this twist serves to increase heterogeneity in Spy by providing more binding poses. Additionally, we observed that the linker region (residues 47–57) of Spy, which participates in substrate interaction, becomes mostly disordered upon binding the substrate. This increased disorder might explain how Spy is able to recognize and bind different substrates and/or differing conformations of the same substrate. Importantly, we observed the same structural changes in Spy regardless of which of the four substrates was bound (Fig. 5b, Table 1). The RMSD between the well-folded sections of Spy in the four chaperone-substrate complexes was very small, less than 0.3 Å. Combined with competition experiments showing that the substrates compete in solution for Spy binding (Fig. 5c and Supplementary Fig. 8), we conclude that all the tested substrates share the same overall Spy binding site. DISCUSSION To shed light on how chaperones interact with their substrates, we developed a novel structural biology method (READ) and applied it to determine a conformational ensemble of the chaperone Spy bound to substrate. As a substrate, we used Im76-45, the chaperone-interacting portion of the protein-folding model protein Im7. In the chaperone-bound ensemble, Im76-45 samples unfolded, partially folded, and native-like states. The ensemble provides an unprecedented description of the conformations that a substrate assumes while exploring its chaperone-associated folding landscape. This substrate-chaperone ensemble helps accomplish the longstanding goal of obtaining a detailed view of how a chaperone aids protein folding. We recently showed that Im7 can fold while remaining continuously bound to Spy. The high-resolution ensemble obtained here now provides insight into exactly how this occurs. The structures of our ensemble agree well with lower-resolution crosslinking data, which indicate that chaperone-substrate interactions primarily occur on the concave surface of Spy. The ensemble suggests a model in which Spy provides an amphipathic surface that allows substrate proteins to assume different conformations while bound to the chaperone. This model is consistent with previous studies postulating that the flexible binding of chaperones allows for substrate protein folding. The amphipathic concave surface of Spy likely facilitates this flexible binding and may be a crucial feature for Spy and potentially other chaperones, allowing them to bind multiple conformations of many different substrates. In contrast to Spy’s binding hotspots, Im76-45 displays substantially less specificity in its binding sites. Nearly all Im76-45 residues come in contact with Spy. Unfolded substrate conformers interact with Spy through both hydrophobic and hydrophilic interactions, whereas the binding of native-like states is mainly hydrophilic. This trend suggests that complex formation between an ATP-independent chaperone and its unfolded substrate may initially involve hydrophobic interactions, effectively shielding the exposed aggregation-sensitive hydrophobic regions in the substrate. Once the substrate begins to fold within this protected environment, it progressively buries its own hydrophobic residues, and its interactions with the chaperone shift towards becoming more electrostatic. Notably, the most frequent contacts between Spy and Im76-45 are charge-charge interactions. The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states. Residues Asp32 and Asp35 are close to each other in the folded state of Im7. This proximity likely causes electrostatic repulsion that destabilizes Im7’s native state. Interaction with Spy’s positively-charged residues likely relieves the charge repulsion between Asp32 and Asp35, promoting their compaction into a helical conformation. As inter-molecular hydrophobic interactions between Spy and the substrate become progressively replaced by intra-molecular interactions within the substrate, the affinity between chaperone and substrates could decrease, eventually leading to release of the folded client protein. Recently, we employed a genetic selection system to improve the chaperone activity of Spy. This selection resulted in “Super Spy” variants that were more effective at both preventing aggregation and promoting protein folding. In conjunction with our bound Im76-45 ensemble, these mutants now allowed us to investigate structural features important to chaperone function. Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both. Our ensemble revealed that two of the Super Spy mutations (H96L and Q100L) form part of the chaperone contact surface that binds to Im76-45 (Fig. 4a). Moreover, our co-structure suggests that the L32P substitution, which increases Spy’s flexibility, could operate by unhinging the N-terminal helix and effectively expanding the size of the disordered linker. This possibility is supported by the Spy:substrate structures, in which the linker region becomes more flexible compared to the apo state (Fig. 6a). This expansion would increase the structural plasticity for substrate binding. By sampling multiple conformations, this linker region may allow diverse substrate conformations to be accommodated. Other Super Spy mutations (F115I and F115L) caused increased flexibility but not tighter substrate binding. This residue does not directly contact Im76-45 in our READ-derived ensemble. Instead, when Spy is bound to substrate, F115 engages in close CH⋯π hydrogen bonds with Tyr104 (Fig. 6b). This interaction presumably reduces the mobility of the C-terminal helix. The F115I/L substitutions would replace these hydrogen bonds with hydrophobic interactions that have little angular dependence. As a result, the C-terminus, and possibly also the flexible linker, is likely to become more flexible and thus more accommodating of different conformations of substrates. Overall, comparison of our ensemble to the Super Spy variants provides specific examples to corroborate the importance of conformational flexibility in chaperone-substrate interactions. Despite extensive studies, exactly how complex chaperone machines help proteins fold remains controversial. Our study indicates that the chaperone Spy employs a simple surface binding approach that allows the substrate to explore various conformations and form transiently favorable interactions while being protected from aggregation. We speculate that many other chaperones could utilize a similar strategy. ATP and co-chaperone dependencies may have emerged later through evolution to better modulate and control chaperone action. In addition to insights into chaperone function, this work presents a new method for determining heterogeneous structural ensembles via a hybrid methodology of X-ray crystallography and computational modeling. Heterogeneous dynamic complexes or disordered regions of single proteins, once considered solely approachable by NMR spectroscopy, can now be visualized through X-ray crystallography. Consequently, this technique could enable structural characterization of many important dynamic and heterogeneous biomolecular systems. ONLINE METHODS For computational methods, including simulations of Spy-substrate interactions, binning the residual Im7 electron density, ensemble selection, validation tests, and contact map generation, please see Supplementary Note 1. Spy truncation mutants’ construction and in vitro and in vivo activity measurements To facilitate crystallization, we used Spy 29-124, a truncated Spy version that removes the unstructured N- and C-terminal tails (full length Spy is 138 amino acids). To determine if these alterations impact Spy’s chaperone activity in vitro, we performed in vitro chaperone activity assays and found that they had no significant effect; these deletions also had only a minor effect on Spy’s ability to stabilize Im7 in vivo (Supplementary Fig. 1). The in vitro activity of Spy 29-124 was assessed using the aldolase refolding assay as previously described. Briefly, in the denaturing step, 100 μM aldolase was denatured in buffer containing 6.6 M GdmCl, 40 mM HEPES pH 7.5, and 50 mM NaCl overnight at 22 °C (room temperature). In the refolding step, denatured aldolase was diluted to 3 μM in refolding buffer (40 mM HEPES, 150 mM NaCl, 5 mM DTT pH 7.5) in the presence of 6 μM WT Spy or Spy 29-124 (Spy:aldolase = 2:1). As a control, an identical experiment without Spy added was also performed. The refolding temperature was 37 °C with continuous shaking. The refolding status was monitored at different time points (1 min, 4 min, 10 min, and 20 min) and tested by diluting the refolding sample by 15-fold into the reaction buffer (0.15 mM NADH, 2 mM F1,6-DP, 1.8 U/ml GDH/TPI, 40 mM HEPES, and 150 mM NaCl pH 7.5) at 28 °C. The absorbance was monitored for 1.5 min at 340 nm. The percentage refolding was calculated and averaged over three repeats. To determine the in vivo activity of the Spy mutants, the quantity of the unstable Im7 variant L53A I54A expressed in the periplasm was compared during Spy variant co-expression as previously described. Plasmid Spy (pTrc-spy) was used as the template for the construction of the variant plasmids of Spy for in vivo chaperone activity measurement (Supplementary Table 3). To use the native signal sequence of spy for the periplasmic export of the Spy variants, an NheI site was first introduced between the signal sequence and the mature protein coding region of Spy. The vector was then digested with NheI and BamHI, purified, and ligated with the linear fragments corresponding to truncated sequences (21–130, 24–130, 27–130, 30–130, and 33–130) of Spy. Cells containing a strain that expressed the unstable Im7 mutant IL53A I54A (pCDFTrc-ssIm7L53A I54A) were transformed with plasmids that expressed either WT or one of the five truncated Spy mutants and grown to mid-log phase in LB medium at 37 °C. Im7 L53A I54A and Spy expression were induced with various concentrations of IPTG for 2 h to compare the in vivo chaperone activity of WT Spy and the truncated Spy mutants at similar expression levels. Periplasmic fractions were prepared as previously described and were separated on 16% Tricine gel (Life Technologies Inc.). The bands corresponding to Spy and the C-terminal His-tagged Im7 were either directly visualized on Coomassie stained gels or determined by western blot using anti-His antibody (Abcam ab1187; validation provided on manufacturer’s website). Protein expression and purification The gene for spy 29-124 was amplified from plasmid pET28sumo-spy with primer 1 (5′-CGC GGG ATC CTT CAA AGA CCT GAA CCT GAC CG-3′) and primer 2 (5′-CGC GCT CGA GTT ATG TCA GAC GCT TCT CAA AAT TAG C-3′), and was cloned into pET28sumo via BamHI and XhoI sites. The H96L variant was made by Phusion site-directed mutagenesis (New England Biolabs). WT and H96L Spy 29-124 were expressed and purified as described previously with the exception that Ni-HisTrap columns (GE Healthcare) were utilized instead of the Ni-NTA beads and mini-chromatography column. ULP1 cleavage occurred following elution from the Ni-HisTrap column overnight at 4 °C while dialyzing to 40 mM Tris, 300 mM NaCl, pH 8.0. After dialysis, Spy was passed over the HisTrap column to remove the cleaved SUMO tag (20 mM imidazole was left over from the dialysis). Cleavage of the SUMO tag leaves a single serine in position 28 of Spy. The flow-through was then concentrated and diluted 5 times with 20 mM Tris, pH 8 for further purification on a HiTrap Q column. Spy has an isoelectric point of 9.5 and therefore was collected in the flow-through. The flow-through containing Spy was concentrated and diluted 5-fold with 50 mM sodium phosphate at pH 6.5 and passed over a HiTrap SP column. Spy was then eluted with a gradient from 0 M to 1 M NaCl. Re-buffering to the final reaction buffer was accomplished by gel filtration, passing the pooled and concentrated fractions containing Spy over a HiLoad 75 column in 40 mM HEPES, 100 mM NaCl, pH 7.5. Fractions containing Spy were then concentrated, frozen in liquid nitrogen, and stored at −80 °C. WT Im7, Im7 L18A L19A L37A H40W, and Im7 L18A L19A L37A were purified by the same protocol as Spy, but without the SP column step. In addition to WT Im7 and these various Im7 mutants, co-crystallization experiments extensively utilized Im76-45, a minimal Spy-binding segment that encompasses the first two helices of Im7 and contains 46% of the total Im7 sequence. It displays partial helicity when free in solution (Supplementary Fig. 3). The 6-45 portion of Im7 (H2N-SISDYTEAEFVQLLKEIEKENVAATDDVLD VLLEHFVKIT-OH), 4-iodophenlyalanine variants, and a peptide corresponding to a portion of bovine alpha casein S1 148-177 (Ac-ELFRQFYQLDAYPSGAWYYVPLGTQYTDAP-amide) were obtained from New England Peptide at ≥ 95% purity. Anomalous signals for residues E12, E14, L19, and E21 substitutions were determined using a peptide containing Im7 6–26, which was also obtained from New England Peptide at ≥ 95% purity. Protein crystallization Co-crystals of WT Spy 29-124 and Spy H96L 29-124 in complex with Im7 variants and casein were grown by vapor diffusion. 25–130 mg/ml dimer Spy was incubated with various Im7 or casein substrates at concentrations ranging from equimolar to three-fold excess substrate in 22%–33% PEG 3000, 0.88–1.0 M imidazole pH 8.0, and 40–310 mM zinc acetate at 20 °C. Crystals were flash frozen in liquid nitrogen using 35% PEG 3000 as a cryo-protectant. It is worthwhile to note that the flash freezing could somewhat bias the conformations observed in the crystal structure. However, we chose to freeze the crystal to provide us with the maximum capability to identify and interpret the iodine anomalous signals. Assessing presence of substrate in crystals Crystals were washed by sequential transfer between three to six 2 μl drops of mother liquor, incubating in each wash solution for 2–10 s in an effort to remove all surface bound and precipitated substrate protein before being dissolved for visualization by SDS-PAGE. Before loading, samples were boiled for 10 min in reducing loading buffer, and then loaded onto 16% Tricine gels. Wash samples and dissolved crystal samples were analyzed by Lumetein staining (Biotium) and Flamingo staining (Bio-Rad) per manufacturer’s instructions, and imaged using a FluorChem M Imager (ProteinSimple). X-ray crystallography Data were collected at the LS-CAT beamlines at the Advanced Photon Source at 100 K. SeMet and native Spy:Im76-45 crystals were collected at 12.7 keV and 9.7 keV, respectively. Spy:Casein 148-177, and Spy H96L:WT Im7 crystals were collected also collected at 12.7 keV. Data integration and scaling were performed with iMosflm and AIMLESS, respectively. As molecular replacement attempts using the previously published apo Spy structures (PDB IDs: 3O39 and 3OEO) were unsuccessful, the Spy:Im76-45 complex was solved using Se-SAD phasing with SeMet-Spy, followed by density modification and initial model building by AutoSol in Phenix. The initial model was completed and refined using the native Spy:Im76-45 complex data. The rest of the structures were built using the native Spy:Im76-45 structure as a molecular replacement search model. Refinements, including TLS refinement, were performed using COOT and Phenix. All refined structures were validated using the Molprobity server, with Clashscores ranking better than the 90th percentile for all structures. Structural figures were rendered using PyMOL and UCSF Chimera, and movies generated using UCSF Chimera. Several partially occupied zinc atoms were observed in the crystal structure. Although some of these zinc atoms could also potentially modelled as water molecules, doing so resulted in an increase in the RFree. Additionally, a section of density near His A96 that is potentially partially occupied by a combination of water, Spy linker region, and possibly zinc, was modelled as containing water molecules. Spy H96L:Im76-45 was employed for iodine anomalous scattering experiments due to increased robustness and reproducibility of the crystals. The expected anomalous scatterers in the structures were S in methionine residues of Spy, Zn from the crystallization buffer, and I in the single pI-Phe residue of each synthetic Im76-45 peptide. Each I site is expected to be partially occupied as Im76-45 had diffuse density corresponding to multiple, partially occupied conformers; the Zn sites also may be partially occupied. To identify I, S, and Zn atomic positions using anomalous scattering, datasets were collected at 6.5 keV and 14.0 keV at 100 K using the ID-D beamline at LS-CAT. Anomalous difference maps for initial anomalous signal screening were calculated with phases from a molecular replacement search using the native Spy:Im76-45 (with no Im76-45 built in) complex as the search model. Anomalous difference maps calculated with the 14.0 keV data were used as controls to distinguish iodine from zinc atoms, as the iodine and zinc anomalous scattering factors are comparable at 14.0 keV, whereas at 6.5 keV, f″ is ~9-fold greater for iodine than for zinc. Anomalous differences were also collected and analyzed for a crystal of WT Spy 29-124:Im76-45 containing no iodine. The resulting anomalous difference map was inspected for peaks corresponding to sulfur, which were then excluded when selecting iodine peaks. Also, peaks that overlapped with Spy in the crystal lattice were excluded from analysis. As an initial screen for placing iodine atoms in the 6.5 keV anomalous difference maps, the median methionine sulfur signal was used as a cutoff for each individual map to control for varying data quality between crystals. Then, all anomalous atoms were refined in Phenix using anomalous group refinement. Refined B-factor of placed iodine ions was then used to estimate the positional fluctuation of the anomalous signals. This positional fluctuation was used as estimated error in the ensuing selection. A summary of all the anomalous signal heights (Supplementary Table 1) and anomalous difference maps (Supplemental Dataset 1) are displayed at varying contour levels for maximum clarity of iodine and methionine peak heights. Substrate binding to Spy The dissociation constant of Im76-45 was determined via a fluorescence-based competition experiment with Im7 L18A L19A L37A H40W, and its ability to compete with casein 148-177 for Spy binding was tested. Im7 L18A L19A L37A H40W was chosen for competition experiments due to its tight binding (Supplementary Fig. 8) and substantial fluorescence change upon binding. This mutant binds to Spy tighter than Im7 L18A L19A L37A. 10 μM Spy 29-124 dimer was mixed with 10 μM Im7 L18A L19A L37A H40W or casein 148-177 to form a 1:1 complex in a buffer containing 40 mM HEPES pH 7.5 and 100 mM NaCl at 22 °C. Complex formation was monitored with a QuantaMaster 400 (Photon Technology International) using the tryptophan fluorescence of Im7 L18A L19A L37A H40W. Naturally tryptophan-free Im76-45 was then titrated into the complex to compete with Im7 L18A L19A L37A H40W for Spy binding. The observed fluorescence intensity at 350 nm was plotted as a function of the logarithm of the Im76-45 or casein 148-177 concentration. The data were fit for a one-site-binding competition model (OriginLab 9.1):   where A1 and A2 are the maximum and minimum asymptotes, respectively, and x is the concentration of Im76-45. x0 is the apparent KD for Im76-45 based on its ability to compete with Im7 L18A L19A L37A H40W. Using the KD of Im7 L18A L19A L37A H40W binding to Spy 29-124, we then calculated the KD for Im76-45 binding to Spy 29-124 using the Cheng-Prusoff equation:   where L is the concentration of Im7 L18A L19A L37A H40W and KD is the dissociation constant for Im7 L18A L19A L37A H40W binding to Spy. Due to interaction between higher oligomer states of Im76-45 and casein 148-177 (Supplementary Fig. 8), the competition curve was unable to be fit for casein 148-177 competing with Im76-45. The stoichiometry of binding of casein 148-177 and Spy was determined by tryptophan fluorescence of the casein upon Spy 29-124 addition. Increasing concentrations of Spy 29-124 were titrated to 20 μM of casein 148-177 in 40 mM HEPES (pH 7.5), 100 mM NaCl, at 22 °C. Complex formation was monitored with a QuantaMaster 400 (Photon Technology International) using the tryptophan fluorescence of casein 148-177. The observed fluorescence intensity at 339 nm was plotted as a function of the Spy 29-124 dimer concentration and fit with a quadratic equation using Origin 9.1 (OriginLab). To determine the dissociation constant, increasing concentrations of Spy 29-124 were titrated to 0.25 μM of casein in 40 mM HEPES (pH 7.5), 100 mM NaCl, at 22 °C. Complex formation was monitored with a QuantaMaster 400 (Photon Technology International) using the tryptophan fluorescence of casein 148-177. The observed fluorescence intensity at 339 nm was corrected for dilution due to the titration and then plotted as a function of the Spy 29-124 dimer concentration. The data were fit using a square hyperbola function in Origin 9.1 (OriginLab):   where F is the recorded fluorescence signal, Fmax is the maximum fluorescence reached upon saturation of the complex, L is the concentration of free Spy in solution, KD is the dissociation constant, and C is a parameter for the offset. The calculated KD is an average of three independent repetitions. The measured dissociation constants for the different substrates ranged from 0.1 to 1 μM. Isothermal titration calorimetry (ITC) Spy 29-124 and Im7-L18A L19A L37A H40W were dialyzed overnight against 40 mM HEPES, 100 mM NaCl, pH 7.5. 165 μM Spy dimer was loaded into a syringe and titrated into a cell containing 15 μM Im7 L18A L19 AL37A H40W at 25 °C in an iTC200 (Malvern Instruments) with an injection interval of 120 s and an initial delay time of 60 s. The solution was stirred at 1000 rpm, and the reference power was set to 6 μcal s−1 in high feedback mode. Data analysis was conducted using a plugin for Origin 7 (OriginLab), the software provided by the manufacturer. Analytical ultracentrifugation Sedimentation velocity experiments for the Im76-45 and the bovine α-S1-casein peptide were performed using a Beckman Proteome Lab XL-I analytical ultracentrifuge (Beckman Coulter). Both peptides were first dialyzed against 40 mM HEPES, 100 mM NaCl, pH 7.5, then diluted to a concentration of 10 μM using the dialysis buffer. Samples were loaded into cells containing standard sector shaped 2-channel Epon centerpieces with 1.2 cm path-length (Beckman Coulter) and equilibrated to 22 °C for at least 1 h prior to sedimentation. All samples were spun at 48,000 rpm in a Beckman AN-50 Ti rotor, and the sedimentation of the protein was monitored continuously using interference optics, since the Im76-45 does not absorb strongly at 280 nm. Data analysis was conducted with SEDFIT (version 14.1), using the continuous c(s) distribution model. The confidence level for the maximum entropy (ME) regularization was set to 0.95. Buffer density and viscosity were calculated using SEDNTERP (http://sednterp.unh.edu/). Supplementary Material ACCESSION CODES Structures and datasets in this work have been deposited in the PDB under the IDs 5INA, 5IOG, 5IOE, and 5IOA. AUTHOR CONTRIBUTIONS Overall concept was conceived by S.H. and J.B. Experiments were designed by S.H., S.Q., J.B., R.T., H.B., and P.K. Experiments were performed by S.H., S.Q., P.K., R.M., and L.W. Analysis and computational modeling was designed by C.B., L.S., P.A., L.A., H.B., and S.H. Computational analysis was carried out by Q.X., S.H., L.S., L.A., P.A., P.K., and R.M. The manuscript was written primarily by S.H. and J.B., with assistance from L.S., L.A. and all other authors. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Principles of protein-protein interactions: what are the preferred ways for proteins to interact? Accessing protein conformational ensembles using room-temperature X-ray crystallography New Views of Functionally Dynamic Proteins by Solution NMR Spectroscopy Investigating protein conformational energy landscapes and atomic resolution dynamics from NMR dipolar couplings: a review Conformational Backbone Dynamics of the Cyclic Decapeptide Antamanide - Application of a New Multiconformational Search Algorithm-Based on Nmr Data Mapping the Population of Protein Conformational Energy Sub-States from NMR Dipolar Couplings Reconciling theories of chaperonin accelerated folding with experimental evidence Reshaping of the conformational search of a protein by the chaperone trigger factor A structural model for GroEL-polypeptide recognition Promiscuous substrate recognition in folding and assembly activities of the trigger factor chaperone Structural basis for protein antiaggregation activity of the trigger factor chaperone The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT Visualizing GroEL/ES in the act of encapsulating a folding protein Hsp90-Tau complex reveals molecular basis for specificity in chaperone action The crystal structure of yeast CCT reveals intrinsic asymmetry of eukaryotic cytosolic chaperonins Crystal structure of the open conformation of the mammalian chaperonin CCT in complex with tubulin Topologies of a substrate protein bound to the chaperonin GroEL Structure of GroEL in complex with an early folding intermediate of alanine glyoxylate aminotransferase Genetic selection designed to stabilize proteins uncovers a chaperone called Spy The mechanism of folding of Im7 reveals competition between functional and kinetic evolutionary constraints Conformational dynamics is more important than helical propensity for the folding of the all alpha-helical protein Im7 Substrate protein folds while it is bound to the ATP-independent chaperone Spy The crystal structure Escherichia coli Spy Super Spy variants implicate flexibility in chaperone action Secondary structure of bovine alpha s1- and beta-casein in solution The crystal structure of the immunity protein of colicin E7 suggests a possible colicin-interacting surface Conformational Properties of the Unfolded State of Im7 in Nondenaturing Conditions Direct observation of protein solvation and discrete disorder with experimental crystallographic phases Modeling discrete heterogeneity in X-ray diffraction data by fitting multi-conformers Capturing a dynamic chaperone-substrate interaction using NMR-informed molecular modeling A Suite of Programs for Calculating X-Ray Absorption, Reflection, and Diffraction Performance for a Variety of Materials at Arbitrary Wavelengths The origins of asymmetry in the folding transition states of protein L and protein G Folding on the chaperone: Yield enhancement through loose binding Conditional disorder in chaperone action Isolation of bacteria envelope proteins One Crystal, Two Temperatures: Cryocooling Penalties Alter Ligand Binding to Transient Protein Sites iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM Overview of the CCP4 suite and current developments PHENIX: a comprehensive Python-based system for macromolecular structure solution Features and development of Coot MolProbity: all-atom structure validation for macromolecular crystallography  UCSF chimera - A visualization system for exploratory research and analysis Towards automated crystallographic structure refinement with phenix.refine Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling Crystallographic data and ensemble selection. (a) 2mFo−DFc omit map of residual Im76-45 and flexible linker electron density contoured at 0.5 σ. This is the residual density that is used in the READ selection. (b) Composites of iodine positions detected from anomalous signals using pI-Phe substitutions, colored and numbered by sequence. Multiple iodine positions were detected for most residues. Agreement to the residual Im76-45 electron density (c) and anomalous iodine signals (d) for ensembles of varying size generated by randomly choosing from the MD pool (blue) and from the selection procedure (black). The agreement from back-calculating a subset of data excluded from the selection procedure is shown by the red curve (cross-validation). The cost function, χ2, decreases as the agreement to the experimental data increases and is defined in the Online Methods. Flowchart of the READ sample-and-select process. Spy:Im76-45 ensemble, arranged by RMSD to native state of Im76-45. Although the six-membered ensemble from the READ selection should be considered only as an ensemble, for clarity, the individual conformers are shown separately here. Spy is depicted as a gray surface and the Im76-45 conformer is shown as orange balls. Atoms that were either not directly selected in the READ procedure, or whose position could not be justified based on agreement with the residual electron density were removed, leading to non-contiguous sections. Dashed lines connect non-contiguous segments of the Im76-45 substrate. Residues of the Spy flexible linker region that fit the residual electron density are shown as larger gray spheres. Shown below each ensemble member is the RMSD of each conformer to the native state of Im76-45, as well as the percentage of contacts between Im76-45 and Spy that are hydrophobic. Contact maps of Spy:Im76-45 complex. (a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures. For clarity, Im76-45 is represented with a single conformation. The frequency plotted is calculated as the average contact frequency from Spy to every residue of Im76-45 and vice-versa. As the residues involved in contacts are more evenly distributed in Im76-45 compared to Spy, its contact map was amplified. (b) Detailed contact maps of Spy:Im76-45. Contacts to the two Spy monomers are depicted separately. Note that the flexible linker region of Spy (residues 47–57) is not represented in the 2D contact maps. Spy conformation changes upon substrate binding. (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). Error bars depict standard deviations of n=3 technical replicates. Flexibility of Spy linker region and effect of Super Spy mutants. (a) The Spy linker region adopts one dominant conformation in its apo state (PDB ID 3039, gray), but expands and adopts multiple conformations in bound states (green). (b) F115 and L32 tether Spy’s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility. The Super Spy mutants F115L, F115I, and L32P are proposed to gain activity by increasing the flexibility or size of this linker region. L32, F115, and Y104 are rendered in purple to illustrate residues that are most affected by Super Spy mutations; CH⋯π hydrogen bonds are depicted by orange dashes. Crystallography Statistics \tSeMet Spy:Im76-45\tSpy:Im76-45\tSpy:Casein 148-177, substrate not modeled\tSpy H96L:Im7 L18A L19A L37A, substrate not modeled\tSpy H96L:WT Im7, substrate not modeled\t \tPDB ID\t\t5INA\t5IOG\t5IOE\t5IOA\t \tData collection\t \tSpace group\tP4122\tP4122\tP4122\tP4122\tP4122\t \tCell dimensions\t \t a, b, c (Å)\t42.9, 42.9, 259.3\t42.9, 42.9, 260.2\t43.0, 43.0, 258.2\t43.1, 43.1, 258.7\t43.1, 43.14, 260.2\t \t α, β, γ (°)\t90, 90, 90\t90, 90, 90\t90, 90, 90\t90, 90, 90\t90, 90, 90\t \tResolution (Å)\t64.82–2.44(2.53–2.44)\t30.50–1.79(1.83–1.79)\t36.88–1.77 (1.80–1.77)\t30.48–1.87(1.91–1.87)\t33.21–1.87(1.91–1.87)\t \tRmerge (%)\t10.6(36)\t8.2(108)\t6.2(134)\t8.4(152)\t9.6(249)\t \tI/σ(I)\t15.1(6.8)\t7.0(1.1)\t15.3(1.6)\t13.8(1.8)\t13.2(1.3)\t \tCompleteness (%)\t100(100)\t94.0(90.1)\t99.9(99.5)\t100(100)\t96.8(93.1)\t \tRedundancy\t15.6(15.6)\t4.3(4.2)\t8.7(8.2)\t9.6(9.4)\t8.2(8.2)\t \tCC1/2\t\t0.998(0.689)\t0.999(0.745)\t0.999(0.676)\t0.998(0.606)\t \tRefinement\t \tResolution (Å)\t\t1.79\t1.77\t1.87\t1.87\t \tNo. of Reflections\t\t22583\t25052\t21505\t20838\t \tRwork/Rfree\t\t0.22/0.23\t0.21/0.24\t0.22/0.24\t0.21/0.25\t \tNo. of Atoms\t\t1765\t1669\t1715\t1653\t \t Protein\t\t1586\t1493\t1541\t1444\t \t Ligand/ion\t\t30\t56\t60\t30\t \t Water\t\t149\t120\t114\t179\t \tB-factors\t\t49.4\t48.5\t47.4\t39.2\t \t Protein\t\t49.0\t47.5\t46.3\t38.3\t \t Ligand/ion\t\t48.6\t65.9\t80.4\t62.9\t \t Water\t\t54.2\t51.9\t44.5\t42.1\t \tr.m.s. Deviations\t\t\t\t\t\t \t Bond lengths (Å)\t\t0.013\t0.013\t0.013\t0.014\t \t Bond angles (º)\t\t1.24\t1.30\t1.24\t1.39\t 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+[{"sourceid":"4968113","sourcedb":"","project":"","target":"","text":"Structural diversity in a human antibody germline library ABSTRACT To support antibody therapeutic development, the crystal structures of a set of 16 germline variants composed of 4 different kappa light chains paired with 4 different heavy chains have been determined. All four heavy chains of the antigen-binding fragments (Fabs) have the same complementarity-determining region (CDR) H3 that was reported in an earlier Fab structure. The structure analyses include comparisons of the overall structures, canonical structures of the CDRs and the VH:VL packing interactions. The CDR conformations for the most part are tightly clustered, especially for the ones with shorter lengths. The longer CDRs with tandem glycines or serines have more conformational diversity than the others. CDR H3, despite having the same amino acid sequence, exhibits the largest conformational diversity. About half of the structures have CDR H3 conformations similar to that of the parent; the others diverge significantly. One conclusion is that the CDR H3 conformations are influenced by both their amino acid sequence and their structural environment determined by the heavy and light chain pairing. The stem regions of 14 of the variant pairs are in the ‘kinked’ conformation, and only 2 are in the extended conformation. The packing of the VH and VL domains is consistent with our knowledge of antibody structure, and the tilt angles between these domains cover a range of 11 degrees. Two of 16 structures showed particularly large variations in the tilt angles when compared with the other pairings. The structures and their analyses provide a rich foundation for future antibody modeling and engineering efforts. Introduction At present, therapeutic antibodies are the largest class of biotherapeutic proteins that are in clinical trials. The use of monoclonal antibodies as therapeutics began in the early 1980s, and their composition has transitioned from murine antibodies to generally less immunogenic humanized and human antibodies. The technologies currently used to obtain human antibodies include transgenic mice containing human antibody repertoires, cloning directly from human B cells, and in vitro selection from antibody libraries using various display technologies. Once a candidate antibody is identified, protein engineering is usually required to produce a molecule with the right biophysical and functional properties. All engineering efforts are guided by our understanding of the atomic structures of antibodies. In such efforts, the crystal structure of the specific antibody may not be available, but modeling can be used to guide the engineering efforts. Today's antibody modeling approaches, which normally focus on the variable region, are being developed by the application of structural principles and insights that are evolving as our knowledge of antibody structures continues to expand. Our current structural knowledge of antibodies is based on a multitude of studies that used many techniques to gain insight into the functional and structural properties of this class of macromolecule. Five different antibody isotypes occur, IgG, IgD, IgE, IgA and IgM, and each isotype has a unique role in the adaptive immune system. IgG, IgD and IgE isotypes are composed of 2 heavy chains (HCs) and 2 light chains (LCs) linked through disulfide bonds, while IgA and IgM are double and quintuple versions of antibodies, respectively. Isotypes IgG, IgD and IgA each have 4 domains, one variable (V) and 3 constant (C) domains, while IgE and IgM each have the same 4 domains along with an additional C domain. These multimeric forms are linked with an additional J chain. The LCs that associate with the HCs are divided into 2 functionally indistinguishable classes, κ and λ. Both κ and λ polypeptide chains are composed of a single V domain and a single C domain. The heavy and light chains are composed of structural domains that have ∼110 amino acid residues. These domains have a common folding pattern often referred to as the “immunoglobulin fold,” formed by the packing together of 2 anti-parallel β-sheets. All immunoglobulin chains have an N-terminal V domain followed by 1 to 4 C domains, depending upon the chain type. In antibodies, the heavy and light chain V domains pack together forming the antigen combining site. This site, which interacts with the antigen (or target), is the focus of current antibody modeling efforts. This interaction site is composed of 6 complementarity-determining regions (CDRs) that were identified in early antibody amino acid sequence analyses to be hypervariable in nature, and thus are responsible for the sequence and structural diversity of our antibody repertoire. The sequence diversity of the CDR regions presents a substantial challenge to antibody modeling. However, an initial structural analysis of the combining sites of the small set of structures of immunoglobulin fragments available in the 1980s found that 5 of the 6 hypervariable loops or CDRs had canonical structures (a limited set of main-chain conformations). A CDR canonical structure is defined by its length and conserved residues located in the hypervariable loop and framework residues (V-region residues that are not part of the CDRs). Furthermore, studies of antibody sequences revealed that the total number of canonical structures are limited for each CDR, indicating possibly that antigen recognition may be affected by structural restrictions at the antigen-binding site. Later studies found that the CDR loop length is the primary determining factor of antigen-binding site topography because it is the primary factor for determining a canonical structure. Additional efforts have led to our current understanding that the LC CDRs L1, L2, and L3 have preferred sets of canonical structures based on length and amino acid sequence composition. This was also found to be the case for the H1 and H2 CDRs. Classification schemes for the canonical structures of these 5 CDRs have emerged and evolved as the number of depositions in the Protein Data Bank of Fab fragments of antibodies grow. Recently, a comprehensive CDR classification scheme was reported identifying 72 clusters of conformations observed in antibody structures. The knowledge and predictability of these CDR canonical structures have greatly advanced antibody modeling efforts. In contrast to CDRs L1, L2, L3, H1 and H2, no canonical structures have been observed for CDR H3, which is the most variable in length and amino acid sequence. Some clustering of conformations was observed for the shortest lengths; however, for the longer loops, only the portions nearest the framework (torso, stem or anchor region) were found to have defined conformations. In the torso region, 2 primary groups could be identified, which led to sequence-based rules that can predict with some degree of reliability the conformation of the stem region. The “kinked” or “bulged” conformation is the most prevalent, but an “extended” or “non-bulged” conformation is also, but less frequently, observed. The cataloging and development of the rules for predicting the conformation of the anchor region of CDR H3 continue to be refined, producing new insight into the CDR H3 conformations and new tools for antibody engineering. Current antibody modeling approaches take advantage of the most recent advances in homology modeling, the evolving understanding of the CDR canonical structures, the emerging rules for CDR H3 modeling and the growing body of antibody structural data available from the PDB. Recent antibody modeling assessments show continued improvement in the quality of the models being generated by a variety of modeling methods. Although antibody modeling is improving, the latest assessment revealed a number of challenges that need to be overcome to provide accurate 3-dimensional models of antibody V regions, including accuracies in the modeling of CDR H3. The need for improvement in this area was also highlighted in a recent study reporting an approach and results that may influence future antibody modeling efforts. One important finding of the antibody modeling assessments was that errors in the structural templates that are used as the basis for homology models can propagate into the final models, producing inaccuracies that may negatively influence the predictive nature of the V region model. To support antibody engineering and therapeutic development efforts, a phage library was designed and constructed based on a limited number of scaffolds built with frequently used human germ-line IGV and IGJ gene segments that encode antigen combining sites suitable for recognition of peptides and proteins. This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). Selection of these genes was based on the high frequency of their use and their cognate canonical structures that were found binding to peptides and proteins, as well as their ability to be expressed in bacteria and displayed on filamentous phage. The implementation of the library involves the diversification of the human germline genes to mimic that found in natural human libraries. The crystal structure determinations and structural analyses of all germline Fabs in the library described above along with the structures of a fourth HC germline, IGHV3-53 (H3-53), paired with the 4 LCs of the library have been carried out to support antibody therapeutic development. All 16 HCs of the Fabs have the same CDR H3 that was reported in an earlier Fab structure. This is the first systematic study of the same VH and VL structures in the context of different pairings. The structure analyses include comparisons of the overall structures, canonical structures of the L1, L2, L3, H1 and H2 CDRs, the structures of all CDR H3s, and the VH:VL packing interactions. The structures and their analyses provide a foundation for future antibody engineering and structure determination efforts. Results Crystal structures Crystal data, X-ray data, and refinement statistics. Fab\tH1-69:L1-39\tH1-69:L3-11\tH1-69:L3-20\tH1-69:L4-1\t \tPDB identifier\t5I15\t5I16\t5I17\t5I18\t \tCrystal Data\t \t \t \t \t \tCrystallization Solution\t \t \t \t \t \t Buffer, pH\t0.1 M MES- pH 6.5\t0.1 M MES pH 6.5\t0.1 M MES, pH 6.5\t0.1 M HEPES, pH 7.5\t \t Precipitant1\t5 M Na Formate\t25% PEG 3350\t2.0 M Amm Sulfate\t10% PEG 8000\t \t Additive1\t \t0.2 M Na Formate\t5% MPD\t8% EG\t \t Space Group\tP3121\tC2\tP422\tP4212\t \t Molecules/AU\t1\t2\t2\t1\t \t Unit Cell\t \t \t \t \t \t a(Å)\t129.2\t212.0\t152.5\t120.0\t \t b(Å)\t129.2\t55.1\t152.5\t120.0\t \t c(Å)\t91.8\t80.3\t123.4\t64.2\t \t β(°)\t90.0\t97.8\t90.0\t90.0\t \t γ(°)\t120.0\t90.0\t90.0\t90.0\t \t Vm (Å3/Da)\t4.67\t2.44\t3.77\t2.39\t \t Solvent Content (%)\t74\t50\t67\t48\t \tX-Ray Data2\t \t \t \t \t \t Resolution (Å)\t30-2.6 (2.7-2.6)\t30.0-1.9 (1.95-1.9)\t30.0-3.3 (3.4-3.3)\t30-1.9 (2.0-1.9)\t \t Measured Reflections\t136,745 (8,650)\t241,145 (16,580)\t237,504 (15,007)\t801,080 (19,309)\t \t Unique Reflections\t27,349 (1,730)\t71,932 (5,198)\t22,379 (1,590)\t35,965 (2,194)\t \t Completeness (%)\t99.3 (98.7)\t99.0 (97.3)\t99.5 (96.8)\t98.5 (82.8)\t \t Redundancy\t5.0 (5.0)\t3.4 (3.2)\t10.6 (9.4)\t22.3 (8.8)\t \t Rmerge\t0.048 (0.522)\t0.044 (0.245)\t0.086 (0.536)\t0.093 (0.231)\t \t \u003c I/σ \u003e\t21.2 (3.9)\t17.8 (4.7)\t25.5 (4.5)\t29.2 (8.1)\t \t B-factor (Å2)\t60.5\t33.2\t61.0\t19.6\t \tRefinement\t \t \t \t \t \t Resolution (Å)\t15-2.6\t15-1.9\t15-3.3\t15-1.9\t \t Number of Reflections\t26,238\t70,346\t21,197\t34,850\t \t Number of All Atoms\t3,224\t6,975\t6,398\t3,695\t \t Number of Waters\t2\t472\t0\t399\t \t R-factor (%)\t20.5\t19.2\t20.2\t16.7\t \t R-free (%)\t24.1\t22.2\t24.7\t21.3\t \tRMSD\t \t \t \t \t \t Bond Lengths (Å)\t0.006\t0.005\t0.005\t0.008\t \t Bond Angles (°)\t1.2\t1.1\t1.0\t1.1\t \t Mean B-factor (Å2)\t65.3\t34.4\t80.1\t20.0\t \tRamachandran Plot (%)\t \t \t \t \t \t Outliers\t0.0\t0.0\t0.9\t0.0\t \t Favored\t92.3\t96.9\t93.1\t96.9\t \t Abbreviations: Amm, ammonium;EG, ethylene glycol; PEG, polyethylene glycol. Values for high-resolution shell are in parentheses. (Continued) Crystal data, X-ray data, and refinement statistics. Fab\tH3-23:L1-39\tH3-23:L3-11\tH3-23:L3-20\tH3-23:L4-1\t \t PDB identifier\t5I19\t5I1A\t5I1C\t5I1D\t \tCrystal Data\t \t \t \t \t \tCrystallization Solution\t \t \t \t \t \t Buffer, pH\tNo Buffer\t0.1 M Na Acetate, pH 4.5\t0.1 M MES, pH 6.5\t0.1 M HEPES, pH 7.5\t \t Precipitant1\t20% PEG 3350\t2.0 M Amm Sulfate\t16% PEG 3350\t2.0 M Amm Sulfate\t \t Additive1\t0.2 M Li Citrate\t5% PEG 400\t0.2 M Amm Acetate\t2% PEG 400\t \t Space Group\tP41212\tP212121\tP6222\tP212121\t \t Molecules/AU\t1\t2\t1\t2\t \tUnit Cell\t \t \t \t \t \t a(Å)\t96.6\t60.9\t121.5\t62.7\t \t b(Å)\t96.6\t110.6\t121.5\t111.0\t \t c(Å)\t105.4\t158.9\t160.4\t160.0\t \t β(°)\t90\t90\t90\t90\t \t Vm (Å3/Da)\t2.60\t2.82\t3.60\t2.90\t \t Solvent Content (%)\t53\t56\t66\t57\t \tX-Ray Data2\t \t \t \t \t \t Resolution (Å)\t30-2.8 (2.9-2.8)\t30-2.0 (2.1-2.0)\t30-2.25 (2.3-2.25)\t30-2.0 (2.1-2.0)\t \t Measured Reflections\t177,681 (12,072)\t351,312 (8,634)\t887,349 (59,919)\t873,523 (49,118)\t \t Unique Reflections\t12,678 (899)\t58,989 (2,870)\t32,572 (2,300)\t75,540 (5,343)\t \t Completeness (%)\t99.5 (97.4)\t80.9 (54.2)\t96.9 (94.8)\t99.7 (96.9)\t \t Redundancy\t14.0 (13.4)\t6.0 (3.0)\t27.2 (26.1)\t11.6 (9.2)\t \t Rmerge\t0.091 (0.594)\t0.066 (0.204)\t0.086 (0.478)\t0.094 (0.488)\t \t \u003c I/σ \u003e\t31.2 (5.1)\t20.4 (4.6)\t37.0 (10.4)\t21.6 (5.0)\t \t B-factor (Å2)\t42.8\t27.1\t33.7\t29.4\t \tRefinement\t \t \t \t \t \t Resolution (Å)\t15-2.8\t15-2.0\t15-2.25\t15-2.0\t \t Number of Reflections\t11,972\t57,599\t31,411\t74,238\t \t Number of All Atoms\t3,234\t6,948\t3,472\t7,210\t \t Number of Waters\t0\t416\t222\t635\t \t R-factor (%)\t23.9\t20.5\t22.0\t21.6\t \t R-free (%)\t31.5\t25.5\t26.6\t25.1\t \tRMSD\t \t \t \t \t \t Bond Lengths (Å)\t0.009\t0.010\t0.005\t0.008\t \t Bond Angles (°)\t1.3\t1.3\t1.0\t1.1\t \t Mean B-factor (Å2)\t48.4\t36.7\t47.7\t46.4\t \tRamachandran Plot (%)\t \t \t \t \t \t Outliers\t0.0\t0.0\t0.0\t0.0\t \t Favored\t92.3\t96.8\t97.5\t97.6\t \t Abbreviations: Amm, ammonium; PEG, polyethylene glycol. Values for high-resolution shell are in parentheses. (Continued) Crystal data, X-ray data, and refinement statistics. Fab\tH3-53:L1-39\tH3-53:L3-11\tH3-53:L3-20\tH3-53:L4-1\t \tPDB indentifier\t5I1E\t5I1G\t5I1H\t5I1I\t \tCrystal Data\t \t \t \t \t \tCrystallization Solution\t \t \t \t \t \t Buffer, pH\tNo buffer\t0.1 M Na Acetate pH 4.5\t0.1 M Na Acetate pH 4.5\t0.1M MES, pH 6.5\t \t Precipitant1\t16% PEG 3350\t25% PEG 3350\t19% PEG 4000\t17% PEG 3350\t \t Additive1\t0.2 M Amm Sulfate 5% Dioxane\t0.2 M Li2SO4\t0.2 M Amm Sulfate\t0.2 M Na Formate, 5% MPD\t \t Space Group\tP6522\tP6522\tP6522\tP31\t \t Molecules/AU\t1\t1\t1\t1\t \tUnit Cell\t \t \t \t \t \t a(Å)\t89.4\t88.1\t89.4\t68.1\t \t b(Å)\t89.4\t88.1\t89.4\t68.1\t \t c(Å)\t212.4\t219.6\t211.7\t95.6\t \t β(°)\t90\t90\t90\t90\t \t γ(°)\t120\t120\t120\t120\t \t Vm (Å3/Da)\t2.57\t2.64\t2.57\t2.64\t \t Solvent Content (%)\t52\t53\t52\t53\t \tX-Ray Data2\t \t \t \t \t \t Resolution (Å)\t30-2.7 (2.8-2.7)\t30-2.3 (2.4-2.3)\t30-2.2 (2.3-2.0)\t30-2.5 (2.6-2.5)\t \t Measured Reflections\t297,367 (19,369)\t333,739 (8,008)\t381,125 (1,591)\t137,992 (9,883)\t \t Unique Reflections\t14,402 (1,003)\t21,683 (1,135)\t24,323 (964)\t16,727 (1,227)\t \t Completeness (%)\t99.6 (96.8)\t93.8 (68.4)\t95.3 (52.0)\t98.6 (98.1)\t \t Redundancy\t20.6 (19.3)\t15.4 (7.1)\t15.7 (1.7)\t8.2 (8.1)\t \t Rmerge\t0.095 (0.451)\t0.057 (0.324)\t0.062 (0.406)\t0.047 (0.445)\t \t \u003c I/σ \u003e\t38.3 (8.1)\t36.7 (5.5)\t36.2 (1.6)\t31.6 (5.6)\t \t B-factor (Å2)\t33.2\t37.3\t33.7\t54.8\t \tRefinement\t \t \t \t \t \t Resolution (Å)\t15-2.7\t15-2.3\t15-2.2\t15-2.5\t \t Number of Reflections\t13,583\t20,255\t24,962\t15,811\t \t Number of All Atoms\t3,335\t3,271\t3,298\t3,239\t \t Number of Waters\t88\t70\t71\t21\t \t R-factor (%)\t19.1\t29.8\t22.8\t25.0\t \t R-free (%)\t26.4\t38.3\t26.6\t33.7\t \tRMSD\t \t \t \t \t \t Bond Lengths (Å)\t0.008\t0.005\t0.005\t0.006\t \t Bond Angles (°)\t1.2\t1.0\t1.0\t1.1\t \t Mean B-factor (Å2)\t49.1\t46.3\t51.7\t88.9\t \tRamachandran Plot (%)\t \t \t \t \t \t Outliers\t0.2\t0.2\t0.2\t1.2\t \t Favored\t96.7\t97.1\t96.5\t90.9\t \t Abbreviations: Amm, ammonium; PEG, polyethylene glycol. Values for high-resolution shell are in parentheses. (Continued) Crystal data, X-ray data, and refinement statistics. Fab\tH5-51:L1-39\tH5-51:L3-11\tH5-51:L3-20\tH5-51:L4-1\t \t PDB identifier\t4KMT\t5I1J\t5I1K\t5I1L\t \tCrystal Data\t \t \t \t \t \tCrystallization Solution\t \t \t \t \t \t Buffer, pH\t0.1 M CHES, pH 9.5\t0.1 M Tris, pH 8.5\t0.1 M CHES, pH 9.5\t0.1 M Tris, pH 8.5\t \t Precipitant1\t1.8 M Amm Sulfate\t25% PEG 3350\t1.0 M Amm Sulfate\t24% PEG 3350\t \t Additive1\t5% dioxane\t0.2 M MgCl2\t \t0.2 M Amm Sulfate\t \t Space Group\tP212121\tP212121\tP212121\tP21\t \t Molecules/AU\t1\t1\t1\t2\t \tUnit Cell\t \t \t \t \t \t a(Å)\t63.7\t64.1\t63.8\t106.0\t \t b(Å)\t73.8\t73.8\t74.1\t38.0\t \t c(Å)\t103.1\t103.0\t103.0\t112.3\t \t β(°)\t90\t90\t90\t100.4\t \t Vm (Å3/Da)\t2.53\t2.56\t2.54\t2.28\t \t Solvent Content (%)\t51\t52\t51\t46\t \tX-Ray Data2\t \t \t \t \t \t Resolution (Å)\t30-2.1 (2.2-2.1)\t30-2.5 (2.6-2.5)\t30-1.65 (1.7-1.65)\t30-1.95 (2.0-1.95)\t \t Measured Reflections\t131,839 (6,655)\t120,521 (7,988)\t246,750 (4,142)\t320,324 (12,119)\t \t Unique Reflections\t27,026 (1,885)\t17,286 (1,236)\t53,058 (2,141)\t61,554 (3,243)\t \t Completeness (%)\t93.6 (89.8)\t99.7 (97.3)\t89.8 (49.8)\t94.4 (67.1)\t \t Redundancy\t4.9 (3.5)\t7.0 (6.5)\t4.7 (1.9)\t5.2 (3.7)\t \t Rmerge\t0.079 (0.278)\t0.080 (0.281)\t0.034 (0.131)\t0.060 (0.395)\t \t \u003c I/σ \u003e\t16.8 (5.7)\t21.1(6.9)\t27.5 (5.8)\t19.7 (3.1)\t \t B-factor (Å2)\t26.0\t27.0\t21.6\t31.4\t \tRefinement\t \t \t \t \t \t Resolution (Å)\t15-2.1\t15-2.5\t15-1.65\t15-1.95\t \t Number of Reflections\t25,857\t16,328\t51,882\t60,181\t \t Number of All Atoms\t3,676\t3,454\t3,814\t7,175\t \t Number of Waters\t302\t196\t527\t445\t \t R-factor (%)\t17.1\t17.7\t17.2\t19.4\t \t R-free (%)\t22.0\t25.8\t19.7\t25.8\t \tRMSD\t \t \t \t \t \t Bond Lengths (Å)\t0.006\t0.009\t0.005\t0.009\t \t Bond Angles (°)\t1.0\t1.3\t1.3\t1.3\t \t Mean B-factor (Å2)\t25.2\t38.2\t20.0\t19.5\t \tRamachandran Plot (%)\t \t \t \t \t \t Outliers\t0.0\t0.0\t0.0\t0.0\t \t Favored\t98.4\t97.9\t98.1\t98.0\t \t Abbreviations: Amm, ammonium; PEG, polyethylene glycol. Values for high-resolution shell are in parentheses. The crystal structures of a germline library composed of 16 Fabs generated by combining 4 HCs (H1-69, H3-23, H3-53 and H5-51) and 4 LCs (L1-39, L3-11, L3-20 and L4-1) have been determined. The Fab heavy and light chain sequences for the variants numbered according to Chothia are shown in Fig. S1. The four different HCs all have the same CDR H3 sequence, ARYDGIYGELDF. Crystallization of the 16 Fabs was previously reported. Three sets of the crystals were isomorphous with nearly identical unit cells (Table 1). These include (1) H3-23:L3-11 and H3-23:L4-1 in P212121, (2) H3-53:L1-39, H3-53:L3-11 and H3-53:L3-20 in P6522, and (3) H5-51:L1-39, H5-51:L3-11 and H5-51:L3-20 in P212121. Crystallization conditions for the 3 groups are also similar, but not identical (Table 1). Variations occur in the pH (buffer) and the additives, and, in group 3, PEG 3350 is the precipitant for one variants while ammonium sulfate is the precipitant for the other two. The similarity in the crystal forms is attributed in part to cross-seeding using the microseed matrix screening for groups 2 and 3. The crystal structures of the 16 Fabs have been determined at resolutions ranging from 3.3 Å to 1.65 Å (Table 1). The number of Fab molecules in the crystallographic asymmetric unit varies from 1 (for 12 Fabs) to 2 (for 4 Fabs). Overall the structures are fairly complete, and, as can be expected, the models for the higher resolution structures are more complete than those for the lower resolution structures (Table S1). Invariably, the HCs have more disorder than the LCs. For the LC, the disorder is observed at 2 of the C-terminal residues with few exceptions. Apart from the C-terminus, only a few surface residues in LC are disordered. The HCs feature the largest number of disordered residues, with the lower resolution structures having the most. The C-terminal residues including the 6xHis tags are disordered in all 16 structures. In addition to these, 2 primary disordered stretches of residues are observed in a number of structures (Table S1). One involves the loop connecting the first 2 β-strands of the constant domain (in all Fabs except H3-23:L1-39, H3-23:L3-11 and H3-53:L1-39). The other is located in CDR H3 (in H5-51:L3-11, H5-51:L3-20 and in one of 2 copies of H3-23:L4-1). CDR H1 and CDR H2 also show some degree of disorder, but to a lesser extent. CDR canonical structures Several CDR definitions have evolved over decades of antibody research. Depending on the focus of the study, the CDR boundaries differ slightly between various definitions. In this work, we use the CDR definition of North et al., which is similar to that of Martin with the following exceptions: 1) CDRs H1 and H3 begin immediately after the Cys; and 2) CDR L2 includes an additional residue at the N-terminal side, typically Tyr. CDR H1 The superposition of CDR H1 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. Canonical structures. Pairs\tPDB\tCDR H1\tCDR H2\tCDR H3\t \tH1-69\t \tKASGGTFSSYAIS\tGIIPIFGTAN\tARYDGIYGELDF\t \tH1-69:L1-39\t5I15\tH1-13-4\tH2-10-1\tH3-12-1\t \tH1-69:L3-11\t5I16\tH1-13-1/H1-13-1\tH2-10-1/H2-10-1\tH3-12-1/H3-12-1\t \tH1-69:L3-20\t5I17\tH1-13-3/H1-13-6\tH2-10-1/NA\tH3-12-1/H3-12-1\t \tH1-69:L4-1\t5I18\tH1-13-10\tH2-10-1\tH3-12-1\t \tH3-23\t \tAASGFTFSSYAMS\tAISGSGGSTY\tAKYDGIYDGIYGELDF\t \tH3-23:L1-39\t5I19\tH1-13-1\tH2-10-2\tH3-12-1\t \tH3-23:L3-11\t5I1A\tH1-13-1/H1-13-1\tH2-10-2/H2-10-2\tH3-12-1/H3-12-1\t \tH3-23:L3-20\t5I1C\tH1-13-1\tH2-10-2\tH3-12-1\t \tH3-23:L4-1\t5I1D\tH1-13-1/H1-13-1\tH2-10-2/H2-10-2\tH3-12-1/NA\t \tH3-53\t \tAASGFTVSSNYMS\tVIYSGGSTY\tARYDGIYGELDF\t \tH3-53:L1-39\t5I1E\tH1-13-1\tH2-9-3\tH3-12-1\t \tH3-53:L3-11\t5I1G\tH1-13-1\tH2-9-3\tH3-12-1\t \tH3-53:L3-20\t5I1H\tH1-13-1\tH2-9-3\tH3-12-1\t \tH3-53:L4-1\t5I1I\tH1-13-1\tH2-9-3\tNA\t \tH5-51\t \tKGSGYSFTSYWIG\tIIYPGDSDTR\tARYDGIYGELDF\t \tH5-51:L1-39\t4KMT\tH1-13-1\tH2-10-1\tH3-12-1\t \tH5-51:L3-11\t5I1J\tH1-13-1\tH2-10-1\tNA\t \tH5-51:L3-20\t5I1K\tH1-13-1\tH2-10-1\tNA\t \tH5-51:L4-1\t5I1L\tH1-13-1/H1-13-1\tH2-10-1/H2-10-1\tH3-12-1/H3-12-1\t \t \t \tCDR L1\tCDR L2\tCDR L3\t \tL1-39\t \tRASQSISSYLN\tYAASSLQS\tQQSYSTPLT\t \tH1-69:L1-39\t5I15\tL1-11-1\tL2-8-1\tL3-9-cis7-1\t \tH3-23:L1-39\t5I19\tL1-11-1\tL2-8-1\tL3-9-cis7-1\t \tH3-53:L1-39\t5I1E\tL1-11-1\tL2-8-1\tL3-9-cis7-1\t \tH5-51:L1-39\t4KMT\tL1-11-1\tL2-8-1\tL3-9-cis7-1\t \tL3-11\t \tRASQSVSSYLA\tYDASNRAT\tQQRSNWPLT\t \tH1-69:L3-11\t5I16\tL1-11-1/L1-11-1\tL2-8-1/L2-8-1\tL3-9-cis7-1/L3-9-cis7-1\t \tH3-23:L3-11\t5I1A\tL1-11-1/L1-11-1\tL2-8-1/L2-8-1\tL3-9-cis7-1/L3-9-cis7-1\t \tH3-53:L3-11\t5I1G\tL1-11-1\tL2-8-1\tL3-9-cis7-1\t \tH5-51:L3-11\t5I1J\tL1-11-1\tL2-8-1\tL3-9-cis7-1\t \tL3-20\t \tRASQSVSSSYLA\tYGASSRAT\tQQYGSSPLT\t \tH1-69:L3-20\t5I17\tL1-12-2/L1-12-1\tL2-8-1/L2-8-1\tL3-9-cis7-1/L3-9-cis7-1\t \tH3-23:L3-20\t5I1C\tL1-12-2\tL2-8-1\tL3-9-cis7-1\t \tH3-53:L3-20\t5I1H\tL1-12-1\tL2-8-1\tL3-9-cis7-1\t \tH5-51:L3-20\t5I1K\tL1-12-1\tL2-8-1\tL3-9-cis7-1\t \tL4-1\t \tKSSQSVLYSSNNKNYLA\tYWASTRES\tQQYYSTPLT\t \tH1-69:L4-1\t5I18\tL1-17-1\tL2-8-1\tL3-9-cis7-1\t \tH3-23:L4-1\t5I1D\tL1-17-1/L1-17-1\tL2-8-1/L2-8-1\tL3-9-cis7-1/L3-9-cis7-1\t \tH3-53:L4-1\t5I1I\tL1-17-1\tL2-8-1\tL3-9-cis7-1\t \tH5-51:L4-1\t5I1L\tL1-17-1/L1-17-1\tL2-8-1/L2-8-1\tL3-9-cis7-1/L3-9-cis7-1\t \t CDRs are defined using the Dunbrack convention [12]. Assignments for 2 copies of the Fab in the asymmetric unit are given for 5 structures. No assignment (NA) for CDRs with missing residues. The four HCs feature CDR H1 of the same length, and their sequences are highly similar (Table 2). The CDR H1 backbone conformations for all variants for each of the HCs are shown in Fig. 1. Three of the HCs, H3-23, H3-53 and H5-51, have the same canonical structure, H1-13-1, and the backbone conformations are tightly clustered for each set of Fab structures as reflected in the rmsd values (Fig. 1B-D). Some deviation is observed for H3-53, mostly due to H3-53:L4-1, which exhibits a significant degree of disorder in CDR H1. The electron density for the backbone is weak and discontinuous, and completely missing for several side chains.    The CDR H1 structures with H1-69 shown in Fig. 1A are quite variable, both for the structures with different LCs and for the copies of the same Fab in the asymmetric unit, H1-69:L3-11 and H1-69:L3-20. In total, 6 independent Fab structures produce 5 different canonical structures, namely H1-13-1, H1-13-3, H1-13-4, H1-13-6 and H1-13-10. A major difference of H1-69 from the other germlines in the experimental data set is the presence of Gly instead of Phe or Tyr at position 27 (residue 5 of 13 in CDR H1). Glycine introduces the possibility of a higher degree of conformational flexibility that undoubtedly translates to the differences observed, and contributes to the elevated thermal parameters for the atoms in the amino acid residues in this region. CDR H2 The superposition of CDR H2 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. The canonical structures of CDR H2 have fairly consistent conformations (Table 2, Fig. 2). Each of the 4 HCs adopts only one canonical structure regardless of the pairing LC. Germlines H1-69 and H5-51 have the same canonical structure assignment H2-10-1, H3-23 has H2-10-2, and H3-53 has H2-9-3. The conformations for all of these CDR H2s are tightly clustered (Fig. 2). In one case, in the second Fab of H1-69:L3-20, CDR H2 is partially disordered (Δ55-60).   Although three of the germlines have CDR H2 of the same length, 10 residues, they adopt 2 distinctively different conformations depending mostly on the residue at position 71 from the so-called CDR H4. Arg71 in H3-23 fills the space between CDRs H2 and H4, and defines the conformation of the tip of CDR H2 so that residue 54 points away from the antigen binding site. Germlines H1-69 and H5-51 are unique in the human repertoire in having an Ala at position 71 that leaves enough space for H-Pro52a to pack deeper against CDR H4 so that the following residues 53 and 54 point toward the putative antigen. Conformations of CDR H2 in H1-69 and H5-51, both of which have canonical structure H2-10-1, show little deviation within each set of 4 structures. However, there is a significant shift of the CDR as a rigid body when the 2 sets are superimposed. Most likely this is the result of interaction of CDR H2 with CDR H1, namely with the residue at position 33 (residue 11 of 13 in CDR H1). Germline H1-69 has Ala at position 33 whereas in H5-51 position 33 is occupied by a bulky Trp, which stacks against H-Tyr52 and drives CDR H2 away from the center. CDR L1 The superposition of CDR L1 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. The four LC CDRs L1 feature 3 different lengths (11, 12 and 17 residues) having a total of 4 different canonical structure assignments. Of these LCs, L1-39 and L3-11 have the same canonical structure, L1-11-1, and superimpose very well (Fig. 3A, B). For the remaining 2, L3-20 has 2 different assignments, L1-12-1 and L1-12-2, while L4-1 has a single assignment, L1-17-1.   L4-1 has the longest CDR L1, composed of 17 amino acid residues (Fig. 3D). Despite this, the conformations are tightly clustered (rmsd is 0.20 Å). The backbone conformations of the stem regions superimpose well. Some changes in conformation occur between residues 30a and 30f (residues 8 and 13 of 17 in CDR L1). This is the tip of the loop region, which appears to have similar conformations that fan out the structures because of the slight differences in torsion angles in the backbone near Tyr30a and Lys30f. L3-20 is the most variable in CDR L1 among the 4 germlines as indicated by an rmsd of 0.54 Å (Fig. 3C). Two structures, H3-53:L3-20 and H5-51:L3-20 are assigned to canonical structure L1-12-1 with virtually identical backbone conformations. The third structure, H3-23:L3-20, has CDR L1 as L1-12-2, which deviates from L1-12-1 at residues 29-32, i.e., at the site of insertion with respect to the 11-residue CDR. The fourth member of the set, H1-69:L3-20, was crystallized with 2 Fabs in the asymmetric unit. The conformation of CDR L1 in these 2 Fabs is slightly different, and both conformations fall somewhere between L1-12-1 and L1-12-2. This reflects the lack of accuracy in the structure due to low resolution of the X-ray data (3.3 Å). CDR L2 The superposition of CDR L2 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. All four LCs have CDR L2 of the same length and canonical structure, L2-8-1 (Table 2). The CDR L2 conformations for each of the LCs paired with the 4 HCs are clustered more tightly than any of the other CDRs (rmsd values are in the range 0.09-0.16 Å), and all 4 sets have virtually the same conformation despite the sequence diversity of the loop. No significant conformation outliers are observed (Fig. 4).   CDR L3 The superposition of CDR L3 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. As with CDR L2, all 4 LCs have CDR L3 of the same length and canonical structure, L3-9-cis7-1 (Table 2). The conformations of CDR L3 for L1-39, L3-11, and particularly for L320, are not as tightly clustered as those of L4-1 (Fig. 5). The slight conformational variability occurs in the region of amino acid residues 90-92, which is in contact with CDR H3.   CDR H3 conformational diversity As mentioned earlier, all 16 Fabs have the same CDR H3, for which the amino acid sequence is derived from the anti-CCL2 antibody CNTO 888. The loop and the 2 β-strands of the CDR H3 in this ‘parent’ structure are stabilized by H-bonds between the carbonyl oxygen and peptide nitrogen atoms in the 2 strands. An interesting feature of these CDR H3 structures is the presence of a water molecule that interacts with the peptide nitrogens and carbonyl oxygens near the bridging loop connecting the 2 β-strands. This water is present in both the bound (4DN4) and unbound (4DN3) forms of CNTO 888. The stem region of CDR H3 in the parental Fab is in a ‘kinked’ conformation, in which the indole nitrogen of Trp103 forms a hydrogen bond with the carbonyl oxygen of Leu100b. The carboxyl group of Asp101 forms a salt bridge with Arg94. These interactions are illustrated in Fig. S2. Ribbon representations of (A) the superposition of all CDR H3s of the structures with complete backbone traces. (B) The CDR H3s rotated 90° about the y axis of the page. The structure of each CDR H3 is represented with a different color. Despite having the same amino acid sequence in all variants, CDR H3 has the highest degree of structural diversity and disorder of all of the CDRs in the experimental set. Three of the 21 Fab structures (including multiple copies in the asymmetric unit), H5-51:L3-11, H551:L3-20 and H3-23:L4-1 (one of the 2 Fabs), have missing (disordered) residues at the apex of the CDR loop. Another four of the Fabs, H3-23:L1-39, H3-53:L1-39, H3-53:L3-11 and H3-53:L4-1 have missing side-chain atoms. The variations in CDR H3 conformation are illustrated in Fig. 6 for the 18 Fab structures that have ordered backbone atoms.   A comparison of representatives of the “kinked” and “extended” structures. (A) The “kinked” CDR H3 of H1-69:L3-11 with purple carbon atoms and yellow dashed lines connecting the H-bond pairs for Leu100b O and Trp103 NE1, Arg94 NE and Asp101 OD1, and Arg94 NH2 and Asp101 OD2. (B) The “extended” CDR H3 of H1-69:L3-20 with green carbon atoms and yellow dashed lines connecting the H-bond pairs for Asp101 OD1 and OD2 and Trp103 NE1. In 10 of the 18 Fab structures, H1-69:L1-39, H1-69:L3-11 (2 Fabs), H1-69:L4-1, H3-23:L3-11 (2 Fabs), H3-23:L3-20, H3-53:L3-11, H3-53:L3-20 and H5-51:L1-39, the CDRs have similar conformations to that found in 4DN3. The bases of these structures have the ‘kinked’ conformation with the H-bond between Trp103 and Leu100b. A representative CDR H3 structure for H1-69:L1-39 illustrating this is shown in Fig. 7A. The largest backbone conformational deviation for the set is at Tyr99, where the C=O is rotated by 90° relative to that observed in 4DN3. Also, it is worth noting that only one of these structures, H1-69:L4-1, has the conserved water molecule in CDR H3 observed in the 4DN3 and 4DN4 structures. In fact, it is the only Fab in the set that has a water molecule present at this site. The CDR H3 for this structure is shown in Fig. S3.   The remaining 8 Fabs can be grouped into 5 different conformational classes. Three of the Fabs, H3-23:L1-39, H3-23:L4-1 and H3-53:L1-39, have distinctive conformations. The stem regions in these 3 cases are in the ‘kinked’ conformation consistent with that observed for 4DN3. The five remaining Fabs, H5-51:L4-1 (2 copies), H1-69:L3-20 (2 copies) and H3-53:L4-1, have 3 different CDR H3 conformations (Fig. S4). The stem regions of CDR H3 for the H5-51:L4-1 Fabs are in the ‘kinked’ conformation while, surprisingly, those of the H1-69:L3-20 pair and H3-53:L4-1 are in the ‘extended’ conformation (Fig. 7B). VH:VL domain packing The VH and VL domains have a β-sandwich structure (also often referred as a Greek key motif) and each is composed of a 4-stranded and a 5-stranded antiparallel β-sheets. The two domains pack together such that the 5-stranded β-sheets, which have hydrophobic surfaces, interact with each other bringing the CDRs from both the VH and VL domains into close proximity. The domain packing of the variants was assessed by computing the domain interface interactions, the VH:VL tilt angles, the buried surface area and surface complementarity. The results of these analyses are shown in Tables 3, 4 and S2. VH:VL interface amino acid residue interactions The conserved VH:VL interactions as viewed along the VH/VL axis. The VH residues are in blue, the VL residues are in orange. The VH:VL interface is pseudosymmetric, and involves 2 stretches of the polypeptide chain from each domain, namely CDR3 and the framework region between CDRs 1 and 2. These stretches form antiparallel β-hairpins within the internal 5-stranded β-sheet. There are a few principal inter-domain interactions that are conserved not only in the experimental set of 16 Fabs, but in all human antibodies. They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). With the exception of L-Ala43, all other residues are conserved in human germlines. Position 43 may be alternatively occupied by Ser, Val or Pro (as in L4-1), but the hydrophobic interaction with H-Tyr91 is preserved. These core interactions provide enough stability to the VH:VL dimer so that additional VH-VL contacts can tolerate amino acid sequence variations in CDRs H3 and L3 that form part of the VH:VL interface.   In total, about 20 residues are involved in the VH:VL interactions on each side (Fig. S5). Half of them are in the framework regions and those residues (except residue 61 in HC, which is actually in CDR2 in Kabat's definition) are conserved in the set of 16 Fabs. The side chain conformations of these conserved residues are also highly similar. One notable exception is H-Trp47, which exhibits 2 conformations of the indole ring. In most of the structures, it has the χ2 angle of ∼80°, while the ring is flipped over (χ2 = −100°) in H5-51:L3:11 and H5-51:L3-20. Interestingly, these are the only 2 structures with residues missing in CDR H3 because of disorder, although both structures are determined at high resolution and the rest of the structure is well defined. Apparently, residues flanking CDR H3 in the 2 VH:VL pairings are inconsistent with any stable conformation of CDR H3, which translates into a less restricted conformational space for some of them, including H-Trp47. VH:VL tilt angles The relative orientation of VH and VL has been measured in a number of different ways. Presented here are the results of 2 different approaches for determining the orientation of one domain relative to the other. The first approach uses ABangles, the results of which are shown in Table S2. The four LCs all are classified as Type A because they have a proline at position 44, and the results for each orientation parameter are within the range of values of this type reported by Dunbar and co-workers. In fact, the parameter values for the set of 16 Fabs are in the middle of the distribution observed for 351 non-redundant antibody structures determined at 3.0 Å resolution or better. The only exception is HC1, which is shifted toward smaller angles with the mean value of 70.8° as compared to the distribution centered at 72° for the entire PDB. This probably reflects the invariance of CDR H3 in the current set as opposed to the CDR H3 diversity in the PDB. The second approach used for comparing tilt angles involved computing the difference in the tilt angles between all pairs of structures. For structures with 2 copies of the Fab in the asymmetric unit, only one structure was used. The differences between independent Fabs in the same structure are 4.9° for H1-69:L3-20, 1.6° for H1-69:L3-11, 1.4° for H3-23:L4-1, 3.3° for H3-23:L3-11, and 2.5° for H5-51:L4-1. With the exception of H1-69:L3-20, the angles are within the range of 2-3° as are observed in the identical structures in the PDB. In H1-69:L3-20, one of the Fabs is substantially disordered so that part of CDR H2 (the outer β-strand, residues 55-60) is completely missing. This kind of disorder may compromise the integrity of the VH domain and its interaction with the VL. Indeed, this Fab has the largest twist angle HC2 within the experimental set that exceeds the mean value by 2.5 standard deviations (Table S2). An illustration of the difference in tilt angle for 2 pairs of variants by the superposition of the VH domains of (A) H1-69:L3-20 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 10.5°) and (B) H1-69:L4-1 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 1.6°). Differences in VH:VL tilt angles.  \tH1-69:L1-39\tH1-69:L3-11\tH1-69:L3-20\tH1-69:L4-1\tH3-23:L1-39\tH3-23:L3-11\tH3-23:L3-20\tH3-23:L4-1\tH3-53:L1-39\tH3-53:L3-11\tH3-53:L3-20\tH3-53:L4-1\tH5-51:L1-39\tH5-51:L3-11\tH5-51:L3-20\tH5-51:L4-1\t \tH1-69:L1-39\t0\t2.1\t8.9\t1.1\t4.2\t3.0\t9.5\t1.5\t3.3\t3.6\t3.1\t1.6\t1.8\t2.9\t2.4\t5.2\t \tH1-69:L3-11\t \t0\t7.3\t2.9\t2.5\t2.0\t8.4\t1.3\t2.6\t2.9\t3.2\t1.8\t3.9\t4.6\t4.4\t5.0\t \tH1-69:L3-20\t \t \t0\t9.2\t5.0\t8.7\t7.4\t7.6\t8.9\t8.6\t9.4\t7.9\t10.5\t10.1\t11.0\t9.7\t \tH1-69:L4-1\t \t \t \t0\t4.6\t3.9\t10.1\t1.8\t4.4\t4.7\t4.1\t2.3\t1.6\t2.5\t2.3\t6.2\t \tH3-23:L1-39\t \t \t \t \t0\t4.0\t8.0\t2.8\t4.8\t4.8\t5.4\t3.6\t6.0\t6.2\t6.6\t6.6\t \tH3-23:L3-11\t \t \t \t \t \t0\t9.3\t3.0\t2.1\t2.9\t3.3\t3.3\t4.6\t5.8\t5.0\t5.2\t \tH3-23:L3-20\t \t \t \t \t \t \t0\t8.9\t7.9\t7.0\t7.6\t7.9\t10.5\t9.7\t10.7\t6.2\t \tH3-23:L4-1\t \t \t \t \t \t \t \t0\t3.6\t3.8\t3.7\t1.5\t3.2\t3.7\t3.8\t5.6\t \tH3-53:L1-39\t \t \t \t \t \t \t \t \t0\t1.0\t1.6\t2.9\t4.6\t5.3\t4.8\t3.1\t \tH3-53:L3-11\t \t \t \t \t \t \t \t \t \t0\t1.3\t2.9\t4.8\t5.2\t5.0\t2.3\t \tH3-53:L3-20\t \t \t \t \t \t \t \t \t \t \t0\t2.5\t3.8\t4.2\t3.9\t2.2\t \tH3-53:L4-1\t \t \t \t \t \t \t \t \t \t \t \t0\t2.9\t3.0\t3.3\t4.2\t \tH5-51:L1-39\t \t \t \t \t \t \t \t \t \t \t \t \t0\t1.9\t0.6\t5.8\t \tH5-51:L3-11\t \t \t \t \t \t \t \t \t \t \t \t \t \t0\t1.9\t5.7\t \tH5-51:L3-20\t \t \t \t \t \t \t \t \t \t \t \t \t \t \t0\t5.8\t \tH5-51:L4-1\t \t \t \t \t \t \t \t \t \t \t \t \t \t \t \t0\t \t The differences in the tilt angle are shown for all pairs of V regions in Table 3. They range from 0.6° to 11.0°. The smallest differences in the tilt angle are between the Fabs in isomorphous crystal forms. The largest deviations in the tilt angle, up to 11.0°, are found for 2 structures, H1-69:L3-20 and H3-23:L3-20, that stand out from the other Fabs. One of the 2 structures, H1-69:L3-20, has its CDR H3 in the ‘extended’ conformation; the other structure has it in the ‘kinked’ conformation. Two examples illustrating large (10.5°) and small (1.6°) differences in the tilt angles are shown in Fig. 9.    VH:VL buried surface area and complementarity VH:VL surface areas and surface complementarity. Chain Pairs\tPDB\tContact surfaceVH (Å2)\tContact surfaceVL (Å2)\tInterface(Å2)\tSurface complementarity\t \tH1-69:L1-39\t5I15\t727\t771\t749\t0.743\t \tH1-69:L3-11\t5I16\t802\t870\t836\t0.762\t \tH1-69:L3-20\t5I17\t713\t736\t725\t0.723\t \tH1-69:L4-1\t5I18\t729\t736\t733\t0.734\t \tH3-23:L1-39\t5I19\t795\t817\t806\t0.722\t \tH3-23:L3-11\t5I1A\t822\t834\t828\t0.725\t \tH3-23:L3-20\t5I1C\t670\t698\t684\t0.676\t \tH3-23:L4-1\t5I1D\t743\t770\t757\t0.708\t \tH3-53:L1-39\t5I1E\t698\t719\t709\t0.712\t \tH3-53:L3-11\t5I1G\t747\t758\t753\t0.690\t \tH3-53:L3-20\t5I1H\t743\t735\t739\t0.687\t \tH3-53:L4-1\t5I1I\t689\t693\t691\t0.711\t \tH5-51:L1-39\t4KMT\t761\t808\t785\t0.728\t \tH5-51:L3-11\t5I1J\t648\t714\t681\t0.717\t \tH5-51:L3-20\t5I1K\t622\t643\t633\t0.740\t \tH5-51:L4-1\t5I1L\t790\t792\t791\t0.704\t \t Some side chain atoms in CDR H3 are missing. Residues in CDR H3 are missing: YGE in H5-51:L3-11, GIY in H5-51:L3-20. The results of the PISA contact surface calculation and surface complementarity calculation are shown in Table 4. The interface areas are calculated as the average of the VH and VL contact surfaces. Six of the 16 structures have CDR H3 side chains or complete residues missing, and therefore their interfaces are much smaller than in the other 10 structures with complete CDRs (the results are provided for all Fabs for completeness). Among the complete structures, the interface areas range from 684 to 836 Å2. Interestingly, the 2 structures that have the largest tilt angle differences with the other variants, H3-23:L3-20 and H1-69:L3-20, have the smallest VH:VL interfaces, 684 and 725 Å2, respectively. H3-23:L3-20 is also unique in that it has the lowest value (0.676) of surface complementarity.   Stability of germline pairings Melting temperatures for the 16 Fabs.  \tL3-20\tL4-1\tL3-11\tL1-39\tHC average\t \tH1-69\t73.6\t74.8\t75.6\t80.3\t76.1\t \tH3-23\t74.8\t75.2\t4.8\t81.5\t76.6\t \tH3-53\t68.4\t68.0\t71.5\t73.9\t70.5\t \tH5-51\t68.4\t68.4\t71.9\t77.0\t71.4\t \tLC average\t71.3\t71.6\t73.5\t78.2\t \t \t Colors: blue (Tm \u003c 70°C), green (70°C \u003c Tm \u003c 73°C), yellow (73°C \u003c Tm \u003c 78°C), orange (Tm \u003e 78°C). Melting temperatures (Tm) were measured for all Fabs using differential scanning calorimetry (Table 5). It appears that for each given LC, the Fabs with germlines H1-69 and H3-23 are substantially more stable than those with germlines H3-53 and H5-51. In addition, L1-39 provides a much higher degree of stabilization than the other 3 LC germlines when combined with any of the HCs. As a result, the Tm for pairs H1-69:L1-39 and H3-23:L1-39 is 12-13° higher than for pairs H3-53:L3-20, H3-53:L4-1, H5-51:L3-20 and H5-51:L4-1.   These findings correlate well with the degree of conformational disorder observed in the crystal structures. Parts of CDR H3 main chain are completely disordered, and were not modeled in Fabs H5-51:L3-20 and H5-51:L3-11 that have the lowest Tms in the set. No electron density is observed for a number of side chains in CDRs H3 and L3 in all Fabs with germline H3-53, which indicates loose packing of the variable domains. All those molecules are relatively unstable, as is reflected in their low Tms. Discussion This is the first report of a systematic structural investigation of a phage germline library. The 16 Fab structures offer a unique look at all pairings of 4 different HCs (H1-69, H3-23, H3-53, and H5-51) and 4 different LCs (L1-39, L3-11, L3-20 and L4-1), all with the same CDR H3. The structural data set taken as a whole provides insight into how the backbone conformations of the CDRs of a specific heavy or light chain vary when it is paired with 4 different light or heavy chains, respectively. A large variability in the CDR conformations for the sets of HCs and LCs is observed. In some cases the CDR conformations for all members of a set are virtually identical, for others subtle changes occur in a few members of a set, and in some cases larger deviations are observed within a set. The five variants that crystallized with 2 copies of the Fab in the asymmetric unit serve somewhat as controls for the influence of crystal packing on the conformations of the CDRs. In four of the 5 structures the CDR conformations are consistent. In only one case, that of H1-69:L3-20 (the lowest resolution structure), do we see differences in the conformations of the 2 copies of CDRs H1 and L1. This variability is likely a result of 2 factors, crystal packing interactions and internal instability of the variable domain. For the CDRs with canonical structures, the largest changes in conformation occur for CDR H1 of H1-69 and H3-53. The other 2 HCs, H3-23 and H5-51, have canonical structures that are remarkably well conserved (Fig. 1). Of the 4 HCs, H1-69 has the greatest number of canonical structure assignments (Table 2). H1-69 is unique in having a pair of glycine residues at positions 26 and 27, which provide more conformational freedom in CDR H1. Besides IGHV1-69, only the germlines of the VH4 family possess double glycines in CDR H1, and it will be interesting to see if they are also conformationally unstable. Having all 16 VH:VL pairs with the same CDR H3 provides some insights into why molecular modeling efforts of CDR H3 have proven so difficult. As mentioned in the Results section, this data set is composed of 21 Fabs, since 5 of the 16 variants have 2 Fab copies in the asymmetric unit. For the 18 Fabs with complete backbone atoms for CDR H3, 10 have conformations similar to that of the parent, while the others have significantly different conformations (Fig. 6). Thus, it is likely that the CDR H3 conformation is dependent upon 2 dominating factors: 1) amino acid sequence; and 2) VH and VL context. More than half of the variants retain the conformation of the parent despite having differences in the VH:VL pairing. This subset includes 2 structures with 2 copies of the Fab in the asymmetric unit, all of which are nearly identical in conformation. This provides an internal control showing a consistency in the conformations. The remaining 8 structures exhibit “non-parental” conformations, indicating that the VH and VL context can also be a dominating factor influencing CDR H3. Importantly, there are 5 distinctive conformations in this subset. This subset also has 2 structures with 2 Fab copies in the asymmetric unit. Each pair has nearly identical conformations providing an internal check on the consistency of the conformations. Interestingly, as described earlier, these 2 pairs differ in the stem regions with the H1-69:L3-20 pair in the ‘extended’ conformation and H5-51:L4-1 pair in the ‘kinked’ conformation. The conformations are different from each other, as well as from the parent. The CDR H3 conformational analysis shows that, for each set of variants of one HC paired with the 4 different LCs, both “parental” and “non-parental” conformations are observed. The same variability is observed for the sets of variants composed of one LC paired with each of the 4 HCs. Thus, no patterns of conformational preference for a particular HC or LC emerge to shed any direct light on what drives the conformational differences. This finding supports the hypothesis of Weitzner et al. that the H3 conformation is controlled both by its sequence and its environment. In looking at a possible correlation between the tilt angle and the conformation of CDR H3, no clear trends are observed. Two variants, H1-69:L3-20 and H3-23:L3-20, have the largest differences in the tilt angles compared to other variants as seen in Table 3. The absolute VH:VL orientation parameters for the 2 Fabs (Table S2) show significant deviation in HL, LC1 and HC2 values (2-3 standard deviations from the mean). One of the variants, H3-23:L3-20, has the CDR H3 conformation similar to the parent, but the other, H1-69:L3-20, is different. As noted in the Results section, the 2 variants, H1-69:L3-20 and H3-23:L3-20, are outliers in terms of the tilt angle; at the same time, both have the smallest VH:VL interface. These smaller interfaces may perhaps translate to a significant deviation in how VH is oriented relative to VL than the other variants. These deviations from the other variants can also be seen to some extent in VH:VL orientation parameters in Table S2, as well as in the smaller number of residues involved in the VH:VL interfaces of these 2 variants (Fig. S5). These differences undoubtedly influence the conformation of the CDRs, in particular CDR H1 (Fig. 1A) and CDR L1 (Fig. 3C), especially with the tandem glycines and multiple serines present, respectively. Pairing of different germlines yields antibodies with various degrees of stability. As indicated by the melting temperatures, germlines H1-69 and H3-23 for HC and germline L1-39 for LC produce more stable Fabs compared to the other germlines in the experimental set. Structural determinants of the differential stability are not always easy to decipher. One possible explanation of the clear preference of LC germline L1-39 is that CDR L3 has smaller residues at positions 91 and 94, allowing for more room to accommodate CDR H3. Other germlines have bulky residues, Tyr, Arg and Trp, at these positions, whereas L1-39 has Ser and Thr. Various combinations of germline sequences for VL and VH impose certain constraints on CDR H3, which has to adapt to the environment. A more compact CDR L3 may be beneficial in this situation. At the other end of the stability range is LC germline L3-20, which yields antibodies with the lowest Tms. While pairings with H3-53 and H5-51 may be safely called a mismatch, those with H1-69 and H3-23 have Tms about 5-6° higher. Curiously, the 2 Fabs, H1-69:L3-20 and H3-23:L3-20, deviate markedly in their tilt angles from the rest of the panel. It is possible that by adopting extreme tilt angles the structure modulates CDR H3 and its environment, which apparently cannot be achieved solely by conformational rearrangement of the CDR. Note that most of the VH:VL interface residues are invariant; therefore, significant change of the tilt angle must come with a penalty in free energy. Yet, for the 2 antibodies, the total gain in stability merits the domain repacking. Overall, the stability of the Fab, as measured by Tm, is a result of the mutual adjustment of the HC and LC variable domains and adjustment of CDR H3 to the VH:VL cleft. The final conformation represents an energetic minimum; however, in most cases it is very shallow, so that a single mutation can cause a dramatic rearrangement of the structure. In summary, the analysis of this structural library of germline variants composed of all pairs of 4 HCs and 4LCs, all with the same CDR H3, offers some unique insights into antibody structure and how pairing and sequence may influence, or not, the canonical structures of the L1, L2, L3, H1 and H2 CDRs. Comparison of the CDR H3s reveals a large set of variants with conformations similar to the parent, while a second set has significant conformational variability, indicating that both the sequence and the structural context define the CDR H3 conformation. Quite unexpectedly, 2 of the variants, H1-69:L3-20 and H3-53:L4-1, have the ‘extended’ stem region differing from the other 14 that have a ‘kinked’ stem region. Why this is the case is unclear at present. These data reveal the difficulty of modeling CDR H3 accurately, as shown again in Antibody Modeling Assessment II. Furthermore, antibody CDRs, H3 in particular, may go through conformational changes upon binding their targets, making structural prediction for docking purposes an even more difficult task. Fortunately, for most applications of antibody modeling, such as engineering affinity and biophysical properties, an accurate CDR H3 structure is not always necessary. For those applications where accurate CDR structures are essential, such as docking, the results in this work demonstrate the importance of experimental structures. With the recent advances in expression and crystallization methods, Fab structures can be obtained rapidly. The set of 16 germline Fab structures offers a unique dataset to facilitate software development for antibody modeling. The results essentially support the underlying idea of canonical structures, indicating that most CDRs with germline sequences tend to adopt predefined conformations. From this point of view, a novel approach to design combinatorial antibody libraries would be to cover the range of CDR conformations that may not necessarily coincide with the germline usage in the human repertoire. This would insure more structural diversity, leading to a more diverse panel of antibodies that would bind to a broad spectrum of targets. Materials and methods Fab production, purification and crystallization The production, purification and crystallization of the Fabs reported in this article were described previously. Briefly, the 16 Fabs were produced by combining 4 different HC and 4 different LC germline constructs. The human HC germlines were IGHV1-69 (H1-69), IGHV3-23 (H3-23), IGHV3-53 (H3-53) and IGHV5-51 (H5-51) in the IMGT nomenclature. The human LC germlines were IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1) corresponding to O12, L6, A27 and B3 in the V-BASE nomenclature. CDR H3 of the anti-CCL2 antibody CNTO 888 with the amino acid sequence ARYDGIYGELDF  was used in all Fab constructs. The J region genes were IGHJ1 for the HC and IGKJ1 for the LC for all Fabs. Human IgG1 and κ constant regions were used in all Fab constructs. A 6xHis tag was added to the C-terminus of the HC to facilitate purification. The Fabs were expressed in HEK 293E cells and purified by affinity and size-exclusion chromatography. For crystallization, the Fabs were dialyzed into 20 mM Tris buffer, pH 7.4, with 50 mM NaCl and concentrated to 12-18 mg/mL. Automated crystallization screening was carried out using the vapor diffusion method at 20°C with an Oryx4 (Douglas Instruments) or a Mosquito (TTP Labtech) crystallization robot in a sitting drop format using Corning 3550 plates. Initial screening was carried out with an in-house 192-well screen optimized for Fab crystallization and the Hampton 96-well Crystal Screen HT (Hampton Research). For the majority of the Fabs, the crystallization protocol employed microseed matrix screening using self-seeding or cross-seeding approaches. A summary of the final crystallization conditions for each of the Fabs is presented in Table 1. X-ray data collection For 13 of the Fab crystals, X-ray data collection was carried out at Janssen Research and Development, LLC using a Rigaku MicroMax™-007HF microfocus X-ray generator equipped with a Saturn 944 CCD detector and an X-stream™ 2000 cryocooling system (Rigaku), and for the remaining 3, X-ray data collection was carried out at the Advanced Photon Source (APS) synchrotron at Argonne National Laboratory using the IMCA 17-ID beamline with a Pilatus 6M detector. For X-ray data collection, the Fab crystals were soaked for a few seconds in a cryo-protectant solution containing the corresponding mother liquor supplemented with 17-25% glycerol (Table S1). The crystals for which data were collected in-house were flash cooled in the stream of nitrogen at 100 K. Crystals sent to the APS were flash cooled in liquid nitrogen prior to shipping them to the synchrotron. Diffraction data for all variants were processed with the program XDS. X-ray data statistics are given in Table 1. Structure determination A summary of the methods used in the structure solution and refinement of the 16 Fabs is presented in Table S1. Twelve of the structures were solved by molecular replacement with Phaser using different combinations of search models for the VH, VL and constant domains. Four of the structures, H3-53:L1-39, H3-53:L3-11, H5-51:L1-39 and H5-51:L3-11, were solved by direct replacement followed by rigid body refinement with REFMAC. All structures were refined using REFMAC. Model adjustments were carried out using the program Coot. The refinement statistics are given in Table 1. Other crystallographic calculations were performed with the CCP4 suite of programs. The structural figures were prepared using the PyMOL Molecular Graphics System, Version 1.0 (Schrödinger, LLC). Structural analysis The canonical structure assignments (Table 2) were made using PyIgClassify, an online canonical structure classification tool (http://dunbrack2.fccc.edu/pyigclassify/) that uses the rules set forth by Dunbrack and coworkers. The conformational variability within the CDRs was assessed by calculating the root-mean-square deviation (rmsd) from the average structure that was generated after superposition of all structures of the set using the main-chain atoms of the CDR in question. The rmsd was calculated for all main-chain atoms (N, CA, C, O) of the CDR. The contact surface areas of the VH and VL domains at the VH:VL inteface were computed with the CCP4 program PISA. The surface complementarity of the VH and VL domains was computed using the CCP4 program SC. VH:VL tilt angles The orientation of the VH domain with respect to the VL domain was assessed using 2 different approaches. The first approach calculates the 6 VH:VL orientation parameters that describe the VH:VL relationship according to Dunbar and co-workers using a script downloaded from the website (http://opig.stats.ox.ac.uk/webapps/abangle). The six parameters include 5 angles, HL, H1, H2, L1 and L2, and a distance, dc. These parameters are derived by first defining 2 planes, one for each domain, based on core residues in the domains. The distance between the planes, dc, is determined along a vector between the planes that is used to establish a consistent coordinate system. The torsion angle between the domains, HL, is much like the VH:VL packing angle defined by Abhinandan and Martin. The tilt of one domain relative to the other is defined by the HC1 and LC1 angles, and the twist of one domain relative to the other is defined by the HC2 and LC2 angles. The second approach calculates the difference in the tilt angle between pairs of Fvs, which reflects the relative orientation between the VH and VL domains. The difference with respect to the reference structure is calculated by sequential root-mean-square superposition of the VL and VH domains using β-sheet core Cα positions (Chothia numbering scheme): 3–13, 18–25, 33–38, 43–49, 61–67, 70–76, 85–90, 97–103 for VL; 3–7, 18–24, 34–40, 44–51, 56–59, 67–72, 77–82a, 87–94, 102–110 for VH. The κ angle in the spherical polar angular system (ω, ϕ, κ) of the latter transformation is the difference in the tilt angle. Differential scanning calorimetry DSC experiments were performed on a VP-capillary DSC system (MicroCal Inc., Northampton, MA) in which temperature differences between the reference and sample cell are continuously measured and calibrated to power units. Samples were heated from 10°C to 95°C at a heating rate of 60°C/hour. The pre-scan time was 15 minutes and the filtering period was 10 seconds. The concentration used in the DSC experiments was about 0.4 mg/mL in phosphate-buffered saline. Analysis of the resulting thermograms was performed using MicroCal Origin 7 software. Melting temperature of proteins was determined by deconvolution of the DSC scans using non-2 state model in the MicroCal Origin 7 software. Scans were deconvoluted using a non-2 state model with either 1-step transition or 2-step transition depending on the number of resolved peaks observed in a scan. Accession numbers Atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers 4KMT, 5I15, 5I16, 5I17, 5I18, 5I19, 5I1A, 5I1C, 5I1D, 5I1E, 5I1G, 5I1H, 5I1I, 5I1J, 5I1K and 5I1L. Supplementary Material Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed. References Antibodies to watch in 2016 An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity Canonical structures for the hypervariable regions of immunoglobulins Conformations of immunoglobulin hypervariable regions Structural repertoire of the human VH segments The structural repertoire of the human V kappa domain Canonical structure repertoire of the antigen-binding site of immunoglobulins suggests strong geometrical restrictions associated to the mechanism of immune recognition Standard conformations for the canonical structures of immunoglobulins Analysis of the antigen combining site: correlations between length and sequence composition of the hypervariable loops and the nature of the antigen The Protein Data Bank Structural families in loops of homologous proteins: automatic classification, modelling and application to antibodies A new clustering of antibody CDR loop conformations PyIgClassify: a database of antibody CDR structural classifications Structural classification of CDR-H3 in antibodies Antibody structure, prediction and redesign Conformations of the third hypervariable region in the VH domain of immunoglobulins Structural classification of CDR-H3 revisited: a lesson in antibody modeling The origin of CDR H3 structural diversity Antibody modeling assessment Second Antibody Modeling Assessment (AMA-II) Antibody modeling assessment II. Structures and models De novo selection of high-affinity antibodies from synthetic fab libraries displayed on phage as pIX fusion proteins Analysis of heavy and light chain pairings indicates that receptor editing shapes the human antibody repertoire Structural basis for high selectivity of anti-CCL2 neutralizing antibody CNTO 888 Protein crystallization with microseed matrix screening: application to human germline antibody Fabs Framework residue-71 is a major determinant of the position and conformation of the 2nd hypervariable region in the VH domains of immunoglobulins Modeling the anti-CEA antibody combining site by homology and conformational search Major antigen-induced domain rearrangements in an antibody Energy-based analysis and prediction of the orientation between light- and heavy-chain antibody variable domains Analysis and prediction of VH/VL packing in antibodies ABangle: characterising the VH-VL orientation in antibodies Inference of macromolecular assemblies from crystalline state Shape complementarity at protein/protein interfaces Structural evidence for induced fit as a mechanism for antibody-antigen recognition Nomenclature of the human immunoglobulin genes  Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: papain digestion of mAb and transient expression in mammalian cells An automated microseed matrix-screening method for protein crystallization Promoting crystallization of antibody-antigen complexes via microseed matrix screening XDS Phaser crystallographic software REFMAC5 for the refinement of macromolecular crystal structures Features and development of Coot Overview of the CCP4 suite and current 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similarity index 100%
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diff --git a/BioC_XML/4772114_v1.xml b/annotated_BioC_XML/PMC4772114_ann.xml
similarity index 100%
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diff --git a/BioC_XML/4784909_v0.xml b/annotated_BioC_XML/PMC4784909_ann.xml
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similarity index 100%
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similarity index 100%
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+<collection><source>PMC</source><date>20201220</date><key>pmc.key</key><document><id>4772114</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/srep22324</infon><infon key="article-id_pii">srep22324</infon><infon key="article-id_pmc">4772114</infon><infon key="article-id_pmid">26927947</infon><infon key="elocation-id">22324</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Yokogawa;given-names:Mariko</infon><infon key="name_1">surname:Tsushima;given-names:Takashi</infon><infon key="name_2">surname:Noda;given-names:Nobuo N.</infon><infon key="name_3">surname:Kumeta;given-names:Hiroyuki</infon><infon key="name_4">surname:Enokizono;given-names:Yoshiaki</infon><infon key="name_5">surname:Yamashita;given-names:Kazuo</infon><infon key="name_6">surname:Standley;given-names:Daron M.</infon><infon key="name_7">surname:Takeuchi;given-names:Osamu</infon><infon key="name_8">surname:Akira;given-names:Shizuo</infon><infon key="name_9">surname:Inagaki;given-names:Fuyuhiko</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">6</infon><infon key="year">2016</infon><offset>0</offset><text>Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>101</offset><text>Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses. Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). The PIN domain harbors the RNase catalytic center; however, it is insufficient for enzymatic activity. We found that the NTD associates with the PIN domain and significantly enhances its RNase activity. The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site. Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro. These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1084</offset><text>The initial sensing of infection is mediated by a set of pattern-recognition receptors (PRRs) such Toll-like receptors (TLRs) and the intracellular signaling cascades triggered by TLRs evoke transcriptional expression of inflammatory mediators that coordinate the elimination of pathogens and infected cells. Since aberrant activation of this system leads to auto immune disorders, it must be tightly regulated. Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase whose expression level is stimulated by lipopolysaccharides and prevents autoimmune diseases by directly controlling the stability of mRNAs of inflammatory genes such as interleukin (IL)-6, IL-1β, IL-2, and IL-12p40. Regnase-1 accelerates target mRNA degradation via their 3′-terminal untranslated region (3′UTR), and also degrades its own mRNA.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1904</offset><text>Regnase-1 is a member of Regnase family and is composed of a PilT N-terminus like (PIN) domain followed by a CCCH-type zinc–finger (ZF) domain, which are conserved among Regnase family members. Recently, the crystal structure of the Regnase-1 PIN domain derived from Homo sapiens was reported. The structure combined with functional analyses revealed that four catalytically important Asp residues form the catalytic center and stabilize Mg2+ binding that is crucial for RNase activity. Several CCCH-type ZF motifs in RNA-binding proteins have been reported to directly bind RNA. In addition, Regnase-1 has been predicted to possess other domains in the N- and C- terminal regions. However, the structure and function of the ZF domain, N-terminal domain (NTD) and C-terminal domain (CTD) of Regnase-1 have not been solved.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2729</offset><text>Here, we performed structural and functional analyses of individual domains of Regnase-1 derived from Mus musculus in order to understand the catalytic activity in vitro. Our data revealed that the catalytic activity of Regnase-1 is regulated through both intra and intermolecular domain interactions in vitro. The NTD plays a crucial role in efficient cleavage of target mRNA, through intramolecular NTD-PIN interactions. Moreover, Regnase-1 functions as a dimer through intermolecular PIN-PIN interactions during cleavage of target mRNA. Our findings suggest that Regnase-1 cleaves its target mRNA by an NTD-activated functional PIN dimer, while the ZF increases RNA affinity in the vicinity of the PIN dimer.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>3441</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>3449</offset><text>Domain structures of Regnase-1</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>3480</offset><text>We analyzed Rengase-1 derived from Mus musculus and solved the structures of the four domains; NTD, PIN, ZF, and CTD individually by X-ray crystallography or NMR (Fig. 1a–e). X-ray crystallography was attempted for the fragment containing both the PIN and ZF domains, however, electron density was observed only for the PIN domain (Fig. 1c), consistent with a previous report on Regnase-1 derived from Homo sapiens. This suggests that the PIN and ZF domains exist independently without interacting with each other. The domain structures of NTD, ZF, and CTD were determined by NMR (Fig. 1b,d,e). The NTD and CTD are both composed of three α helices, and structurally resemble ubiquitin conjugating enzyme E2 K (PDB ID: 3K9O) and ubiquitin associated protein 1 (PDB ID: 4AE4), respectively, according to the Dali server.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>4303</offset><text>Contribution of each domain of Regnase-1 to the mRNA binding activity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4373</offset><text>Although the PIN domain is responsible for the catalytic activity of Regnase-1, the roles of the other domains are largely unknown. First, we evaluated a role of the NTD and ZF domains for mRNA binding by an in vitro gel shift assay (Fig. 1f). Fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1—hereafter referred to as the DDNN mutant—were utilized. Upon addition of a larger amount of Regnase-1, the fluorescence of free RNA decreased, indicating that Regnase-1 bound to the RNA. Based on the decrease in the free RNA fluorescence band, we evaluated the contribution of each domain of Regnase-1 to RNA binding. While the RNA binding ability was not significantly changed in the presence of NTD, it increased in the presence of the ZF domain (Fig. 1f,g and Supplementary Fig. 1). Direct binding of the ZF domain and RNA were confirmed by NMR spectral changes. The fitting of the titration curve of Y314 resulted in an apparent dissociation constant (Kd) of 10 ± 1.1 μM (Supplementary Fig. 2). These results indicate that not only the PIN but also the ZF domain contribute to RNA binding, while the NTD is not likely to be involved in direct interaction with RNA.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>5661</offset><text>Contribution of each domain of Regnase-1 to RNase activity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5720</offset><text>In order to characterize the role of each domain in the RNase activity of Regnase-1, we performed an in vitro cleavage assay using fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR (Fig. 1g). Regnase-1 constructs consisting of NTD-PIN-ZF completely cleaved the target mRNA and generated the cleaved products. The apparent half-life (T1/2) of the RNase activity was about 20 minutes. Regnase-1 lacking the ZF domain generated a smaller but appreciable amount of cleaved product (T1/2 ~ 70 minutes), while those lacking the NTD did not generate cleaved products (T1/2 &gt; 90 minutes). It should be noted that NTD-PIN(DDNN)-ZF, which possesses the NTD but lacks the catalytic residues in PIN, completely lost all RNase activity (Fig. 1g, right panel), as expected, confirming that the RNase catalytic center is located in the PIN domain. Taken together with the results in the previous section, we conclude that the NTD is crucial for the RNase activity of Regnase-1 in vitro, although it does not contribute to the direct mRNA binding.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>6810</offset><text>Dimer formation of the PIN domains</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6845</offset><text>During purification by gel filtration, the PIN domain exhibited extremely asymmetric elution peaks in a concentration dependent manner (Fig. 2a). By comparison with the elution volume of standard marker proteins, the PIN domain was assumed to be in equilibrium between a monomer and a dimer in solution at concentrations in the 20–200 μM range. The crystal structure of the PIN domain has been determined in three distinct crystal forms with a space group of P3121 (form I in this study and PDB ID 3V33), P3221 (form II in this study), and P41 (PDB ID 3V32 and 3V34), respectively. We found that the PIN domain formed a head-to-tail oligomer that was commonly observed in all three crystal forms in spite of the different crystallization conditions (Supplementary Fig. 3). Mutation of Arg215, whose side chain faces to the opposite side of the oligomeric surface, to Glu preserved the monomer/dimer equilibrium, similar to the wild type. On the other hand, single mutations of side chains involved in the PIN–PIN oligomeric interaction resulted in monomer formation, judging from gel filtration (Fig. 2a,b). Wild type and monomeric PIN mutants (P212A and D278R) were also analyzed by NMR. The spectra indicate that the dimer interface of the wild type PIN domain were significantly broadened compared to the monomeric mutants (Supplementary Fig. 4). These results indicate that the PIN domain forms a head-to-tail oligomer in solution similar to the crystal structure. Interestingly, the monomeric PIN mutants P212A, R214A, and D278R had no significant RNase activity for IL-6 mRNA in vitro (Fig. 2c). The side chains of these residues point away from the catalytic center on the same molecule (Fig. 2b). Therefore, we concluded that head-to-tail PIN dimerization, together with the NTD, are required for Regnase-1 RNase activity in vitro.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8692</offset><text>Domain-domain interaction between the NTD and the PIN domain</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8753</offset><text>While the NTD does not contribute to RNA binding (Fig. 1f,g, and Supplementary Fig. 1), it increases the RNase activity of Regnase-1 (Fig. 1h). In order to gain insight into the molecular mechanism of the NTD-mediated enhancement of Regnase-1 RNase activity, we further investigated the domain-domain interaction between the NTD and the PIN domain using NMR. We used the catalytically inactive monomeric PIN mutant possessing both the DDNN and D278R mutations to avoid dimer formation of the PIN domain. The NMR signals from the PIN domain (residues V177, F210-T211, R214, F228-L232, and F234-S236) exhibited significant chemical shift changes upon addition of the NTD (Fig. 3a). Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). These results clearly indicate a direct interaction between the PIN domain and the NTD. Based on the titration curve for the chemical shift changes of L58, the apparent Kd between the isolated NTD and PIN was estimated to be 110 ± 5.8 μM. Considering the fact that the NTD and PIN domains are attached by a linker, the actual binding affinity is expected much higher in the native protein. Mapping the residues with chemical shift changes reveals the putative PIN/NTD interface, which includes a helix that harbors catalytic residues D225 and D226 on the PIN domain (Fig. 3a). Interestingly, the putative binding site for the NTD overlaps with the PIN-PIN dimer interface, implying that NTD binding can “terminate” PIN-PIN oligomerization (Fig. 2b). An in silico docking of the NTD and PIN domains using chemical shift restraints provided a model consistent with the NMR experiments (Fig. 3c).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10692</offset><text>Residues critical for Regnase-1 RNase activity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10739</offset><text>To gain insight into the residues critical for Regnase-1 RNase activity, each basic or aromatic residue located around the catalytic site of the PIN oligomer was mutated to alanine, and the oligomerization and RNase activity were investigated (Fig. 4). From the gel filtration assays, all mutants except R214A formed dimers, suggesting that any lack of RNase activity in the mutants, except R214A, was directly due to mutational effects of the specific residues and not to abrogation of dimer formation. The W182A, R183A, and R214A mutants markedly lost cleavage activity for IL-6 mRNA as well as for Regnase-1 mRNA. The K184A, R215A, and R220A mutants moderately but significantly decreased the cleavage activity for both target mRNAs. The importance of K219 and R247 was slightly different for IL-6 and Regnase-1 mRNA; both K219 and R247 were more important in the cleavage of IL-6 mRNA than for Regnase-1 mRNA. The other mutated residues—K152, R158, R188, R200, K204, K206, K257, and R258—were not critical for RNase activity. The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the “secondary” chain’s residue K184 is positioned near the “primary” chain’s catalytic site (Fig. 4). In contrast, R214 is important for oligomerization of the PIN domain and the “secondary” chain’s residue R214 is also positioned near the “primary” chain’s active site within the dimer interface. It should be noted that the putative-RNA binding residues K184 and R214 are unique to Regnase-1 among PIN domains.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>12527</offset><text>Molecular mechanism of target mRNA cleavage by the PIN dimer</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12588</offset><text>Our mutational experiments indicated that the observed dimer is functional and that the role of the secondary PIN domain is to position Regnase-1-unique RNA binding residues near the active site of the primary PIN domain. If this model is correct, then we reasoned that a catalytically inactive PIN and a PIN lacking the putative RNA-binding residues ought to be inactive in isolation but become active when mixed together. In order to test this hypothesis, we performed in vitro cleavage assays using combinations of Regnase-1 mutants that had no or decreased RNase activities by themselves (Fig. 5). One group consisted of catalytically active PIN domains with mutation of basic residues found in the previous section to confer decreased RNase activity (Fig. 4). These were paired with a DDNN mutant that had no RNase activity by itself. When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). When we compared the fluorescence intensity of uncleaved IL-6 mRNA, basic residue mutants W182A, K184A, R214A, and R220A were rescued upon addition of the DDNN mutant (Fig. 5b). Consistently, when we compared the fluorescence intensity of the uncleaved Regnase-1 mRNA, basic residue mutants K184A and R214A were rescued upon addition of the DDNN mutant (Fig. 5c). Rescue of K184A and R214A by the DDNN mutant was also confirmed by a significant increase in the cleaved products. This is particularly significant because the side chains of K184 and R214 in the primary PIN are oriented away from their own catalytic center, while those in the secondary PIN face toward the catalytic center of the primary PIN. R214 is an important residue for dimer formation as shown in Fig. 2, therefore, R214A in the secondary PIN cannot dimerize. According to the proposed model, an R214A PIN domain can only form a dimer when the DDNN PIN acts as the secondary PIN. Taken together, the rescue experiments above support the proposed model in which the head-to-tail dimer is functional in vitro.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>14859</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>14870</offset><text>We determined the individual domain structures of Regnase-1 by NMR and X-ray crystallography. Although the function of the CTD remains elusive, we revealed the functions of the NTD, PIN, and ZF domains. A Regnase-1 construct consisting of PIN and ZF domains derived from Mus musculus was crystallized; however, the electron density of the ZF domain was low, indicating that the ZF domain is highly mobile in the absence of target mRNA or possibly other protein-protein interactions. Our NMR experiments confirmed direct binding of the ZF domain to IL-6 mRNA with a Kd of 10 ± 1.1 μM. Furthermore, an in vitro gel shift assay indicated that Regnase-1 containing the ZF domain enhanced target mRNA-binding, but the protein-RNA complex remained in the bottom of the well without entering into the polyacrylamide gel. These results indicate that Regnase-1 directly binds to RNA and precipitates under such experimental conditions. Due to this limitation, it is difficult to perform further structural analyses of mRNA-Regnase-1 complexes by X-ray crystallography or NMR.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>15945</offset><text>The previously reported crystal structure of the Regnase-1 PIN domain derived from Homo sapiens is nearly identical to the one derived from Mus musculus in this study, with a backbone RMSD of 0.2 Å. The amino acid sequences corresponding to PIN (residues 134–295) are the two non-identical residues are substituted with similar amino acids. Both the mouse and human PIN domains form head-to-tail oligomers in three distinct crystal forms. Rao and co-workers previously argued that PIN dimerization is likely to be a crystallographic artifact with no physiological significance, since monomers were dominant in their analytical ultra-centrifugation experiments. In contrast, our gel filtration data, mutational analyses, and NMR spectra all indicate that the PIN domain forms a head-to-tail dimer in solution in a manner similar to the crystal structure. This inconsistency might be due to difference in the analytical methods and/or protein concentrations used in each experiment, since the oligomer formation of PIN was dependent on the protein concentration in our study.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>17024</offset><text>Single mutations to residues involved in the putative oligomeric interaction of PIN monomerized as expected and these mutants lost their RNase activity as well. Since the NMR spectra of monomeric mutants overlaps with those of the oligomeric forms, it is unlikely that the tertiary structure of the monomeric mutants were affected by the mutations. (Supplementary Fig. 4b,c). Based on these observations, we concluded that PIN-PIN dimer formation is critical for Regnase-1 RNase activity in vitro. Within the crystal structure of the PIN dimer, the Regnase-1 specific basic regions in both the “primary” and “secondary” PINs are located around the catalytic site of the primary PIN (Supplementary Fig. 6). Moreover, our structure-based mutational analyses showed these two Regnase-1 specific basic regions were essential for target mRNA cleavage in vitro.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>17888</offset><text>The cleavage assay also showed that the NTD is crucial for efficient mRNA cleavage. Moreover, we found that the NTD associates with the oligomeric surface of the primary PIN, docking to a helix that harbors its catalytic residues (Figs 2b and 3a). Taken together, this suggests that the NTD and the PIN domain compete for a common binding site. The affinity of the domain-domain interaction between two PIN domains (Kd = ~10−4 M) is similar to that of the NTD-PIN (Kd = 110 ± 5.8 μM) interactions; however, the covalent connection corresponding to residues 90–133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1. While further analyses are necessary to prove this point, our preliminary docking and molecular dynamics simulations indicate that NTD-binding rearranges the catalytic residues of the PIN domain toward an active conformation suitable for binding Mg2+. In this context, it is interesting that, in response to TCR stimulation, Malt1 cleaves Regnase-1 at R111 to control immune responses in vivo. This result is consistent with a model in which the NTD acts as an enhancer, and cleavage of the linker lowers enzymatic activity dramatically.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>19145</offset><text>Based on these structural and functional analyses of Regnase-1 domain-domain interactions, we performed docking simulations of the NTD, PIN dimer, and IL-6 mRNA. We incorporated information from the cleavage site of IL-6 mRNA in vitro is indicated by denaturing polyacrylamide gel electrophoresis (Supplementary Fig. 7a,b). The docking result revealed multiple RNA binding modes that satisfied the experimental results in vitro (Supplementary Fig. 7c,d), however, it should be noted that, in vivo, there would likely be many other RNA-binding proteins that would protect loop regions from cleavage by Regnase-1.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>19757</offset><text>The overall model of regulation of Regnase-1 RNase activity through domain-domain interactions in vitro is summarized in Fig. 6. In the absence of target mRNA, the PIN domain forms head-to-tail oligomers at high concentration. A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). While further investigations on the domain-domain interactions of Regnase-1 in vivo are necessary, these intramolecular and intermolecular domain interactions of Regnase-1 appear to structurally constrain Regnase-1activity, which, in turn, enables tight regulation of immune responses.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>20555</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>20563</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20599</offset><text>The DNA fragment encoding Regnase-1 derived from Mus musculus was cloned into pGEX6p vector (GE Healthcare). All the mutants were generated by PCR-mediated site-directed mutagenesis and confirmed by the DNA sequence analyses. As a catalytically deficient mutant, both Asp226 and Asp244 at the catalytic center of PIN were mutated to Asn, which is referred to as DDNN mutant. Regnase-1 was expressed at 16 °C using the Escherichia coli RosettaTM(DE3)pLysS strain. After purification with a GST-affinity resin, an N-terminal GST tag was digested by HRV-3 C protease. NTD was further purified by gel filtration chromatography using a HiLoad 16/60 Superdex 75 pg (GE Healthcare). The other domains were further purified by cation exchange chromatography using Resource S (GE Healthcare), followed by gel filtration chromatography using a HiLoad 16/60 Superdex 75 pg (GE Healthcare). Uniformly 15N or 13C, 15N-double labeled proteins for NMR experiments were prepared by growing E. coli host in M9 minimal medium containing 15NH4Cl, unlabeled glucose and 15N CELTONE® Base Powder (CIL) or 15NH4Cl, 13C6-glucose, and13C, 15N CELTONE® Base Powder (CIL), respectively.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>21771</offset><text>X-ray crystallography</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>21793</offset><text>Crystallization was performed using the sitting drop vapor diffusion method at 20 °C and two crystal forms (I and II) were obtained. In the case of form I crystals, drops (0.5 μl) of 6 mg/ml selenomethionine-labeled Regnase-1 PIN-ZF (residues 134–339 derived from Mus musculus) in 20 mM HEPES-NaOH (pH 6.8), 200 mM NaCl and 5 mM DTT were mixed with reservoir solution consisting of 1 M (NH4)2HPO4, 200 mM NaCl and 100 mM sodium citrate (pH 5.5) whereas in the case of form II crystals, drops (0.5 μl) of 6 mg/ml native Regnase-1 PIN-ZF (residues 134–339) in 20 mM HEPES-NaOH (pH 6.8), 200 mM NaCl and 5 mM DTT were mixed with reservoir solution consisting of 1.7 M NaCl and 100 mM HEPES-NaOH (pH 7.0). Diffraction data were collected at a Photon Factory Advanced Ring beamline NE3A (form I) or at a SPring-8 beamline BL41XU (form II), and were processed with HKL2000. The structure of the form I crystal was determined by the multiple anomalous dispersion (MAD) method. Nine Se sites were found using the program SOLVE; however, the electron density obtained by MAD phases calculated using SOLVE was not good enough to build a model even after density modification using the program RESOLVE. Then the program CNS was used to find additional three Se sites and calculate MAD phases using 12 Se sites. The electron density after density modification using CNS was good enough to build a model. Structure of the form II crystal was determined by the molecular replacement method using CNS and using the structure of the form I crystal as a search model. For all structures, further model building was performed manually with COOT, and TLS and restrained refinement using isotropic individual B factors was performed with REFMAC5 in the CCP4 program suite. Crystallographic parameters are summarized in Supplementary Table 1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>23654</offset><text>NMR measurements</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>23671</offset><text>All NMR experiments were carried out at 298 K on Inova 500-MHz, 600-MHz, and 800-MHz spectrometer (Agilent). The NMR data were processed using the NMRPipe, the Olivia (fermi.pharm.hokudai.ac.jp/olivia/), and the Sparky program (Sparky3, University of California, San Francisco).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>23952</offset><text>For structure calculation, NOE distance restraints were obtained from 3D 15N-NOESY-HSQC (100 ms mixing time for the NTD, 75 ms mixing time for the ZF domain and the CTD) and 13C-NOESY-HSQC spectra (100 ms mixing time for the NTD, 75 ms mixing time for the ZF domain and the CTD). The NMR structures were determined using the CANDID/CYANA2.1. Dihedral restraints were derived from backbone chemical shifts using TALOS.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24378</offset><text>For the domain-domain interaction analyses between the NTD and the PIN domain, 1H-15N HSQC spectra of uniformly 15N-labeled proteins in the concentration of 100 μM were obtained in the presence of 3 or 6 molar equivalents of unlabeled proteins.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24626</offset><text>Preparation of RNAs</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24646</offset><text>The fluorescently labeled RNAs at the 5′-end by 6-FAM were purchased from SIGMA-ALDORICH. The RNA sequences used in this study were shown below.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24793</offset><text>IL-6 mRNA 3′UTR (82–106): 5′-UGUUGUUCUCUACGAAGAACUGACA-3′ (25 nts)</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24868</offset><text>Regnase-1 mRNA 3′UTR (191–211): 5′- CUGUUGAUACACAUUGUAUCU-3′ (21 nts)</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24946</offset><text>Electrophoretic mobility shift assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24983</offset><text>Catalytically deficient Regnase-1 proteins, containing DDNN mutations, and 5′-terminally 6-FAM labeled RNAs were incubated in the RNA-binding buffer (20 mM HEPES-NaOH (pH 6.8), 150 mM NaCl, 1 mM DTT, 10% glycerol (v/v), and 0.1% NP-40 (v/v)) at 4 °C for 30 minutes, then analyzed by non-denaturing polyacrylamide gel electrophoresis. The electrophoreses were performed at 4 °C using the 7.5% polyacrylamide (w/v) gel (monomer : bis = 29 : 1) in the electrophoresis buffer (25 mM Tris-HCl (pH 7.5) and 200 mM glycine). The fluorescence of 6-FAM labeled RNA was directly detected at the excitation wavelength of 460 nm with a fluorescence filter (Y515-Di) using a fluoroimaging analyzer (LAS-4000 (FUJIFILM)). The fluorescence intensity of each sample was quantified using ImageJ software.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>25797</offset><text>In vitro RNA cleavage assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>25825</offset><text>Regnase-1 (2 μM) and 5′-terminally 6-FAM labeled RNA (1 μM) were incubated in the RNA-cleavage buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, and 1 mM DTT) at 37 °C. For the assay using combinations of Regnase-1 mutants, equimolar amounts of Regnase-1 mutants (2 μM each) were mixed with fluorescently labeled RNA (1 μM). After incubation for 30–120 minutes, the reaction was stopped by the addition of 1.5-fold volume of denaturing buffer containing 8 M urea and 100 mM EDTA, and samples were boiled. The electrophoreses were performed at room temperature using the 8 M urea containing denaturing gel with 20% polyacrylamide (w/v) (monomer : bis = 19 : 1) in 0.5 × TBE as the electrophoresis buffer.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>26581</offset><text>Docking calculations</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26602</offset><text>For docking NTD to PIN, OSCAR-star was first used to rebuild sidechains in the head-to-tail PIN dimer. Docking was carried out by surFit (http://sysimm.ifrec.osaka-u.ac.jp/docking/main/) with restraints obtained from NMR data (Fig. 3a,b) as follows. NTD: R56, L58, G59, V86, K87, H88; PIN: V177, F210, T211, R214, F228, I229, V230, K231, L232, F234, D235, S236. Top-scoring model was selected.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26996</offset><text>For docking IL-6 mRNA 3′UTR to the PIN dimer, each domain of the PIN dimer structure was superimposed onto the PIN dimer of the human X-ray structure (PDB ID: 3V34) in order to graft both water molecules and Mg2+ ions to the mouse model. Each IL-6 representative structure was submitted to the HADDOCK 2.0 server, for total of 10 independent jobs. In order to be consistent with the cleavage assay, active residues consisted of all nucleotides in RNA, Mg2+ and W182, R183, K184, R188, R214, R215, K219, R220, and R247 in the protein. Docked models were selected based on the following criteria: one heavy atom within 7, 8, or 9th nucleotide from the 5′ end was &lt;5 Å from the Mg2+ ion on the primary PIN. Further classification was done manually in order to divide the selected models into two clusters.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>27806</offset><text>Additional Information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>27829</offset><text>Accession codes: The crystal structure of the Regnase-1 PIN domain has been deposited in the Protein Data Bank (accession codes: 5H9V (Form I) and 5H9W (Form II)). The chemical shift assignments of the NTD, the ZF domain, and the CTD have been deposited at Biological Magnetic Resonance Bank (accession codes: 25718, 25719, and 25720, respectively), and the coordinates for the ensemble have been deposited in the Protein Data Bank (accession codes: 2N5J, 2N5K, and 2N5L, respectively). </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28317</offset><text>How to cite this article: Yokogawa, M. et al. Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions. Sci. Rep. 6, 22324; doi: 10.1038/srep22324 (2016).</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>28516</offset><text>Supplementary Material</text></passage><passage><infon key="fpage">783</infon><infon key="lpage">801</infon><infon key="name_0">surname:Akira;given-names:S.</infon><infon key="name_1">surname:Uematsu;given-names:S.</infon><infon key="name_2">surname:Takeuchi;given-names:O.</infon><infon key="pub-id_pmid">16497588</infon><infon key="section_type">REF</infon><infon key="source">Cell,</infon><infon key="type">ref</infon><infon key="volume">124</infon><infon key="year">2006</infon><offset>28539</offset><text>Pathogen recognition and innate immunity.</text></passage><passage><infon key="fpage">819</infon><infon key="lpage">26</infon><infon key="name_0">surname:Medzhitov;given-names:R.</infon><infon key="pub-id_pmid">17943118</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">449</infon><infon key="year">2007</infon><offset>28581</offset><text>Recognition of microorganisms and activation of the immune response</text></passage><passage><infon key="fpage">353</infon><infon key="lpage">89</infon><infon key="name_0">surname:Beutler;given-names:B.</infon><infon key="pub-id_pmid">16551253</infon><infon key="section_type">REF</infon><infon key="source">Annu Rev Immunol</infon><infon key="type">ref</infon><infon key="volume">24</infon><infon key="year">2006</infon><offset>28649</offset><text>Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large</text></passage><passage><infon key="fpage">1185</infon><infon key="lpage">90</infon><infon key="name_0">surname:Matsushita;given-names:K.</infon><infon key="pub-id_pmid">19322177</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">458</infon><infon key="year">2009</infon><offset>28737</offset><text>Zc3h12a is an RNase essential for controlling immune responses by regulating mRNA decay</text></passage><passage><infon key="fpage">7386</infon><infon key="lpage">99</infon><infon key="name_0">surname:Mizgalska;given-names:D.</infon><infon key="pub-id_pmid">19909337</infon><infon key="section_type">REF</infon><infon key="source">FEBS J</infon><infon key="type">ref</infon><infon key="volume">276</infon><infon key="year">2009</infon><offset>28825</offset><text>Interleukin-1-inducible MCPIP protein has structural and functional properties of RNase and participates in degradation of IL-1beta mRNA</text></passage><passage><infon key="fpage">e49841</infon><infon key="name_0">surname:Li;given-names:M.</infon><infon key="pub-id_pmid">23185455</infon><infon key="section_type">REF</infon><infon key="source">PLoS One,</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2012</infon><offset>28962</offset><text>MCPIP1 down-regulates IL-2 expression through an ARE-independent pathway</text></passage><passage><infon key="fpage">708</infon><infon key="lpage">13</infon><infon key="name_0">surname:Uehata;given-names:T.</infon><infon key="name_1">surname:Akira;given-names:S.</infon><infon key="pub-id_pmid">23500036</infon><infon key="section_type">REF</infon><infon key="source">Biochim Biophys Acta</infon><infon key="type">ref</infon><infon key="volume">1829</infon><infon key="year">2013</infon><offset>29035</offset><text>mRNA degradation by the endoribonuclease Regnase-1/ZC3H12a/MCPIP-1</text></passage><passage><infon key="fpage">1167</infon><infon key="lpage">75</infon><infon key="name_0">surname:Iwasaki;given-names:H.</infon><infon key="pub-id_pmid">22037600</infon><infon key="section_type">REF</infon><infon key="source">Nat Immunol</infon><infon key="type">ref</infon><infon key="volume">12</infon><infon key="year">2011</infon><offset>29102</offset><text>The IκB kinase complex regulates the stability of cytokine-encoding mRNA induced by TLR-IL-1R by controlling degradation of regnase-1</text></passage><passage><infon key="fpage">6337</infon><infon key="lpage">46</infon><infon key="name_0">surname:Liang;given-names:J.</infon><infon key="pub-id_pmid">18178554</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">283</infon><infon key="year">2008</infon><offset>29240</offset><text>A novel CCCH-zinc finger protein family regulates proinflammatory activation of macrophages</text></passage><passage><infon key="fpage">903</infon><infon key="lpage">10</infon><infon key="name_0">surname:Xu;given-names:J.</infon><infon key="name_1">surname:Fu;given-names:S.</infon><infon key="name_2">surname:Peng;given-names:W.</infon><infon key="name_3">surname:Rao;given-names:Z.</infon><infon key="pub-id_pmid">23132255</infon><infon key="section_type">REF</infon><infon key="source">Protein Cell</infon><infon key="type">ref</infon><infon key="volume">3</infon><infon key="year">2012</infon><offset>29332</offset><text>MCP-1-induced protein-1, an immune regulator</text></passage><passage><infon key="fpage">6957</infon><infon key="lpage">65</infon><infon key="name_0">surname:Xu;given-names:J.</infon><infon key="pub-id_pmid">22561375</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res</infon><infon key="type">ref</infon><infon key="volume">40</infon><infon key="year">2012</infon><offset>29377</offset><text>Structural study of MCPIP1 N-terminal conserved domain reveals a PIN-like RNase</text></passage><passage><infon key="fpage">685</infon><infon key="lpage">93</infon><infon key="name_0">surname:Hake;given-names:L. E.</infon><infon key="name_1">surname:Mendez;given-names:R.</infon><infon key="name_2">surname:Richter;given-names:J. D.</infon><infon key="pub-id_pmid">9447964</infon><infon key="section_type">REF</infon><infon key="source">Mol Cell Biol</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">1998</infon><offset>29457</offset><text>Specificity of RNA binding by CPEB: requirement for RNA recognition motifs and a novel zinc finger</text></passage><passage><infon key="fpage">9606</infon><infon key="lpage">13</infon><infon key="name_0">surname:Lai;given-names:W. S.</infon><infon key="name_1">surname:Kennington;given-names:E. A.</infon><infon key="name_2">surname:Blackshear;given-names:P. J.</infon><infon key="pub-id_pmid">11782475</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">277</infon><infon key="year">2002</infon><offset>29556</offset><text>Interactions of CCCH zinc finger proteins with mRNA: non-binding tristetraprolin mutants exert an inhibitory effect on degradation of AU-rich element-containing mRNAs</text></passage><passage><infon key="fpage">257</infon><infon key="lpage">64</infon><infon key="name_0">surname:Hudson;given-names:B. P.</infon><infon key="name_1">surname:Martinez-Yamout;given-names:M. A.</infon><infon key="name_2">surname:Dyson;given-names:H. J.</infon><infon key="name_3">surname:Wright;given-names:P. E.</infon><infon key="pub-id_pmid">14981510</infon><infon key="section_type">REF</infon><infon key="source">Nat Struct Mol Biol</infon><infon key="type">ref</infon><infon key="volume">11</infon><infon key="year">2004</infon><offset>29723</offset><text>Recognition of the mRNA AU-rich element by the zinc finger domain of TIS11d</text></passage><passage><infon key="fpage">367</infon><infon key="lpage">73</infon><infon key="name_0">surname:Hall;given-names:T. M.</infon><infon key="pub-id_pmid">15963892</infon><infon key="section_type">REF</infon><infon key="source">Curr Opin Struct Biol</infon><infon key="type">ref</infon><infon key="volume">15</infon><infon key="year">2005</infon><offset>29799</offset><text>Multiple modes of RNA recognition by zinc finger proteins</text></passage><passage><infon key="fpage">1177</infon><infon key="lpage">85</infon><infon key="name_0">surname:Zhou;given-names:L.</infon><infon key="pub-id_pmid">16574901</infon><infon key="section_type">REF</infon><infon key="source">Circ Res</infon><infon key="type">ref</infon><infon key="volume">98</infon><infon key="year">2006</infon><offset>29857</offset><text>Monocyte chemoattractant protein-1 induces a novel transcription factor that causes cardiac myocyte apoptosis and ventricular dysfunction</text></passage><passage><infon key="fpage">2959</infon><infon key="lpage">73</infon><infon key="name_0">surname:Liang;given-names:J.</infon><infon key="pub-id_pmid">21115689</infon><infon key="section_type">REF</infon><infon key="source">J Exp Med</infon><infon key="type">ref</infon><infon key="volume">207</infon><infon key="year">2010</infon><offset>29995</offset><text>MCP-induced protein 1 deubiquitinates TRAF proteins and negatively regulates JNK and NF-kappaB signaling</text></passage><passage><infon key="fpage">W545</infon><infon key="lpage">9</infon><infon key="name_0">surname:Holm;given-names:L.</infon><infon key="name_1">surname:Rosenström;given-names:P.</infon><infon key="pub-id_pmid">20457744</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res</infon><infon key="type">ref</infon><infon key="volume">38</infon><infon key="year">2010</infon><offset>30100</offset><text>Dali server: conservation mapping in 3D</text></passage><passage><infon key="fpage">1036</infon><infon key="lpage">49</infon><infon key="name_0">surname:Uehata;given-names:T.</infon><infon key="pub-id_pmid">23706741</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">153</infon><infon key="year">2013</infon><offset>30140</offset><text>Malt1-induced cleavage of regnase-1 in CD4(+) helper T cells regulates immune activation</text></passage><passage><infon key="fpage">307</infon><infon key="lpage">326</infon><infon key="name_0">surname:Otwinowski;given-names:Z.</infon><infon key="name_1">surname:Minor;given-names:W.</infon><infon key="section_type">REF</infon><infon key="source">Methods Enzymol</infon><infon key="type">ref</infon><infon key="volume">276</infon><infon key="year">1997</infon><offset>30229</offset><text>Processing of X-ray Diffraction Data Collected in Oscillation Mode</text></passage><passage><infon key="fpage">849</infon><infon key="lpage">61</infon><infon key="name_0">surname:Terwilliger;given-names:T. C.</infon><infon key="name_1">surname:Berendzen;given-names:J.</infon><infon key="pub-id_pmid">10089316</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">55</infon><infon key="year">1999</infon><offset>30296</offset><text>Automated MAD and MIR structure solution</text></passage><passage><infon key="fpage">965</infon><infon key="lpage">72</infon><infon key="name_0">surname:Terwilliger;given-names:T. C.</infon><infon key="pub-id_pmid">10944333</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">56</infon><infon key="year">2000</infon><offset>30337</offset><text>Maximum-likelihood density modification</text></passage><passage><infon key="fpage">905</infon><infon key="lpage">21</infon><infon key="name_0">surname:Brünger;given-names:A. T.</infon><infon key="pub-id_pmid">9757107</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">54</infon><infon key="year">1998</infon><offset>30377</offset><text>Crystallography &amp; NMR system: A new software suite for macromolecular structure determination</text></passage><passage><infon key="fpage">486</infon><infon key="lpage">501</infon><infon key="name_0">surname:Emsley;given-names:P.</infon><infon key="name_1">surname:Lohkamp;given-names:B.</infon><infon key="name_2">surname:Scott;given-names:W. G.</infon><infon key="name_3">surname:Cowtan;given-names:K.</infon><infon key="pub-id_pmid">20383002</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>30471</offset><text>Features and development of Coot</text></passage><passage><infon key="fpage">240</infon><infon key="lpage">55</infon><infon key="name_0">surname:Murshudov;given-names:G. N.</infon><infon key="name_1">surname:Vagin;given-names:A. A.</infon><infon key="name_2">surname:Dodson;given-names:E. J.</infon><infon key="pub-id_pmid">15299926</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">53</infon><infon key="year">1997</infon><offset>30504</offset><text>Refinement of macromolecular structures by the maximum-likelihood method</text></passage><passage><infon key="fpage">235</infon><infon key="lpage">42</infon><infon key="name_0">surname:Winn;given-names:M. D.</infon><infon key="pub-id_pmid">21460441</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">67</infon><infon key="year">2011</infon><offset>30577</offset><text>Overview of the CCP4 suite and current developments</text></passage><passage><infon key="fpage">277</infon><infon key="lpage">93</infon><infon key="name_0">surname:Delaglio;given-names:F.</infon><infon key="pub-id_pmid">8520220</infon><infon key="section_type">REF</infon><infon key="source">J Biomol NMR</infon><infon key="type">ref</infon><infon key="volume">6</infon><infon key="year">1995</infon><offset>30629</offset><text>NMRPipe: a multidimensional spectral processing system based on UNIX pipes</text></passage><passage><infon key="fpage">353</infon><infon key="lpage">78</infon><infon key="name_0">surname:Guntert;given-names:P.</infon><infon key="pub-id_pmid">15318003</infon><infon key="section_type">REF</infon><infon key="source">Methods Mol Biol</infon><infon key="type">ref</infon><infon key="volume">278</infon><infon key="year">2004</infon><offset>30704</offset><text>Automated NMR structure calculation with CYANA</text></passage><passage><infon key="fpage">289</infon><infon key="lpage">302</infon><infon key="name_0">surname:Cornilescu;given-names:G.</infon><infon key="name_1">surname:Delaglio;given-names:F.</infon><infon key="name_2">surname:Bax;given-names:A.</infon><infon key="pub-id_pmid">10212987</infon><infon key="section_type">REF</infon><infon key="source">J Biomol NMR</infon><infon key="type">ref</infon><infon key="volume">13</infon><infon key="year">1999</infon><offset>30751</offset><text>Protein backbone angle restraints from searching a database for chemical shift and sequence homology</text></passage><passage><infon key="fpage">2913</infon><infon key="lpage">4</infon><infon key="name_0">surname:Liang;given-names:S.</infon><infon key="name_1">surname:Zheng;given-names:D.</infon><infon key="name_2">surname:Zhang;given-names:C.</infon><infon key="name_3">surname:Standley;given-names:D. M.</infon><infon key="pub-id_pmid">21873640</infon><infon key="section_type">REF</infon><infon key="source">Bioinformatics</infon><infon key="type">ref</infon><infon key="volume">27</infon><infon key="year">2011</infon><offset>30852</offset><text>Fast and accurate prediction of protein side-chain conformations</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>30917</offset><text>Author Contributions F.I. supervised the overall project. M.Y., T.T., Y.E., D.M.S., O.T., S.A. and F.I. designed the research; M.Y. and T.T. performed the research; M.Y., T.T., N.N.N., H.K., K.Y., D.M.S. and F.I. analyzed the data; and M.Y., N.N.N., H.K., K.Y., D.M.S. and F.I. wrote the paper. All authors reviewed the manuscript.</text></passage><passage><infon key="file">srep22324-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>31249</offset><text>Structural and functional analyses of Regnase-1.</text></passage><passage><infon key="file">srep22324-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>31298</offset><text>(a) Domain architecture of Regnase-1. (b) Solution structure of the NTD. (c) Crystal structure of the PIN domain. Catalytic Asp residues were shown in sticks. (d) Solution structure of the ZF domain. Three Cys residues and one His residue responsible for Zn2+-binding were shown in sticks. (e) Solution structure of the CTD. All the structures were colored in rainbow from N-terminus (blue) to C-terminus (red). (f) In vitro gel shift binding assay between Regnase-1 and IL-6 mRNA. Fluorescence intensity of the free IL-6 in each sample was indicated as the percentage against that in the absence of Regnase-1. (g) Binding of Regnase-1 and IL-6 mRNA was plotted. The percentage of the bound IL-6 was calculated based on the fluorescence intensities of the free IL-6 quantified in (f). (h) In vitro cleavage assay of Regnase-1 to IL-6 mRNA. Fluorescence intensity of the uncleaved IL-6 mRNA was indicated as the percentage against that in the absence of Regnase-1.</text></passage><passage><infon key="file">srep22324-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>32262</offset><text>Head-to-tail oligomer formation of the PIN domain is crucial for the RNase activity of Regnase-1.</text></passage><passage><infon key="file">srep22324-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>32360</offset><text>(a) Gel filtration analyses of the PIN domain. Elution volumes of the standard marker proteins were indicated by arrows at the upper part. (b) Dimer structure of the PIN domain. Two PIN molecules in the crystal were colored white and green, respectively. Catalytic residues and mutated residues were shown in sticks. Residues important for the oligomeric interaction were colored red, while R215 that was dispensable for the oligomeric interaction was colored blue. (c) RNase activity of monomeric mutants for IL-6 mRNA was analyzed.</text></passage><passage><infon key="file">srep22324-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>32894</offset><text>Domain-domain interaction between the NTD and the PIN domain.</text></passage><passage><infon key="file">srep22324-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>32956</offset><text>(a) NMR analyses of the NTD-binding to the PIN domain. The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red on the surface and ribbon structure of the PIN domain (right). Pro and the residues without analysis were colored black and gray, respectively. (b) NMR analyses of the PIN-binding to the NTD. The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red, yellow, or green on the surface and ribbon structure of the NTD. S62 was colored gray and excluded from the analysis, due to low signal intensity. (c) Docking model of the NTD and the PIN domain. The NTD and the PIN domain are shown in cyan and white, respectively. Residues in close proximity (&lt;5 Å) to each other in the docking structure were colored yellow. Catalytic residues of the PIN domain are shown in sticks, and the residues that exhibited significant chemical shift changes in (a,b) were labeled.</text></passage><passage><infon key="file">srep22324-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>33942</offset><text>Critical residues in the PIN domain for the RNase activity of Regnase-1.</text></passage><passage><infon key="file">srep22324-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>34015</offset><text>(a) In vitro cleavage assay of basic residue mutants for IL-6 mRNA. The results indicate mean ± SD of four independent experiments. (b)
+In vitro cleavage assay of basic residue mutants for Regnase-1 mRNA. The results indicate mean ± SD of three independent experiments. The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer structure. Mutated basic residues were shown in sticks and those with significantly reduced RNase activities were colored red or yellow.</text></passage><passage><infon key="file">srep22324-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>34541</offset><text>Heterodimer formation by combination of the Regnase-1 basic residue mutants and the DDNN mutant restored the RNase activity.</text></passage><passage><infon key="file">srep22324-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>34666</offset><text>(a) Cartoon representation of the concept of the experiment. (b) In vitro cleavage assay of Regnase-1 for IL-6 mRNA. (c) In vitro cleavage assay of Regnase-1 for Regnase-1 mRNA. The results indicate mean ± SD of three independent experiments. The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer. The mutations whose RNase activities were not increased in the presence of DDNN mutant were colored in blue on the primary PIN. The mutations whose RNase activities were restored in the presence of DDNN mutant were colored in red or yellow on the primary PIN.</text></passage><passage><infon key="file">srep22324-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>35284</offset><text>Schematic representation of regulation of the Regnase-1 catalytic activity through the domain-domain interactions.</text></passage></document></collection>
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+<collection><source>PMC</source><date>20201217</date><key>pmc.key</key><document><id>4784909</id><infon key="license">CC BY</infon><passage><infon key="alt-title">The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle</infon><infon key="article-id_doi">10.1371/journal.pbio.1002399</infon><infon key="article-id_pmc">4784909</infon><infon key="article-id_pmid">26959993</infon><infon key="article-id_publisher-id">PBIOLOGY-D-15-02496</infon><infon key="elocation-id">e1002399</infon><infon key="issue">3</infon><infon key="license">This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.</infon><infon key="name_0">surname:Erbilgin;given-names:Onur</infon><infon key="name_1">surname:Sutter;given-names:Markus</infon><infon key="name_2">surname:Kerfeld;given-names:Cheryl A.</infon><infon key="name_3">surname:Petsko;given-names:Gregory A.</infon><infon key="notes">PDB files are available from the Protein Data Bank under accession codes 5CUO and 5CUP.</infon><infon key="section_type">TITLE</infon><infon key="title">Data Availability</infon><infon key="type">front</infon><infon key="volume">14</infon><infon key="year">2016</infon><offset>0</offset><text>The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>70</offset><text>Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). Here, we report two high-resolution PduL crystal structures with bound substrates. The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1219</offset><text>This study describes the structure of a novel phosphotransacylase enzyme that facilitates the recycling of the essential cofactor acetyl-CoA within a bacterial organelle and discusses the properties of the enzyme's active site and how it is packaged into the organelle.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>1489</offset><text>Author Summary</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1504</offset><text>In metabolism, molecules with “high-energy” bonds (e.g., ATP and Acetyl~CoA) are critical for both catabolic and anabolic processes. Accordingly, the retention of these bonds during biochemical transformations is incredibly important. The phosphotransacylase (Pta) enzyme catalyzes the conversion between acyl-CoA and acyl-phosphate. This reaction directly links an acyl-CoA with ATP generation via substrate-level phosphorylation, producing short-chain fatty acids (e.g., acetate), and also provides a path for short-chain fatty acids to enter central metabolism. Due to this key function, Pta is conserved across the bacterial kingdom. Recently, a new type of phosphotransacylase was described that shares no evolutionary relation to Pta. This enzyme, PduL, is exclusively associated with organelles called bacterial microcompartments, which are used to catabolize various compounds. Not only does PduL facilitate substrate level phosphorylation, but it also is critical for cofactor recycling within, and product efflux from, the organelle. We solved the structure of this convergent phosphotransacylase and show that it is completely structurally different from Pta, including its active site architecture. We also discuss features of the protein important to its packaging in the organelle.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>2804</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2817</offset><text>Bacterial Microcompartments (BMCs) are organelles that encapsulate enzymes for sequential biochemical reactions within a protein shell. The shell is typically composed of three types of protein subunits, which form either hexagonal (BMC-H and BMC-T) or pentagonal (BMC-P) tiles that assemble into a polyhedral shell. The facets of the shell are composed primarily of hexamers that are typically perforated by pores lined with highly conserved, polar residues that presumably function as the conduits for metabolites into and out of the shell.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3360</offset><text>The vitamin B12-dependent propanediol-utilizing (PDU) BMC was one of the first functionally characterized catabolic BMCs; subsequently, other types have been implicated in the degradation of ethanolamine, choline, fucose, rhamnose, and ethanol, all of which produce different aldehyde intermediates (Table 1). More recently, bioinformatic studies have demonstrated the widespread distribution of BMCs among diverse bacterial phyla and grouped them into 23 different functional types. The reactions carried out in the majority of catabolic BMCs (also known as metabolosomes) fit a generalized biochemical paradigm for the oxidation of aldehydes (Fig 1). This involves a BMC-encapsulated signature enzyme that generates a toxic and/or volatile aldehyde that the BMC shell sequesters from the cytosol. The aldehyde is subsequently converted into an acyl-CoA by aldehyde dehydrogenase, which uses NAD+ and CoA as cofactors. These two cofactors are relatively large, and their diffusion across the protein shell is thought to be restricted, necessitating their regeneration within the BMC lumen. NAD+ is recycled via alcohol dehydrogenase, and CoA is recycled via phosphotransacetylase (PTAC) (Fig 1). The final product of the BMC, an acyl-phosphate, can then be used to generate ATP via acyl kinase, or revert back to acyl-CoA by Pta for biosynthesis. Collectively, the aldehyde and alcohol dehydrogenases, as well as the PTAC, constitute the common metabolosome core.</text></passage><passage><infon key="file">pbio.1002399.g001.jpg</infon><infon key="id">pbio.1002399.g001</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>4825</offset><text>General biochemical model of aldehyde-degrading BMCs (metabolosomes) illustrating the common metabolosome core enzymes and reactions.</text></passage><passage><infon key="file">pbio.1002399.g001.jpg</infon><infon key="id">pbio.1002399.g001</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>4959</offset><text>Substrates and cofactors involving the PTAC reaction are shown in red; other substrates and enzymes are shown in black, and other cofactors are shown in gray.</text></passage><passage><infon key="file">pbio.1002399.t001.xml</infon><infon key="id">pbio.1002399.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>5118</offset><text>Characterized and predicted catabolic BMC (metabolosome) types that represent the aldehyde-degrading paradigm (for definition of types see Kerfeld and Erbilgin).</text></passage><passage><infon key="file">pbio.1002399.t001.xml</infon><infon key="id">pbio.1002399.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;colgroup span=&quot;1&quot;&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;/colgroup&gt;&lt;thead&gt;&lt;tr&gt;&lt;th align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Name&lt;/th&gt;&lt;th align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PTAC Type&lt;/th&gt;&lt;th align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Sequestered Aldehyde&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PDU&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t001fn001&quot;&gt;*&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PduL&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;propionaldehyde&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;EUT1&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PTA_PTB&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;acetaldehyde&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;EUT2&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PduL&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;acetaldehyde&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ETU&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;None&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;acetaldehyde&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;GRM1/CUT&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PduL&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;acetaldehyde&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;GRM2&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PduL&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;acetaldehyde&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;GRM3&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t001fn001&quot;&gt;*&lt;/xref&gt;,4&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PduL&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;propionaldehyde&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;GRM5/GRP&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PduL&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;propionaldehyde&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PVM&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t001fn001&quot;&gt;*&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PduL&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;lactaldehyde&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;RMM1,2&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;None&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;unknown&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SPU&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PduL&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;unknown&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>5280</offset><text>Name	PTAC Type	Sequestered Aldehyde	 	PDU*	PduL	propionaldehyde	 	EUT1	PTA_PTB	acetaldehyde	 	EUT2	PduL	acetaldehyde	 	ETU	None	acetaldehyde	 	GRM1/CUT	PduL	acetaldehyde	 	GRM2	PduL	acetaldehyde	 	GRM3*,4	PduL	propionaldehyde	 	GRM5/GRP	PduL	propionaldehyde	 	PVM*	PduL	lactaldehyde	 	RMM1,2	None	unknown	 	SPU	PduL	unknown	 	</text></passage><passage><infon key="file">pbio.1002399.t001.xml</infon><infon key="id">pbio.1002399.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>5607</offset><text>* PduL from these functional types of metabolosomes were purified in this study.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5688</offset><text>The activities of core enzymes are not confined to BMC-associated functions: aldehyde and alcohol dehydrogenases are utilized in diverse metabolic reactions, and PTAC catalyzes a key biochemical reaction in the process of obtaining energy during fermentation. The concerted functioning of a PTAC and an acetate kinase (Ack) is crucial for ATP generation in the fermentation of pyruvate to acetate (see Reactions 1 and 2). Both enzymes are, however, not restricted to fermentative organisms. They can also work in the reverse direction to activate acetate to the CoA-thioester. This occurs, for example, during acetoclastic methanogenesis in the archaeal Methanosarcina species.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6366</offset><text> Reaction 1: acetyl-S-CoA + Pi ←→ acetyl phosphate + CoA-SH (PTAC)</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6437</offset><text> Reaction 2: acetyl phosphate + ADP ←→ acetate + ATP (Ack)</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6500</offset><text>The canonical PTAC, Pta, is an ancient enzyme found in some eukaryotes and archaea, and widespread among the bacteria; 90% of the bacterial genomes in the Integrated Microbial Genomes database contain a gene encoding the PTA_PTB phosphotransacylase (Pfam domain PF01515). Pta has been extensively characterized due to its key role in fermentation. More recently, a second type of PTAC without any sequence homology to Pta was identified. This protein, PduL (Pfam domain PF06130), was shown to catalyze the conversion of propionyl-CoA to propionyl-phosphate and is associated with a BMC involved in propanediol utilization, the PDU BMC.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>7136</offset><text>Both pduL and pta genes can be found in genetic loci of functionally distinct BMCs, although the PduL type is much more prevalent, being found in all but one type of metabolosome locus: EUT1 (Table 1). Furthermore, in the Integrated Microbial Genomes Database, 91% of genomes that encode PF06130 also encode genes for shell proteins. As a member of the core biochemical machinery of functionally diverse aldehyde-oxidizing metabolosomes, PduL must have a certain level of substrate plasticity (see Table 1) that is not required of Pta, which has generally been observed to prefer acetyl-CoA. PduL from the PDU BMC of Salmonella enterica favors propionyl-CoA over acetyl-CoA, and it is likely that PduL orthologs in functionally diverse BMCs would have substrate preferences for other CoA derivatives. Another distinctive feature of BMC-associated PduL homologs is an N-terminal encapsulation peptide (EP) that is thought to “target” proteins for encapsulation by the BMC shell. EPs are frequently found on BMC-associated proteins and have been shown to interact with shell proteins. EPs have also been observed to cause proteins to aggregate, and this has recently been suggested to be functionally relevant as an initial step in metabolosome assembly, in which a multifunctional protein core is formed, around which the shell assembles.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>8478</offset><text>Of the three common metabolosome core enzymes, crystal structures are available for both the alcohol and aldehyde dehydrogenases. In contrast, the structure of PduL, the PTAC found in the vast majority of catabolic BMCs, has not been determined. This is a major gap in our understanding of metabolosome-encapsulated biochemistry and cofactor recycling. Structural information will be essential to working out how the core enzymes and their cofactors assemble and organize within the organelle lumen to enhance catalysis. Moreover, it will be useful for guiding efforts to engineer novel BMC cores for biotechnological applications.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>9110</offset><text>The primary structure of PduL homologs is subdivided into two PF06130 domains, each roughly 80 residues in length. No available protein structures contain the PF06130 domain, and homology searches using the primary structure of PduL do not return any significant results that would allow prediction of the structure. Moreover, the evident novelty of PduL makes its structure interesting in the context of convergent evolution of PTAC function; to-date, only the Pta active site and catalytic mechanism is known. Here we report high-resolution crystal structures of a PduL-type PTAC in both CoA- and phosphate-bound forms, completing our understanding of the structural basis of catalysis by the metabolosome common core enzymes. We propose a catalytic mechanism analogous but yet distinct from the ubiquitous Pta enzyme, highlighting the functional convergence of two enzymes with completely different structures and metal requirements. We also investigate the quaternary structures of three different PduL homologs and situate our findings in the context of organelle biogenesis in functionally diverse BMCs.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>10220</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10228</offset><text>Structure Determination of PduL</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10260</offset><text>We cloned, expressed, and purified three different PduL homologs from functionally distinct BMCs (Table 1): from the well-studied pdu locus in S. enterica Typhimurium LT2 (sPduL), from the recently characterized pvm locus in Planctomyces limnophilus (pPduL), and from the grm3 locus in Rhodopseudomonas palustris BisB18 (rPduL). While purifying full-length sPduL, we observed a tendency to aggregation as described previously, with a large fraction of the expressed protein found in the insoluble fraction in a white, cake-like pellet. Remarkably, after removing the N-terminal putative EP (27 amino acids), most of the sPduLΔEP protein was in the soluble fraction upon cell lysis. Similar differences in solubility were observed for pPduL and rPduL when comparing EP-truncated forms to the full-length protein, but none were quite as dramatic as for sPduL. We confirmed that all homologs were active (S1a and S1b Fig). Among these, we were only able to obtain diffraction-quality crystals of rPduL after removing the N-terminal putative EP (33 amino acids, also see Fig 2a) (rPduLΔEP). Truncated rPduLΔEP had comparable enzymatic activity to the full-length enzyme (S1a Fig).</text></passage><passage><infon key="file">pbio.1002399.g002.jpg</infon><infon key="id">pbio.1002399.g002</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>11449</offset><text>Structural overview of R. palustris PduL from the grm3 locus.</text></passage><passage><infon key="file">pbio.1002399.g002.jpg</infon><infon key="id">pbio.1002399.g002</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>11511</offset><text>(a) Primary and secondary structure of rPduL (tubes represent α-helices, arrows β-sheets and dashed line residues disordered in the structure. Blocks of ten residues are shaded alternatively black/dark gray. The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). Metal coordination residues are highlighted in light blue and CoA contacting residues in magenta, residues contacting the CoA of the other chain are also outlined. (b) Cartoon representation of the structure colored by domains and including secondary structure numbering. The N-and C-termini are in close proximity. Coenzyme A is shown in magenta sticks and Zinc (grey) as spheres.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12438</offset><text>We collected a native dataset from rPduLΔEP crystals diffracting to a resolution of 1.54 Å (Table 2). Using a mercury-derivative crystal form diffracting to 1.99 Å (Table 2), we obtained high quality electron density for model building and used the initial model to refine against the native data to Rwork/Rfree values of 18.9/22.1%. There are two PduL molecules in the asymmetric unit of the P212121 unit cell. We were able to fit all of the primary structure of PduLΔEP into the electron density with the exception of three amino acids at the N-terminus and two amino acids at the C-terminus (Fig 2a); the model is of excellent quality (Table 2). A CoA cofactor as well as two metal ions are clearly resolved in the density (for omit maps of CoA see S2 Fig).</text></passage><passage><infon key="file">pbio.1002399.t002.xml</infon><infon key="id">pbio.1002399.t002</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>13207</offset><text>Data collection and refinement statistics</text></passage><passage><infon key="file">pbio.1002399.t002.xml</infon><infon key="id">pbio.1002399.t002</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;colgroup span=&quot;1&quot;&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;/colgroup&gt;&lt;thead&gt;&lt;tr&gt;&lt;th align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;th align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PduL native&lt;/th&gt;&lt;th align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PduL mercury derivative&lt;/th&gt;&lt;th align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PduL phosphate soaked&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;bold&gt;Data collection&lt;/bold&gt;
+&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Space group&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;P 2&lt;sub&gt;1&lt;/sub&gt; 2&lt;sub&gt;1&lt;/sub&gt; 2&lt;sub&gt;1&lt;/sub&gt;
+&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;P 2&lt;sub&gt;1&lt;/sub&gt; 2&lt;sub&gt;1&lt;/sub&gt; 2&lt;sub&gt;1&lt;/sub&gt;
+&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;P 2&lt;sub&gt;1&lt;/sub&gt; 2&lt;sub&gt;1&lt;/sub&gt; 2&lt;sub&gt;1&lt;/sub&gt;
+&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Cell dimensions&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;italic&gt;a&lt;/italic&gt;, &lt;italic&gt;b&lt;/italic&gt;, &lt;italic&gt;c&lt;/italic&gt; (Å)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;57.7, 56.4, 150.4&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;55.6, 57.7, 150.2&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;57.1, 58.8, 136.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;italic&gt;α&lt;/italic&gt;, &lt;italic&gt;β&lt;/italic&gt;, &lt;italic&gt;γ&lt;/italic&gt; (°)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90, 90, 90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution (Å)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;31.4 − 1.54 (1.60 − 1.54)&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t002fn001&quot;&gt;*&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;35.3 − 1.99 (2.07 − 1.99)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;39.2 − 2.10 (2.21 − 2.10)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;italic&gt;R&lt;/italic&gt;
+&lt;sub&gt;merge&lt;/sub&gt;
+&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.169 (1.223)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.084 (0.299)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.122 (0.856)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;I/σ(I)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;12.9 (1.7)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;22.1 (7.1)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;12.6 (2.0)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Completeness (%)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.4 (94.4)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.3 (93.3)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100 (99.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Redundancy&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;13.9 (12.1)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;7.2 (7.0)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.5 (6.1)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;bold&gt;Refinement&lt;/bold&gt;
+&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution (Å)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;31.4 − 1.54 (1.60 − 1.54)&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t002fn001&quot;&gt;*&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;39.2 − 2.10 (2.18 − 2.1)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;No. reflections&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;72,698&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;27,554&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;italic&gt;R&lt;/italic&gt;
+&lt;sub&gt;work/&lt;/sub&gt;
+&lt;italic&gt;R&lt;/italic&gt;
+&lt;sub&gt;free&lt;/sub&gt; (%)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;18.9 (30.7) / 22.1 (34.7)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;17.5 (24.2) / 22.6 (30.0)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;No. atoms&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3,453&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3,127&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Protein&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2,841&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2,838&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Ligand/ion&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;24&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Water&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;512&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;265&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;B-factors&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;22.8&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;34.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Protein&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;21.5&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;24.3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Ligand/ion&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;21.9&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;40.6&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Water&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;30.3&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;37.9&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;R.m.s deviations&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Bond lengths (Å)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.006&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.013&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Bond angles (°)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.26&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.30&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Ramachandran Plot&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;favored (%)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;allowed (%)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;disallowed (%)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>13249</offset><text>	PduL native	PduL mercury derivative	PduL phosphate soaked	 	Data collection				 	Space group	P 21 21 21	P 21 21 21	P 21 21 21	 	Cell dimensions				 	a, b, c (Å)	57.7, 56.4, 150.4	55.6, 57.7, 150.2	57.1, 58.8, 136.7	 	α, β, γ (°)	90, 90, 90	90, 90, 90	90, 90, 90	 	Resolution (Å)	31.4 − 1.54 (1.60 − 1.54)*	35.3 − 1.99 (2.07 − 1.99)	39.2 − 2.10 (2.21 − 2.10)	 	Rmerge	0.169 (1.223)	0.084 (0.299)	0.122 (0.856)	 	I/σ(I)	12.9 (1.7)	22.1 (7.1)	12.6 (2.0)	 	Completeness (%)	99.4 (94.4)	99.3 (93.3)	100 (99.9)	 	Redundancy	13.9 (12.1)	7.2 (7.0)	6.5 (6.1)	 	Refinement				 	Resolution (Å)	31.4 − 1.54 (1.60 − 1.54)*		39.2 − 2.10 (2.18 − 2.1)	 	No. reflections	72,698		27,554	 	Rwork/Rfree (%)	18.9 (30.7) / 22.1 (34.7)		17.5 (24.2) / 22.6 (30.0)	 	No. atoms	3,453		3,127	 	Protein	2,841		2,838	 	Ligand/ion	100		24	 	Water	512		265	 	B-factors	22.8		34.7	 	Protein	21.5		24.3	 	Ligand/ion	21.9		40.6	 	Water	30.3		37.9	 	R.m.s deviations				 	Bond lengths (Å)	0.006		0.013	 	Bond angles (°)	1.26		1.30	 	Ramachandran Plot				 	favored (%)	99		99	 	allowed (%)	1		1	 	disallowed (%)	0		0	 	</text></passage><passage><infon key="file">pbio.1002399.t002.xml</infon><infon key="id">pbio.1002399.t002</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>14364</offset><text>*Highest resolution shell is shown in parentheses.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14415</offset><text>Structurally, PduL consists of two domains (Fig 2, blue/red), each a beta-barrel that is capped on both ends by short α-helices. β-Barrel 1 consists of the N-terminal β strand and β strands from the C-terminal half of the polypeptide chain (β1, β10-β14; residues 37–46 and 155–224). β-Barrel 2 consists mainly of the central segment of primary structure (β2, β5–β9; residues 47–60 and 82–154) (Fig 2, red), but is interrupted by a short two-strand beta sheet (β3-β4, residues 61–81). This β-sheet is involved in contacts between the two domains and forms a lid over the active site. Residues in this region (Gln42, Pro43, Gly44), covering the active site, are strongly conserved (Fig 3). This structural arrangement is completely different from the functionally related Pta, which is composed of two domains, each consisting of a central flat beta sheet with alpha-helices on the top and bottom.</text></passage><passage><infon key="file">pbio.1002399.g003.jpg</infon><infon key="id">pbio.1002399.g003</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>15354</offset><text>Primary structure conservation of the PduL protein family.</text></passage><passage><infon key="file">pbio.1002399.g003.jpg</infon><infon key="id">pbio.1002399.g003</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>15413</offset><text>Sequence logo calculated from the multiple sequence alignment of PduL homologs (see Materials and Methods), but not including putative EP sequences. Residues 100% conserved across all PduL homologs in our dataset are noted with an asterisk, and residues conserved in over 90% of sequences are noted with a colon. The sequences aligning to the PF06130 domain (determined by BLAST) are highlighted in red and blue. The position numbers shown correspond to the residue numbering of rPduL; note that some positions in the logo represent gaps in the rPduL sequence.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15974</offset><text>There are two PduL molecules in the asymmetric unit forming a butterfly-shaped dimer (Fig 4c). Consistent with this, results from size exclusion chromatography of rPduLΔEP suggest that it is a dimer in solution (Fig 5e). The interface between the two chains buries 882 Å2 per monomer and is mainly formed by α-helices 2 and 4 and parts of β-sheets 12 and 14, as well as a π–π stacking of the adenine moiety of CoA with Phe116 of the adjacent chain (Fig 4c). The folds of the two chains in the asymmetric unit are very similar, superimposing with a rmsd of 0.16 Å over 2,306 aligned atom pairs. The peripheral helices and the short antiparallel β3–4 sheet mediate most of the crystal contacts.</text></passage><passage><infon key="file">pbio.1002399.g004.jpg</infon><infon key="id">pbio.1002399.g004</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>16683</offset><text>Details of active site, dimeric assembly, and sequence conservation of PduL.</text></passage><passage><infon key="file">pbio.1002399.g004.jpg</infon><infon key="id">pbio.1002399.g004</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>16760</offset><text>(a,b) Proposed active site of PduL with relevant residues shown as sticks in atom coloring (nitrogen blue, oxygen red, sulfur yellow), zinc as grey colored spheres and coordinating ordered water molecules in red. Distances between atom centers are indicated in Å. (a) Coenzyme A containing, (b) phosphate-bound structure. (c) View of the dimer in the asymmetric unit from the side, domains 1 and 2 colored as in Fig 2 and the two chains differentiated by blue/red versus slate/firebrick. The bottom panel shows a top view down the 2-fold axis as indicated by the arrow in the top panel. The asterisk and double arrow marks the location of the π–π interaction between F116 and the CoA base of the other dimer chain. (d) Surface representation of the structure with indicated conservation (red: high, white: intermediate, yellow: low).</text></passage><passage><infon key="file">pbio.1002399.g005.jpg</infon><infon key="id">pbio.1002399.g005</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>17599</offset><text>Size exclusion chromatography of PduL homologs.</text></passage><passage><infon key="file">pbio.1002399.g005.jpg</infon><infon key="id">pbio.1002399.g005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>17647</offset><text>(a)–(c): Chromatograms of sPduL (a), rPduL (b), and pPduL (c) with (orange) or without (blue) the predicted EP, post-nickel affinity purification, applied over a preparative size exclusion column (see Materials and Methods). (d)–(f): Chromatograms of sPduL (d), rPduL (e), and pPduL (f) post-preparative size exclusion chromatography with different size fractions separated, applied over an analytical size exclusion column (see Materials and Methods). All chromatograms are cropped to show only the linear range of separation based on standard runs, shown in black squares with a dashed linear trend line. All y-axes are arbitrary absorbance units except the right-hand axes for panels (a) and (d), which is the log10(molecular weight) of the standards.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>18406</offset><text>Active Site Properties</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18429</offset><text>CoA and the metal ions bind between the two domains, presumably in the active site (Figs 2b and 4a). To identify the bound metals, we performed an X-ray fluorescence scan on the crystals at various wavelengths (corresponding to the K-edges of Mn, Fe, Co, Ni, Cu, and Zn). There was a large signal at the zinc edge, and we tested for the presence of zinc by collecting full data sets before and after the Zn K-edge (1.2861 and 1.2822 Å, respectively). The large differences between the anomalous signals confirm the presence of zinc at both metal sites (S3 Fig).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18992</offset><text>The first zinc ion (Zn1) is in a tetrahedral coordination state with His48, His50, Glu109, and the CoA sulfur (Fig 4a). The second (Zn2) is in octahedral coordination by three conserved histidine residues (His157, His159 and His204) as well as three water molecules (Fig 4a). The nitrogen atom coordinating the zinc is the Nε in each histidine residue, as is typical for this interaction. When the crystals were soaked in a sodium phosphate solution for 2 d prior to data collection, the CoA dissociates, and density for a phosphate molecule is visible at the active site (Table 2, Fig 4b). The phosphate-bound structure aligns well with the CoA-bound structure (0.43 Å rmsd over 2,361 atoms for the monomer, 0.83 Å over 5,259 aligned atoms for the dimer). The phosphate contacts both zinc atoms (Fig 4b) and replaces the coordination by CoA at Zn1; the coordination for Zn2 changes from octahedral with three bound waters to tetrahedral with a phosphate ion as one of the ligands (Fig 4b). Conserved Arg103 seems to be involved in maintaining the phosphate in that position. The two zinc atoms are slightly closer together in the phosphate-bound form (5.8 Å vs 6.3 Å), possibly due to the bridging effect of the phosphate. An additional phosphate molecule is bound at a crystal contact interface, perhaps accounting for the 14 Å shorter c-axis in the phosphate-bound crystal form (Table 2).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>20390</offset><text>Oligomeric States of PduL Orthologs Are Influenced by the EP</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20451</offset><text>Interestingly, some of the residues important for dimerization of rPduL, particularly Phe116, are poorly conserved across PduL homologs associated with functionally diverse BMCs (Figs 4c and 3), suggesting that they may have alternative oligomeric states. We tested this hypothesis by performing size exclusion chromatography on both full-length and truncated variants (lacking the EP, ΔEP) of sPduL, rPduL, and pPduL. These three homologs are found in functionally distinct BMCs (Table 1). Therefore, they are packaged with different signature enzymes and different ancillary proteins. It has been proposed that the catabolic BMCs may assemble in a core-first manner, with the luminal enzymes (signature enzyme, aldehyde, and alcohol dehydrogenases and the BMC PTAC) forming an initial bolus, or prometabolosome, around which a shell assembles. Given the diversity of signature enzymes (Table 1), it is plausible that PduL orthologs may adopt different oligomeric states that reflect the differences in the proteins being packaged with them in the organelle lumen.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21521</offset><text>We found that not only did the different orthologs appear to assemble into different oligomeric states, but that quaternary structure was dependent on whether or not the EP was present. Full-length sPduL was unstable in solution—precipitating over time—and eluted throughout the entire volume of a size exclusion column, indicating it was nonspecifically aggregating. However, when the putative EP (residues 1–27) was removed (sPduL ΔEP), the truncated protein was stable and eluted as a single peak (Fig 5a) consistent with the size of a monomer (Fig 5d, blue curve). In contrast, both full-length rPduL and pPduL appeared to exist in two distinct oligomeric states (Fig 5b and 5c respectively, orange curves), one form of the approximate size of a dimer and the second, a higher molecular weight oligomer (~150 kDa). Upon deletion of the putative EP (residues 1–47 for rPduL, and 1–20 for pPduL), there was a distinct change in the elution profiles (Fig 5b and 5c respectively, blue curves). pPduLΔEP eluted as two smaller forms, possibly corresponding to a trimer and a monomer. In contrast, rPduLΔEP eluted as one smaller oligomer, possibly a dimer. We also analyzed purified rPduL and rPduLΔEP by size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) for a complementary approach to assessing oligomeric state. SEC-MALS analysis of rPdulΔEP is consistent with a dimer (as observed in the crystal structure) with a weighted average (Mw) and number average (Mn) of the molar mass of 58.4 kDa +/− 11.2% and 58.8 kDa +/− 10.9%, respectively (S4a Fig). rPduL full length runs as Mw = 140.3 kDa +/− 1.2% and Mn = 140.5 kDa +/− 1.2%. This corresponds to an oligomeric state of six subunits (calculated molecular weight of 144 kDa). Collectively, these data strongly suggest that the N-terminal EP of PduL plays a role in defining the quaternary structure of the protein.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>23441</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>23452</offset><text>The hallmark attribute of an organelle is that it serves as a discrete subcellular compartment functioning as an isolated microenvironment distinct from the cytosol. In order to create and preserve this microenvironment, the defining barrier (i.e., lipid bilayer membrane or microcompartment shell) must be selectively permeable. The BMC shell not only sequesters specific enzymes but also their cofactors, thereby establishing a private cofactor pool dedicated to the encapsulated reactions. In catabolic BMCs, CoA and NAD+ must be continually recycled within the organelle (Fig 1). Homologs of the predominant cofactor utilizer (aldehyde dehydrogenase) and NAD+ regenerator (alcohol dehydrogenase) have been structurally characterized, but until now structural information was lacking for PduL, which recycles CoA in the organelle lumen. Curiously, while the housekeeping Pta could provide this function, and indeed does so in the case of one type of ethanolamine-utilizing (EUT) BMC, the evolutionarily unrelated PduL fulfills this function for the majority of metabolosomes using a novel structure and active site for convergent evolution of function.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_2</infon><offset>24608</offset><text>The Tertiary Structure of PduL Is Formed by Discontinuous Segments of Primary Structure</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>24696</offset><text>The structure of PduL consists of two β-barrel domains capped by short alpha helical segments (Fig 2b). The two domains are structurally very similar (superimposing with a rmsd of 1.34 Å (over 123 out of 320/348 aligned backbone atoms, S5a Fig). However, the amino acid sequences of the two domains are only 16% identical (mainly the RHxH motif, β2 and β10), and 34% similar. Our structure reveals that the two assigned PF06130 domains (Fig 3) do not form structurally discrete units; this reduces the apparent sequence conservation at the level of primary structure. One strand of the domain 1 beta barrel (shown in blue in Fig 2) is contributed by the N-terminus, while the rest of the domain is formed by the residues from the C-terminal half of the protein. When aligned by structure, the β1 strand of the first domain (Fig 2a and 2b, blue) corresponds to the final strand of the second domain (β9), effectively making the domains continuous if the first strand was transplanted to the C-terminus. Refined domain assignment based on our structure should be able to predict domains of PF06130 homologs much more accurately. The closest structural homolog of the PduL barrel domain is a subdomain of a multienzyme complex, the alpha subunit of ethylbenzene dehydrogenase (S5b Fig, rmsd of 2.26 Å over 226 aligned atoms consisting of one beta barrel and one capping helix). In contrast to PduL, there is only one barrel present in ethylbenzene dehydrogenase, and there is no comparable active site arrangement. The PduL signature primary structure, two PF06130 domains, occurs in some multidomain proteins, most of them annotated as Acks, suggesting that PduL may also replace Pta in variants of the phosphotransacetylase-Ack pathway. These PduL homologs lack EPs, and their fusion to Ack may have evolved as a way to facilitate substrate channeling between the two enzymes.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_2</infon><offset>26587</offset><text>Implications for Metabolosome Core Assembly</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>26631</offset><text>For BMC-encapsulated proteins to properly function together, they must be targeted to the lumen and assemble into an organization that facilitates substrate/product channeling among the different catalytic sites of the signature and core enzymes. The N-terminal extension on PduL homologs may serve both of these functions. The extension shares many features with previously characterized EPs: it is present only in homologs associated with BMC loci, and it is predicted to form an amphipathic α-helix. Moreover, its removal affects the oligomeric state of the protein. EP-mediated oligomerization has been observed for the signature and core BMC enzymes; for example, full-length propanediol dehydratase and ethanolamine ammonia-lyase (signature enzymes for PDU and EUT BMCs) subunits are also insoluble, but become soluble upon removal of the predicted EP. sPduL has also previously been reported to localize to inclusion bodies when overexpressed; we show here that this is dependent on the presence of the EP. This propensity of the EP to cause proteins to form complexes (Fig 5) might not be a coincidence, but could be a necessary step in the assembly of BMCs. Structured aggregation of the core enzymes has been proposed to be the initial step in metabolosome assembly and is known to be the first step of β-carboxysome biogenesis, where the core enzyme Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO) is aggregated by the CcmM protein. Likewise, CsoS2, a protein in the α-carboxysome core, also aggregates when purified and is proposed to facilitate the nucleation and encapsulation of RuBisCO molecules in the lumen of the organelle. Coupled with protein–protein interactions with other luminal components, the aggregation of these enzymes could lead to a densely packed organelle core. This role for EPs in BMC assembly is in addition to their interaction with shell proteins.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>28535</offset><text>Moreover, the PduL crystal structures offer a clue as to how required cofactors enter the BMC lumen during assembly. Free CoA and NAD+/H could potentially be bound to the enzymes as the core assembles and is encapsulated. However, this raises an issue of stoichiometry: if the ratio of cofactors to core enzymes is too low, then the sequestered metabolism would proceed at suboptimal rates. Our PduL crystals contained CoA that was captured from the Escherichia coli cytosol, indicating that the “ground state” of PduL is in the CoA-bound form; this could provide an elegantly simple means of guaranteeing a 1:1 ratio of CoA:PduL within the metabolosome lumen.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_2</infon><offset>29200</offset><text>Active Site Identification and Structural Insights into Catalysis</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>29266</offset><text>The active site of PduL is formed at the interface of the two structural domains (Fig 2b). As expected, the amino acid sequence conservation is highest in the region around the proposed active site (Fig 4d); highly conserved residues are also involved in CoA binding (Figs 2a and 3, residues Ser45, Lys70, Arg97, Leu99, His204, Asn211). All of the metal-coordinating residues (Fig 2a) are absolutely conserved, implicating them in catalysis or the correct spatial orientation of the substrates. Arg103, which contacts the phosphate (Fig 4b), is present in all PduL homologs. The close resemblance between the structures binding CoA and phosphate likely indicates that no large changes in protein conformation are involved in catalysis, and that our crystal structures are representative of the active form. The native substrate for the forward reaction of rPduL and pPduL, propionyl-CoA, most likely binds to the enzyme in the same way at the observed nucleotide and pantothenic acid moiety, but the propionyl group in the CoA-thioester might point in a different direction. There is a pocket nearby the active site between the well-conserved residues Ser45 and Ala154, which could accommodate the propionyl group (S6 Fig). A homology model of sPduL indicates that the residues making up this pocket and the surrounding active site region are identical to that of rPduL, which is not surprising, because these two homologs presumably have the same propionyl-CoA substrate. The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus. Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms. The catalytic mechanism of Pta involves the abstraction of a thiol hydrogen by an aspartate residue, resulting in the nucleophilic attack of thiolate upon the carbonyl carbon of acetyl-phosphate, oriented by an arginine and stabilized by a serine —there are no metals involved. In contrast, in the rPduL structure, there are no conserved aspartate residues in or around the active site, and the only well-conserved glutamate residue in the active site is involved in coordinating one of the metal ions. These observations strongly suggest that an acidic residue is not directly involved in catalysis by PduL. Instead, the dimetal active site of PduL may create a nucleophile from one of the hydroxyl groups on free phosphate to attack the carbonyl carbon of the thioester bond of an acyl-CoA. In the reverse direction, the metal ion(s) could stabilize the thiolate anion that would attack the carbonyl carbon of an acyl-phosphate; a similar mechanism has been described for phosphatases where hydroxyl groups or hydroxide ions can act as a base when coordinated by a dimetal active site.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>32659</offset><text>Our structures provide the foundation for studies to elucidate the details of the catalytic mechanism of PduL. Conserved residues in the active site that may contribute to substrate binding and/or transition state stabilization include Ser127, Arg103, Arg194, Gln107, Gln74, and Gln/Glu77. In the phosphate-bound crystal structure, Ser127 and Arg103 appear to position the phosphate (Fig 4b). Alternatively, Arg103 might act as a base to render the phosphate more nucleophilic. The functional groups of Gln74, Gln/Glu77, and Arg194 are directed away from the active site in both CoA and phosphate-bound crystal structures and do not appear to be involved in hydrogen bonding with these substrates, although they could be important for positioning an acyl-phosphate.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>33425</offset><text>The free CoA-bound form is presumably poised for attack upon an acyl-phosphate, indicating that the enzyme initially binds CoA as opposed to acyl-phosphate. This hypothesis is strengthened by the fact that the CoA-bound crystals were obtained without added CoA, indicating that the protein bound CoA from the E. coli expression strain and retained it throughout purification and crystallization. The phosphate-bound structure indicates that in the opposite reaction direction phosphate is bound first, and then an acyl-CoA enters. The two high-resolution crystal structures presented here will serve as the foundation for mechanistic studies on this noncanonical PTAC enzyme to determine how the dimetal active site functions to catalyze both forward and reverse reactions.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_2</infon><offset>34199</offset><text>Functional, but Not Structural, Convergence of PduL and Pta</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>34259</offset><text>PduL and Pta are mechanistically and structurally distinct enzymes that catalyze the same reaction, a prime example of evolutionary convergence upon a function. There are several examples of such functional convergence of enzymes, although typically the enzymes have independently evolved similar, or even identical active sites; for example, the carbonic anhydrase family. However, apparently less frequent is functional convergence that is supported by distinctly different active sites and accordingly catalytic mechanism, as revealed by comparison of the structures of Pta and PduL. One well-studied example of this is the β-lactamase family of enzymes, in which the active site of Class A and Class C enzymes involve serine-based catalysis, but Class B enzymes are metalloproteins. This is not surprising, as β-lactamases are not so widespread among bacteria and therefore would be expected to have evolved independently several times as a defense mechanism against β-lactam antibiotics. However, nearly all bacteria encode Pta, and it is not immediately clear why the Pta/PduL functional convergence should have evolved: it would seem to be evolutionarily more resourceful for the Pta-encoding gene to be duplicated and repurposed for BMCs, as is apparently the case in one type of BMC—EUT1 (Table 1). There could be some intrinsic biochemical difference between the two enzymes that renders PduL a more attractive candidate for encapsulation in a BMC—for example, PduL might be more amenable to tight packaging, or is better suited for the chemical microenvironment formed within the lumen of the BMC, which can be quite different from the cytosol. Further biochemical comparison between the two PTACs will likely yield exciting results that could answer this evolutionary question.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_2</infon><offset>36058</offset><text>Implications</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>36071</offset><text>BMCs are now known to be widespread among the bacteria and are involved in critical segments of both autotrophic and heterotrophic biochemical pathways that confer to the host organism a competitive (metabolic) advantage in select niches. As one of the three common metabolosome core enzymes, the structure of PduL provides a key missing piece to our structural picture of the shared core biochemistry (Fig 1) of functionally diverse catabolic BMCs. We have observed the oligomeric state differences of PduL to correlate with the presence of an EP, providing new insight into the function of this sequence extension in BMC assembly. Moreover, our results suggest a means for Coenzyme A incorporation during metabolosome biogenesis. A detailed understanding of the underlying principles governing the assembly and internal structural organization of BMCs is a requisite for synthetic biologists to design custom nanoreactors that use BMC architectures as a template. Furthermore, given the growing number of metabolosomes implicated in pathogenesis, the PduL structure will be useful in the development of therapeutics. It is gradually being realized that the metabolic capabilities of a pathogen are also important for virulence, along with the more traditionally cited factors like secretion systems and effector proteins. The fact that PduL is confined almost exclusively to metabolosomes can be used to develop an inhibitor that blocks only PduL and not Pta as a way to selectively disrupt BMC-based metabolism, while not affecting most commensal organisms that require PTAC activity.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>37659</offset><text>Materials and Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>37681</offset><text>Molecular Cloning</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>37699</offset><text>Genes for PduL homologs with and without the EP were amplified via PCR using the primers listed in S1 Table. sPduL was amplified using S. enterica Typhimurium LT2 genomic DNA, and pPduL and rPduL sequences were codon optimized and synthesized by GenScript with the 6xHis tag. All 5’ primers included EcoRI and BglII restriction sites, and all 3’ primers included a BamHI restriction site to facilitate cloning using the BglBricks strategy. 5’ primers also included the sequence TTTAAGAAGGAGATATACCATG downstream of the restriction sites, serving as a strong ribosome binding site. The 6x polyhistidine tag sequence was added to the 3’ end of the gene using the BglBricks strategy and was subcloned into the pETBb3 vector, a pET21b-based vector modified to be BglBricks compatible.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>38488</offset><text>Protein Purification, Size Exclusion Chromatography, and Protein Crystallization</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38569</offset><text> E. coli BL21(DE3) expression strains containing the relevant PduL construct in the pETBb3 vector were grown overnight at 37°C in standard LB medium and then used to inoculate 1L of standard LB medium in 2.8 L Fernbach flasks at a 1:100 dilution, which were then incubated at 37°C shaking at 150 rpm, until the culture reached an OD600 of 0.8–1.0, at which point cultures were induced with 200 μM IPTG (isopropylthio-β-D-galactoside) and incubated at 20°C for 18 h, shaking at 150 rpm. Cells were centrifuged at 5,000 xg for 15 min, and cell pellets were frozen at –20°C.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>39151</offset><text>For protein purifications, cell pellets from 1–3 L cultures were resuspended in 20–30 ml buffer A (50 mM Tris-HCl pH 7.4, 300 mM NaCl) and lysed using a French pressure cell at 20,000 lb/in2. The resulting cell lysate was centrifuged at 15,000 xg. 30 mM imidazole was added to the supernatant that was then applied to a 5 mL HisTrap column (GE Healthcare Bio-Sciences, Pittsburgh, PA). Protein was eluted off the column using a gradient of buffer A from 0 mM to 500 mM imidazole over 20 column volumes. Fractions corresponding to PduL were pooled and concentrated using Amicon Ultra Centrifugal filters (EMD Millipore, Billerica, MA) to a volume of no more than 2.5 mL. The protein sample was then applied to a HiLoad 26/60 Superdex 200 preparative size exclusion column (GE Healthcare Bio-Sciences, Pittsburgh, PA) and eluted with buffer B (20 mM Tris pH 7.4, 50 mM NaCl). Where applicable, fractions corresponding to different oligomeric states were pooled separately, leaving one or two fractions in between to prevent cross contamination. Pooled fractions were concentrated to 1–20 mg/mL protein as determined by the Bradford method prior to applying on a Superdex 200 10/300 GL analytical size exclusion column (GE Healthcare Bio-Sciences, Pittsburgh, PA). Size standards used were Thyroglobulin 670 kDa, γ-globulin 158 kDa, Ovalbumin 44 kDa, and Myoglobin 17 kDa. For light scattering, the proteins were measured in a Protein Solutions Dynapro dynamic light scattering instrument with an acquisition time of 5 s, averaging 10 acquisitions at a constant temperature of 25°C. The radii were calculated assuming a globular particle shape.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>40801</offset><text>Size exclusion chromatography coupled with SEC-MALS was performed on full-length rPduL and rPduL-ΔEP similar to Luzi et al. 2015. A Wyatt DAWN Heleos-II 18-angle light scattering instrument was used in tandem with a GE AKTA pure FPLC with built in UV detector, and a Wyatt Optilab T-Rex refractive index detector. Detector 16 of the DAWN Heleos-II was replaced with a Wyatt Dynapro Nanostar QELS detector for dynamic light scattering. A GE Superdex S200 10/300 GL column was used, with 125–100 μl of protein sample at 1 mg/ml concentration injected, and the column run at 0.5 ml/min in 20 mM Tris, 50 mM NaCl, pH 7.4.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>41424</offset><text>Each detector of the DAWN-Heleos-II was plotted with the Zimm model in the Wyatt ASTRA software to calculate the molar mass. The molar mass was measured at each collected data point across the peaks at ~1 point per 8 μl eluent. Both the Mw and Mn of the molar mass calculations, as well as percent deviations, were also determined using Wyatt software program ASTRA.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>41792</offset><text>For preparing protein for crystallography, expression cells were grown as above, except were induced with 50 μM IPTG. Harvested cells were resuspended in buffer B and lysed using a French Press. Cleared lysate was applied on a 5 ml HisTrap HP column (GE Healthcare) and washed with buffer A containing 20 mM imidazole. Pdul-His was eluted with 2 CV buffer B containing 300 mM imidazole, concentrated and then applied on a HiLoad 26/60 Superdex 200 (GE Healthcare) column equilibrated in buffer B for final cleanup. Protein was then concentrated to 20–30 mg/ml for crystallization. Crystals were obtained from sitting drop experiments at 22°C, mixing 3 μl of protein solution with 3 μl of reservoir solution containing 39%–35% MPD. Crystals were flash frozen in liquid nitrogen after being adding 5 μl of a reservoir solution. For heavy atom derivatives, 0.2 μl of 100 mM Thiomerosal (Hampton Research) was added to the crystallization drop 36 h prior to freezing. For phosphate soaks, 5 μl reservoir and 1.5 μl 200 mM sodium phosphate solution (pH 7.0) were added 2 d prior to flash freezing.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>42897</offset><text>PTAC Activity Assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>42917</offset><text>Enzyme reactions were performed in a 2 mL cuvette containing 50 mM Tris-HCl pH 7.5, 0.2 mM 5,5'-dithiobis-2-nitrobenzoic acid (DTNB; Ellman’s reagent), 0.1 mM acyl-CoA, and 0.5 μg purified PTAC, unless otherwise noted. To initiate the reaction, 5 mM NaH2PO4 was added, the cuvette was inverted to mix, and the absorbance at 412 nm was measured every 2 s over the course of four minutes in a Nanodrop 2000c, in the cuvette holder. 14,150 M-1cm-1 was used as the extinction coefficient of DTNB to determine the specific activity.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>43448</offset><text>PduL Sequence Analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>43471</offset><text>A multiple sequence alignment of 228 PduL sequences associated with BMCs and 20 PduL sequences not associated with BMCs was constructed using MUSCLE. PduL sequences associated with BMCs were determined from Dataset S1 of Reference, and those not associated with BMCs were determined by searching for genomes that encoded PF06130 but not PF03319 nor PF00936 in the IMG database. The multiple sequence alignment was visualized in Jalview, and the nonconserved N- and C-terminal amino acids were deleted. This trimmed alignment was used to build the sequence logo using WebLogo.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>44047</offset><text>Diffraction Data Collection, Structure Determination and Visualization</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>44118</offset><text>Diffraction data were collected at the Advanced Light Source at Lawrence Berkeley National Laboratory beamline 5.0.2 (100 K, 1.0000 Å wavelength for native data, 1.0093 Å for mercury derivative, 1.2861 Å for Zn pre-edge and 1.2822 Å for Zn peak). Diffraction data were integrated with XDS and scaled with SCALA (CCP4). The structure of PduL was solved using phenix.autosol, which found 11 heavy atom sites and produced density suitable for automatic model building. The model was refined with phenix.refine, with refinement alternating with model building using 2Fo-Fc and Fo-Fc maps visualized in COOT. Statistics for diffraction data collection, structure determination and refinement are summarized in Table 2. Figures were prepared using pymol (www.pymol.org) and Raster3D.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>44900</offset><text>Homology Modeling</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>44918</offset><text>Models of S. enterica Typhimurium LT2 and P. limnophilus PduL were generated with Modeller using the align2d and model-default scripts.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>45054</offset><text>Supporting Information</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">title</infon><offset>45077</offset><text>Abbreviations</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45091</offset><text>Ack</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45095</offset><text>acetate kinase</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45110</offset><text>BMC</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45114</offset><text>Bacterial Microcompartment</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45141</offset><text>EP</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45144</offset><text>encapsulation peptide</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45166</offset><text>EUT</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45170</offset><text>ethanolamine-utilizing</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45193</offset><text>Mn</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45196</offset><text>number average</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45211</offset><text>Mw</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45214</offset><text>weighted average</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45231</offset><text>PDU</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45235</offset><text>propanediol-utilizing</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45257</offset><text>Pta</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45261</offset><text>phosphotransacylase</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45281</offset><text>PTAC</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45286</offset><text>phosphotransacylase</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45306</offset><text>RuBisCO</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45314</offset><text>Ribulose Bisphosphate Carboxylase/Oxygenase</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45358</offset><text>SEC-MALS</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>45367</offset><text>multiangle light scattering</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>45395</offset><text>References</text></passage><passage><infon key="fpage">22</infon><infon key="issue">1</infon><infon key="lpage">34</infon><infon key="name_0">surname:Kerfeld;given-names:CA</infon><infon key="name_1">surname:Erbilgin;given-names:O</infon><infon key="pub-id_doi">10.1016/j.tim.2014.10.003</infon><infon key="pub-id_pmid">25455419</infon><infon key="section_type">REF</infon><infon key="source">Trends in microbiology</infon><infon key="type">ref</infon><infon key="volume">23</infon><infon key="year">2015</infon><offset>45406</offset><text>Bacterial microcompartments and the modular construction of microbial metabolism</text></passage><passage><infon key="fpage">e1003898</infon><infon key="issue">10</infon><infon key="name_0">surname:Axen;given-names:SD</infon><infon key="name_1">surname:Erbilgin;given-names:O</infon><infon key="name_2">surname:Kerfeld;given-names:CA</infon><infon key="pub-id_doi">10.1371/journal.pcbi.1003898</infon><infon key="pub-id_pmid">25340524</infon><infon key="section_type">REF</infon><infon key="source">PLoS Comput Biol</infon><infon key="type">ref</infon><infon key="volume">10</infon><infon key="year">2014</infon><offset>45487</offset><text>A taxonomy of bacterial microcompartment loci constructed by a novel scoring method</text></passage><passage><infon key="name_0">surname:Liu;given-names:Y</infon><infon key="name_1">surname:Jorda;given-names:J</infon><infon key="name_2">surname:Yeates;given-names:TO</infon><infon key="name_3">surname:Bobik;given-names:TA</infon><infon key="pub-id_doi">10.1128/JB.00056-15</infon><infon key="pub-id_pmid">25962918</infon><infon key="section_type">REF</infon><infon key="source">Journal of bacteriology</infon><infon key="type">ref</infon><infon key="year">2015</infon><offset>45571</offset><text>The PduL phosphotransacylase is used to recycle coenzyme A within the Pdu microcompartment</text></passage><passage><infon key="fpage">1589</infon><infon key="issue">5</infon><infon key="lpage">96</infon><infon key="name_0">surname:Liu;given-names:Y</infon><infon key="name_1">surname:Leal;given-names:NA</infon><infon key="name_2">surname:Sampson;given-names:EM</infon><infon key="name_3">surname:Johnson;given-names:CLV</infon><infon key="name_4">surname:Havemann;given-names:GD</infon><infon key="name_5">surname:Bobik;given-names:Ta</infon><infon key="pub-id_doi">10.1128/JB.01151-06</infon><infon key="pub-id_pmid">17158662</infon><infon key="section_type">REF</infon><infon key="source">Journal of bacteriology</infon><infon key="type">ref</infon><infon key="volume">189</infon><infon key="year">2007</infon><offset>45662</offset><text>PduL is an Evolutionarily Distinct Phosphotransacylase Involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar typhimurium LT2</text></passage><passage><infon key="fpage">936</infon><infon key="lpage">8</infon><infon key="name_0">surname:Kerfeld;given-names:CA</infon><infon key="name_1">surname:Sawaya;given-names:MR</infon><infon key="name_2">surname:Tanaka;given-names:S</infon><infon key="name_3">surname:Nguyen;given-names:CV</infon><infon key="name_4">surname:Phillips;given-names:M</infon><infon key="name_5">surname:Beeby;given-names:M</infon><infon key="pub-id_doi">10.1126/science.1113397</infon><infon key="section_type">REF</infon><infon key="source">Science (New York, NY)</infon><infon key="type">ref</infon><infon key="volume">309</infon><infon key="year">2005</infon><offset>45818</offset><text>Protein structures forming the shell of primitive bacterial organelles</text></passage><passage><infon key="fpage">1642</infon><infon key="issue">12</infon><infon key="lpage">52</infon><infon key="name_0">surname:Pang;given-names:A</infon><infon key="name_1">surname:Liang;given-names:M</infon><infon key="name_2">surname:Prentice;given-names:MB</infon><infon key="name_3">surname:Pickersgill;given-names:RW</infon><infon key="pub-id_doi">10.1107/S0907444912039315</infon><infon key="pub-id_pmid">23151629</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallographica Section D Biological Crystallography</infon><infon key="type">ref</infon><infon key="volume">68</infon><infon key="year">2012</infon><offset>45889</offset><text>Substrate channels revealed in the trimeric Lactobacillus reuteri bacterial microcompartment shell protein PduB</text></passage><passage><infon key="fpage">5967</infon><infon key="issue">19</infon><infon key="lpage">75</infon><infon key="name_0">surname:Bobik;given-names:TA</infon><infon key="name_1">surname:Havemann;given-names:GD</infon><infon key="name_2">surname:Busch;given-names:RJ</infon><infon key="name_3">surname:Williams;given-names:DS</infon><infon key="name_4">surname:Aldrich;given-names:HC</infon><infon key="pub-id_pmid">10498708</infon><infon key="section_type">REF</infon><infon key="source">Journal of bacteriology</infon><infon key="type">ref</infon><infon key="volume">181</infon><infon key="year">1999</infon><offset>46001</offset><text>The Propanediol Utilization (pdu) Operon of Salmonella enterica serovar Typhimurium LT2 Includes Genes Necessary for Formation of Polyhedral Organelles Involved in coenzyme B12-Dependent 1, 2-Propanediol Degradation</text></passage><passage><infon key="fpage">1000118</infon><infon key="issue">4</infon><infon key="name_0">surname:Abdul-Rahman;given-names:F</infon><infon key="name_1">surname:Petit;given-names:E</infon><infon key="name_2">surname:Blanchard;given-names:JL</infon><infon key="pub-id_doi">10.4172/2329-9002.1000118</infon><infon key="section_type">REF</infon><infon key="source">Phylogenetics and Evolutionary Biology</infon><infon key="type">ref</infon><infon key="volume">1</infon><infon key="year">2013</infon><offset>46217</offset><text>The Distribution of Polyhedral Bacterial Microcompartments Suggests Frequent Horizontal Transfer and Operon Reassembly</text></passage><passage><infon key="fpage">179</infon><infon key="issue">2</infon><infon key="lpage">95</infon><infon key="name_0">surname:Jorda;given-names:J</infon><infon key="name_1">surname:Lopez;given-names:D</infon><infon key="name_2">surname:Wheatley;given-names:NM</infon><infon key="name_3">surname:Yeates;given-names:TO</infon><infon key="pub-id_doi">10.1002/pro.2196</infon><infon key="pub-id_pmid">23188745</infon><infon key="section_type">REF</infon><infon key="source">Protein Science</infon><infon key="type">ref</infon><infon key="volume">22</infon><infon key="year">2013</infon><offset>46336</offset><text>Using comparative genomics to uncover new kinds of protein-based metabolic organelles in bacteria</text></passage><passage><infon key="fpage">3855</infon><infon key="issue">9</infon><infon key="lpage">63</infon><infon key="name_0">surname:Roof;given-names:DM</infon><infon key="name_1">surname:Roth;given-names:JR</infon><infon key="pub-id_pmid">3045078</infon><infon key="section_type">REF</infon><infon key="source">Journal of bacteriology</infon><infon key="type">ref</infon><infon key="volume">170</infon><infon key="year">1988</infon><offset>46434</offset><text>Ethanolamine utilization in Salmonella typhimurium </text></passage><passage><infon key="fpage">353</infon><infon key="issue">5</infon><infon key="lpage">61</infon><infon key="name_0">surname:Leal;given-names:NA</infon><infon key="name_1">surname:Havemann;given-names:GD</infon><infon key="name_2">surname:Bobik;given-names:TA</infon><infon key="pub-id_doi">10.1007/s00203-003-0601-0</infon><infon key="pub-id_pmid">14504694</infon><infon key="section_type">REF</infon><infon key="source">Archives of microbiology</infon><infon key="type">ref</infon><infon key="volume">180</infon><infon key="year">2003</infon><offset>46486</offset><text>PduP is a coenzyme-a-acylating propionaldehyde dehydrogenase associated with the polyhedral bodies involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar Typhimurium LT2</text></passage><passage><infon key="fpage">2864</infon><infon key="issue">12</infon><infon key="lpage">79</infon><infon key="name_0">surname:Huseby;given-names:DL</infon><infon key="name_1">surname:Roth;given-names:JR</infon><infon key="pub-id_doi">10.1128/JB.02179-12</infon><infon key="pub-id_pmid">23585538</infon><infon key="section_type">REF</infon><infon key="source">Journal of bacteriology</infon><infon key="type">ref</infon><infon key="volume">195</infon><infon key="year">2013</infon><offset>46686</offset><text>Evidence that a metabolic microcompartment contains and recycles private cofactor pools</text></passage><passage><infon key="fpage">e47144</infon><infon key="issue">10</infon><infon key="lpage">e</infon><infon key="name_0">surname:Cheng;given-names:S</infon><infon key="name_1">surname:Fan;given-names:C</infon><infon key="name_2">surname:Sinha;given-names:S</infon><infon key="name_3">surname:Bobik;given-names:TA</infon><infon key="pub-id_doi">10.1371/journal.pone.0047144</infon><infon key="pub-id_pmid">23077559</infon><infon key="section_type">REF</infon><infon key="source">PloS ONE</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2012</infon><offset>46774</offset><text>The PduQ enzyme is an alcohol dehydrogenase used to recycle NAD+ internally within the Pdu microcompartment of Salmonella enterica</text></passage><passage><infon key="name_0">surname:White;given-names:D</infon><infon key="section_type">REF</infon><infon key="source">The Physiology and Biochemistry of Prokaryotes</infon><infon key="type">ref</infon><infon key="year">2000</infon><offset>46905</offset></passage><passage><infon key="fpage">18392</infon><infon key="issue">31</infon><infon key="lpage">6</infon><infon key="name_0">surname:Lundie;given-names:LL;suffix:Jr.</infon><infon key="name_1">surname:Ferry;given-names:JG</infon><infon key="pub-id_pmid">2808380</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">264</infon><infon key="year">1989</infon><offset>46906</offset><text>Activation of acetate by Methanosarcina thermophila. Purification and characterization of phosphotransacetylase</text></passage><passage><infon key="fpage">25</infon><infon key="issue">1</infon><infon key="lpage">35</infon><infon key="name_0">surname:Ferry;given-names:JG</infon><infon key="pub-id_pmid">9233537</infon><infon key="section_type">REF</infon><infon key="source">Biofactors</infon><infon key="type">ref</infon><infon key="volume">6</infon><infon key="year">1997</infon><offset>47018</offset><text>Enzymology of the fermentation of acetate to methane by Methanosarcina thermophila</text></passage><passage><infon key="fpage">387</infon><infon key="issue">4</infon><infon key="lpage">97</infon><infon key="name_0">surname:Tielens;given-names:AG</infon><infon key="name_1">surname:van Grinsven;given-names:KW</infon><infon key="name_2">surname:Henze;given-names:K</infon><infon key="name_3">surname:van Hellemond;given-names:JJ</infon><infon key="name_4">surname:Martin;given-names:W</infon><infon key="pub-id_doi">10.1016/j.ijpara.2009.12.006</infon><infon key="pub-id_pmid">20085767</infon><infon key="section_type">REF</infon><infon key="source">Int J Parasitol</infon><infon key="type">ref</infon><infon key="volume">40</infon><infon key="year">2010</infon><offset>47101</offset><text>Acetate formation in the energy metabolism of parasitic helminths and protists</text></passage><passage><infon key="fpage">D115</infon><infon key="issue">Database issue</infon><infon key="lpage">22</infon><infon key="name_0">surname:Markowitz;given-names:VM</infon><infon key="name_1">surname:Chen;given-names:IMA</infon><infon key="name_2">surname:Palaniappan;given-names:K</infon><infon key="name_3">surname:Chu;given-names:K</infon><infon key="name_4">surname:Szeto;given-names:E</infon><infon key="name_5">surname:Grechkin;given-names:Y</infon><infon key="pub-id_doi">10.1093/nar/gkr1044</infon><infon key="pub-id_pmid">22194640</infon><infon key="section_type">REF</infon><infon key="source">Nucleic acids research</infon><infon key="type">ref</infon><infon key="volume">40</infon><infon key="year">2012</infon><offset>47180</offset><text>IMG: the Integrated Microbial Genomes database and comparative analysis system</text></passage><passage><infon key="fpage">D222</infon><infon key="issue">Database issue</infon><infon key="lpage">30</infon><infon key="name_0">surname:Finn;given-names:RD</infon><infon key="name_1">surname:Bateman;given-names:A</infon><infon key="name_2">surname:Clements;given-names:J</infon><infon key="name_3">surname:Coggill;given-names:P</infon><infon key="name_4">surname:Eberhardt;given-names:RY</infon><infon key="name_5">surname:Eddy;given-names:SR</infon><infon key="pub-id_doi">10.1093/nar/gkt1223</infon><infon key="pub-id_pmid">24288371</infon><infon key="section_type">REF</infon><infon key="source">Nucleic acids research</infon><infon key="type">ref</infon><infon key="volume">42</infon><infon key="year">2014</infon><offset>47259</offset><text>Pfam: the protein families database</text></passage><passage><infon key="fpage">D290</infon><infon key="issue">Database issue</infon><infon key="lpage">301</infon><infon key="name_0">surname:Punta;given-names:M</infon><infon key="name_1">surname:Coggill;given-names:PC</infon><infon key="name_2">surname:Eberhardt;given-names:RY</infon><infon key="name_3">surname:Mistry;given-names:J</infon><infon key="name_4">surname:Tate;given-names:J</infon><infon key="name_5">surname:Boursnell;given-names:C</infon><infon key="pub-id_doi">10.1093/nar/gkr1065</infon><infon key="pub-id_pmid">22127870</infon><infon key="section_type">REF</infon><infon key="source">Nucleic acids research</infon><infon key="type">ref</infon><infon key="volume">40</infon><infon key="year">2012</infon><offset>47295</offset><text>The Pfam protein families database</text></passage><passage><infon key="fpage">527</infon><infon key="issue">2</infon><infon key="lpage">34</infon><infon key="name_0">surname:Stadtman;given-names:ER</infon><infon key="pub-id_pmid">12980995</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">196</infon><infon key="year">1952</infon><offset>47330</offset><text>The purification and properties of phosphotransacetylase</text></passage><passage><infon key="fpage">114</infon><infon key="issue">1</infon><infon key="lpage">25</infon><infon key="name_0">surname:Rado;given-names:TA</infon><infon key="name_1">surname:Hoch;given-names:JA</infon><infon key="pub-id_pmid">4201530</infon><infon key="section_type">REF</infon><infon key="source">Biochim Biophys Acta</infon><infon key="type">ref</infon><infon key="volume">321</infon><infon key="year">1973</infon><offset>47387</offset><text>Phosphotransacetylase from Bacillus subtilis: purification and physiological studies</text></passage><passage><infon key="fpage">1657</infon><infon key="issue">11</infon><infon key="lpage">64</infon><infon key="name_0">surname:Smith;given-names:JE</infon><infon key="name_1">surname:Ng;given-names:WS</infon><infon key="pub-id_pmid">4263885</infon><infon key="section_type">REF</infon><infon key="source">Can J Microbiol</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">1972</infon><offset>47472</offset><text>Fluorometric determination of glycolytic intermediates and adenylates during sequential changes in replacement culture of Aspergillus niger</text></passage><passage><infon key="fpage">e1039755</infon><infon key="issue">3</infon><infon key="name_0">surname:Aussignargues;given-names:C</infon><infon key="name_1">surname:Paasch;given-names:BC</infon><infon key="name_2">surname:Gonzales-Esquer;given-names:R</infon><infon key="name_3">surname:Erbilgin;given-names:O</infon><infon key="name_4">surname:Kerfeld;given-names:CA</infon><infon key="pub-id_pmid">26478774</infon><infon key="section_type">REF</infon><infon key="source">Communicative &amp; Integrative Biology</infon><infon key="type">ref</infon><infon key="volume">8</infon><infon key="year">2015</infon><offset>47612</offset><text>Bacterial Microcompartment Assembly: The Key Role of Encapsulation Peptides</text></passage><passage><infon key="fpage">14995</infon><infon key="issue">37</infon><infon key="lpage">5000</infon><infon key="name_0">surname:Fan;given-names:C</infon><infon key="name_1">surname:Cheng;given-names:S</infon><infon key="name_2">surname:Sinha;given-names:S</infon><infon key="name_3">surname:Bobik;given-names:Ta</infon><infon key="pub-id_doi">10.1073/pnas.1207516109</infon><infon key="pub-id_pmid">22927404</infon><infon key="section_type">REF</infon><infon key="source">Proceedings of the National Academy of Sciences of the United States of America</infon><infon key="type">ref</infon><infon key="volume">109</infon><infon key="year">2012</infon><offset>47688</offset><text>Interactions between the termini of lumen enzymes and shell proteins mediate enzyme encapsulation into bacterial microcompartments</text></passage><passage><infon key="fpage">17729</infon><infon key="issue">21</infon><infon key="lpage">36</infon><infon key="name_0">surname:Kinney;given-names:JN</infon><infon key="name_1">surname:Salmeen;given-names:A</infon><infon key="name_2">surname:Cai;given-names:F</infon><infon key="name_3">surname:Kerfeld;given-names:CA</infon><infon key="pub-id_doi">10.1074/jbc.M112.355305</infon><infon key="pub-id_pmid">22461622</infon><infon key="section_type">REF</infon><infon key="source">The Journal of biological chemistry</infon><infon key="type">ref</infon><infon key="volume">287</infon><infon key="year">2012</infon><offset>47819</offset><text>Elucidating the essential role of the conserved carboxysomal protein CcmN reveals a common feature of bacterial microcompartment assembly</text></passage><passage><infon key="fpage">455</infon><infon key="issue">3</infon><infon key="lpage">62</infon><infon key="name_0">surname:Tobimatsu;given-names:T</infon><infon key="name_1">surname:Kawata;given-names:M</infon><infon key="name_2">surname:Toraya;given-names:T</infon><infon key="name_3">surname:Obimatsu;given-names:TT</infon><infon key="name_4">surname:Awata;given-names:MK</infon><infon key="name_5">surname:Oraya;given-names:TT</infon><infon key="pub-id_pmid">15784971</infon><infon key="section_type">REF</infon><infon key="source">Bioscience, Biotechnology, and Biochemistry</infon><infon key="type">ref</infon><infon key="volume">69</infon><infon key="year">2005</infon><offset>47957</offset><text>The N-Terminal Regions of β and γ Subunits Lower the Solubility of Adenosylcobalamin-Dependent Diol Dehydratase</text></passage><passage><infon key="fpage">26484</infon><infon key="issue">34</infon><infon key="lpage">93</infon><infon key="name_0">surname:Shibata;given-names:N</infon><infon key="name_1">surname:Tamagaki;given-names:H</infon><infon key="name_2">surname:Hieda;given-names:N</infon><infon key="name_3">surname:Akita;given-names:K</infon><infon key="name_4">surname:Komori;given-names:H</infon><infon key="name_5">surname:Shomura;given-names:Y</infon><infon key="pub-id_doi">10.1074/jbc.M110.125112</infon><infon key="pub-id_pmid">20519496</infon><infon key="section_type">REF</infon><infon key="source">The Journal of biological chemistry</infon><infon key="type">ref</infon><infon key="volume">285</infon><infon key="year">2010</infon><offset>48076</offset><text>Crystal structures of ethanolamine ammonia-lyase complexed with coenzyme B12 analogs and substrates</text></passage><passage><infon key="fpage">273</infon><infon key="issue">2</infon><infon key="lpage">9</infon><infon key="name_0">surname:Frank;given-names:S</infon><infon key="name_1">surname:Lawrence;given-names:AD</infon><infon key="name_2">surname:Prentice;given-names:MB</infon><infon key="name_3">surname:Warren;given-names:MJ</infon><infon key="pub-id_doi">10.1016/j.jbiotec.2012.09.002</infon><infon key="pub-id_pmid">22982517</infon><infon key="section_type">REF</infon><infon key="source">Journal of biotechnology</infon><infon key="type">ref</infon><infon key="volume">163</infon><infon key="year">2013</infon><offset>48176</offset><text>Bacterial microcompartments moving into a synthetic biological world</text></passage><passage><infon key="name_0">surname:Gonzalez-Esquer;given-names:CR</infon><infon key="name_1">surname:Shubitowski;given-names:TB</infon><infon key="name_2">surname:Kerfeld;given-names:CA</infon><infon key="section_type">REF</infon><infon key="source">Plant Cell</infon><infon key="type">ref</infon><offset>48245</offset><text>Streamlined construction of the cyanobacterial CO2-fixing organelle via protein domain fusions</text></passage><passage><infon key="fpage">1143</infon><infon key="issue">3</infon><infon key="lpage">54</infon><infon key="name_0">surname:Lawrence;given-names:SH</infon><infon key="name_1">surname:Luther;given-names:KB</infon><infon key="name_2">surname:Schindelin;given-names:H</infon><infon key="name_3">surname:Ferry;given-names:JG</infon><infon key="pub-id_doi">10.1128/JB.188.3.1143-1154.2006</infon><infon key="pub-id_pmid">16428418</infon><infon key="section_type">REF</infon><infon key="source">J Bacteriol</infon><infon key="type">ref</infon><infon key="volume">188</infon><infon key="year">2006</infon><offset>48340</offset><text>Structural and functional studies suggest a catalytic mechanism for the phosphotransacetylase from Methanosarcina thermophila</text></passage><passage><infon key="fpage">2193</infon><infon key="issue">7</infon><infon key="lpage">205</infon><infon key="name_0">surname:Erbilgin;given-names:O</infon><infon key="name_1">surname:McDonald;given-names:KL</infon><infon key="name_2">surname:Kerfeld;given-names:Ca</infon><infon key="pub-id_doi">10.1128/AEM.03887-13</infon><infon key="pub-id_pmid">24487526</infon><infon key="section_type">REF</infon><infon key="source">Applied and environmental microbiology</infon><infon key="type">ref</infon><infon key="volume">80</infon><infon key="year">2014</infon><offset>48466</offset><text>Characterization of a planctomycetal organelle: a novel bacterial microcompartment for the aerobic degradation of plant saccharides</text></passage><passage><infon key="fpage">57</infon><infon key="issue">1</infon><infon key="lpage">63</infon><infon key="name_0">surname:Chakrabarti;given-names:P</infon><infon key="pub-id_pmid">2290835</infon><infon key="section_type">REF</infon><infon key="source">Protein engineering</infon><infon key="type">ref</infon><infon key="volume">4</infon><infon key="year">1990</infon><offset>48598</offset><text>Geometry of interaction of metal ions with histidine residues in protein structures</text></passage><passage><infon key="fpage">2392</infon><infon key="issue">14</infon><infon key="lpage">9</infon><infon key="name_0">surname:Liu;given-names:Y</infon><infon key="name_1">surname:Jorda;given-names:J</infon><infon key="name_2">surname:Yeates;given-names:TO</infon><infon key="name_3">surname:Bobik;given-names:TA</infon><infon key="pub-id_doi">10.1128/JB.00056-15</infon><infon key="pub-id_pmid">25962918</infon><infon key="section_type">REF</infon><infon key="source">J Bacteriol</infon><infon key="type">ref</infon><infon key="volume">197</infon><infon key="year">2015</infon><offset>48682</offset><text>The PduL Phosphotransacylase Is Used To Recycle Coenzyme A within the Pdu Microcompartment</text></passage><passage><infon key="fpage">1377</infon><infon key="issue">9</infon><infon key="lpage">88</infon><infon key="name_0">surname:Kloer;given-names:DP</infon><infon key="name_1">surname:Hagel;given-names:C</infon><infon key="name_2">surname:Heider;given-names:J</infon><infon key="name_3">surname:Schulz;given-names:GE</infon><infon key="pub-id_doi">10.1016/j.str.2006.07.001</infon><infon key="pub-id_pmid">16962969</infon><infon key="section_type">REF</infon><infon key="source">Structure</infon><infon key="type">ref</infon><infon key="volume">14</infon><infon key="year">2006</infon><offset>48773</offset><text>Crystal structure of ethylbenzene dehydrogenase from Aromatoleum aromaticum</text></passage><passage><infon key="fpage">1141</infon><infon key="issue">2</infon><infon key="lpage">71</infon><infon key="name_0">surname:Cai;given-names:F</infon><infon key="name_1">surname:Dou;given-names:Z</infon><infon key="name_2">surname:Bernstein;given-names:SL</infon><infon key="name_3">surname:Leverenz;given-names:R</infon><infon key="name_4">surname:Williams;given-names:EB</infon><infon key="name_5">surname:Heinhorst;given-names:S</infon><infon key="pub-id_doi">10.3390/life5021141</infon><infon key="pub-id_pmid">25826651</infon><infon key="section_type">REF</infon><infon key="source">Life (Basel)</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2015</infon><offset>48849</offset><text>Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component</text></passage><passage><infon key="fpage">1131</infon><infon key="issue">5</infon><infon key="lpage">40</infon><infon key="name_0">surname:Cameron;given-names:JC</infon><infon key="name_1">surname:Wilson;given-names:SC</infon><infon key="name_2">surname:Bernstein;given-names:SL</infon><infon key="name_3">surname:Kerfeld;given-names:CA</infon><infon key="pub-id_doi">10.1016/j.cell.2013.10.044</infon><infon key="pub-id_pmid">24267892</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">155</infon><infon key="year">2013</infon><offset>48973</offset><text>Biogenesis of a Bacterial Organelle: The Carboxysome Assembly Pathway</text></passage><passage><infon key="fpage">140130103032007</infon><infon key="name_0">surname:Lawrence;given-names:AD</infon><infon key="name_1">surname:Frank;given-names:S</infon><infon key="name_2">surname:Newnham;given-names:S</infon><infon key="name_3">surname:Lee;given-names:MJ</infon><infon key="name_4">surname:Brown;given-names:IR</infon><infon key="name_5">surname:Xue;given-names:W-F</infon><infon key="pub-id_doi">10.1021/sb4001118</infon><infon key="section_type">REF</infon><infon key="source">ACS Synthetic Biology</infon><infon key="type">ref</infon><infon key="year">2014</infon><offset>49043</offset><text>Solution structure of a bacterial microcompartment targeting peptide and its application in the construction of an ethanol bioreactor</text></passage><passage><infon key="fpage">3252</infon><infon key="issue">8</infon><infon key="lpage">78</infon><infon key="name_0">surname:Cleland;given-names:WW</infon><infon key="name_1">surname:Hengge;given-names:AC</infon><infon key="pub-id_doi">10.1021/cr050287o</infon><infon key="pub-id_pmid">16895327</infon><infon key="section_type">REF</infon><infon key="source">Chem Rev</infon><infon key="type">ref</infon><infon key="volume">106</infon><infon key="year">2006</infon><offset>49177</offset><text>Enzymatic mechanisms of phosphate and sulfate transfer</text></passage><passage><infon key="fpage">1407</infon><infon key="issue">7</infon><infon key="lpage">18</infon><infon key="name_0">surname:Kimber;given-names:MS</infon><infon key="name_1">surname:Pai;given-names:EF</infon><infon key="pub-id_doi">10.1093/emboj/19.7.1407</infon><infon key="pub-id_pmid">10747009</infon><infon key="section_type">REF</infon><infon key="source">EMBO J</infon><infon key="type">ref</infon><infon key="volume">19</infon><infon key="year">2000</infon><offset>49232</offset><text>The active site architecture of Pisum sativum beta-carbonic anhydrase is a mirror image of that of alpha-carbonic anhydrases</text></passage><passage><infon key="fpage">48615</infon><infon key="issue">52</infon><infon key="lpage">8</infon><infon key="name_0">surname:Tripp;given-names:BC</infon><infon key="name_1">surname:Smith;given-names:K</infon><infon key="name_2">surname:Ferry;given-names:JG</infon><infon key="pub-id_doi">10.1074/jbc.R100045200</infon><infon key="pub-id_pmid">11696553</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">276</infon><infon key="year">2001</infon><offset>49357</offset><text>Carbonic anhydrase: new insights for an ancient enzyme</text></passage><passage><infon key="fpage">4914</infon><infon key="issue">20</infon><infon key="lpage">21</infon><infon key="name_0">surname:Carfi;given-names:A</infon><infon key="name_1">surname:Pares;given-names:S</infon><infon key="name_2">surname:Duee;given-names:E</infon><infon key="name_3">surname:Galleni;given-names:M</infon><infon key="name_4">surname:Duez;given-names:C</infon><infon key="name_5">surname:Frere;given-names:JM</infon><infon key="pub-id_pmid">7588620</infon><infon key="section_type">REF</infon><infon key="source">EMBO J</infon><infon key="type">ref</infon><infon key="volume">14</infon><infon key="year">1995</infon><offset>49412</offset><text>The 3-D structure of a zinc metallo-beta-lactamase from Bacillus cereus reveals a new type of protein fold</text></passage><passage><infon key="fpage">160</infon><infon key="issue">1</infon><infon key="lpage">201</infon><infon key="name_0">surname:Drawz;given-names:SM</infon><infon key="name_1">surname:Bonomo;given-names:RA</infon><infon key="pub-id_doi">10.1128/CMR.00037-09</infon><infon key="pub-id_pmid">20065329</infon><infon key="section_type">REF</infon><infon key="source">Clin Microbiol Rev</infon><infon key="type">ref</infon><infon key="volume">23</infon><infon key="year">2010</infon><offset>49519</offset><text>Three decades of beta-lactamase inhibitors</text></passage><passage><infon key="fpage">2455</infon><infon key="issue">6</infon><infon key="lpage">60</infon><infon key="name_0">surname:Peña;given-names:KL</infon><infon key="name_1">surname:Castel;given-names:SE</infon><infon key="name_2">surname:de Araujo;given-names:C</infon><infon key="name_3">surname:Espie;given-names:GS</infon><infon key="name_4">surname:Kimber;given-names:MS</infon><infon key="pub-id_doi">10.1073/pnas.0910866107</infon><infon key="pub-id_pmid">20133749</infon><infon key="section_type">REF</infon><infon key="source">Proceedings of the National Academy of Sciences of the United States of America</infon><infon key="type">ref</infon><infon key="volume">107</infon><infon key="year">2010</infon><offset>49562</offset><text>Structural basis of the oxidative activation of the carboxysomal gamma-carbonic anhydrase, CcmM</text></passage><passage><infon key="fpage">e76127</infon><infon key="issue">9</infon><infon key="lpage">e</infon><infon key="name_0">surname:Chen;given-names:AH</infon><infon key="name_1">surname:Robinson-Mosher;given-names:A</infon><infon key="name_2">surname:Savage;given-names:DF</infon><infon key="name_3">surname:Silver;given-names:Pa</infon><infon key="name_4">surname:Polka;given-names:JK</infon><infon key="pub-id_doi">10.1371/journal.pone.0076127</infon><infon key="pub-id_pmid">24023971</infon><infon key="section_type">REF</infon><infon key="source">PloS ONE</infon><infon key="type">ref</infon><infon key="volume">8</infon><infon key="year">2013</infon><offset>49658</offset><text>The bacterial carbon-fixing organelle is formed by shell envelopment of preassembled cargo</text></passage><passage><infon key="fpage">4105</infon><infon key="issue">10</infon><infon key="lpage">21</infon><infon key="name_0">surname:Harvey;given-names:PC</infon><infon key="name_1">surname:Watson;given-names:M</infon><infon key="name_2">surname:Hulme;given-names:S</infon><infon key="name_3">surname:Jones;given-names:Ma</infon><infon key="name_4">surname:Lovell;given-names:M</infon><infon key="name_5">surname:Berchieri;given-names:A</infon><infon key="pub-id_doi">10.1128/IAI.01390-10</infon><infon key="pub-id_pmid">21768276</infon><infon key="section_type">REF</infon><infon key="source">Infection and immunity</infon><infon key="type">ref</infon><infon key="volume">79</infon><infon key="year">2011</infon><offset>49749</offset><text> Salmonella enterica typhimurium colonizing the lumen of the chicken intestine grows slowly and upregulates a unique set of virulence and metabolism genes</text></passage><passage><infon key="fpage">556</infon><infon key="issue">2</infon><infon key="lpage">68</infon><infon key="name_0">surname:Joseph;given-names:B</infon><infon key="name_1">surname:Przybilla;given-names:K</infon><infon key="name_2">surname:Stu;given-names:C</infon><infon key="name_3">surname:Schauer;given-names:K</infon><infon key="name_4">surname:Fuchs;given-names:TM</infon><infon key="name_5">surname:Goebel;given-names:W</infon><infon key="pub-id_pmid">16385046</infon><infon key="section_type">REF</infon><infon key="source">Journal of bacteriology</infon><infon key="type">ref</infon><infon key="volume">188</infon><infon key="year">2006</infon><offset>49904</offset><text>Identification of Listeria monocytogenes Genes Contributing to Intracellular Replication by Expression Profiling and Mutant Screening</text></passage><passage><infon key="fpage">1</infon><infon key="issue">3</infon><infon key="lpage">10</infon><infon key="name_0">surname:Kendall;given-names:MM</infon><infon key="name_1">surname:Gruber;given-names:CC</infon><infon key="name_2">surname:Parker;given-names:CT</infon><infon key="name_3">surname:Sperandio;given-names:V</infon><infon key="pub-id_doi">10.1128/mBio.00050-12</infon><infon key="section_type">REF</infon><infon key="source">mBio</infon><infon key="type">ref</infon><infon key="volume">3</infon><infon key="year">2012</infon><offset>50038</offset><text>Ethanolamine controls expression of genes encoding components involved in interkingdom signaling and virulence in enterohemorrhagic Escherichia coli O157:H7</text></passage><passage><infon key="fpage">1207</infon><infon key="lpage">20</infon><infon key="name_0">surname:Klumpp;given-names:J</infon><infon key="name_1">surname:Fuchs;given-names:TM</infon><infon key="section_type">REF</infon><infon key="source">Microbiology</infon><infon key="type">ref</infon><infon key="volume">2</infon><infon key="year">2007</infon><offset>50195</offset><text>Identification of novel genes in genomic islands that contribute to Salmonella typhimurium replication in macrophages</text></passage><passage><infon key="fpage">2634</infon><infon key="issue">5</infon><infon key="lpage">7</infon><infon key="name_0">surname:Maadani;given-names:A</infon><infon key="name_1">surname:Fox;given-names:KA</infon><infon key="name_2">surname:Mylonakis;given-names:E</infon><infon key="name_3">surname:Garsin;given-names:DA</infon><infon key="pub-id_doi">10.1128/IAI.01372-06</infon><infon key="pub-id_pmid">17307944</infon><infon key="section_type">REF</infon><infon key="source">Infection and immunity</infon><infon key="type">ref</infon><infon key="volume">75</infon><infon key="year">2007</infon><offset>50313</offset><text> Enterococcus faecalis mutations affecting virulence in the Caenorhabditis elegans model host</text></passage><passage><infon key="fpage">239</infon><infon key="issue">6</infon><infon key="lpage">46</infon><infon key="name_0">surname:Mobley;given-names:H</infon><infon key="section_type">REF</infon><infon key="source">Microbe</infon><infon key="type">ref</infon><infon key="volume">10</infon><infon key="year">2015</infon><offset>50407</offset><text>Redefining Virulence of Bacterial Pathogens</text></passage><passage><infon key="fpage">248</infon><infon key="lpage">54</infon><infon key="name_0">surname:Bradford;given-names:MM</infon><infon key="pub-id_pmid">942051</infon><infon key="section_type">REF</infon><infon key="source">Anal Biochem</infon><infon key="type">ref</infon><infon key="volume">72</infon><infon key="year">1976</infon><offset>50451</offset><text>A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding</text></passage><passage><infon key="fpage">45</infon><infon key="issue">2</infon><infon key="lpage">52</infon><infon key="name_0">surname:Luzi;given-names:S</infon><infon key="name_1">surname:Kondo;given-names:Y</infon><infon key="name_2">surname:Bernard;given-names:E</infon><infon key="name_3">surname:Stadler;given-names:LK</infon><infon key="name_4">surname:Vaysburd;given-names:M</infon><infon key="name_5">surname:Winter;given-names:G</infon><infon key="pub-id_doi">10.1093/protein/gzu055</infon><infon key="pub-id_pmid">25614525</infon><infon key="section_type">REF</infon><infon key="source">Protein Eng Des Sel</infon><infon key="type">ref</infon><infon key="volume">28</infon><infon key="year">2015</infon><offset>50583</offset><text>Subunit disassembly and inhibition of TNFalpha by a semi-synthetic bicyclic peptide</text></passage><passage><infon key="fpage">1792</infon><infon key="issue">5</infon><infon key="lpage">7</infon><infon key="name_0">surname:Edgar;given-names:RC</infon><infon key="pub-id_doi">10.1093/nar/gkh340</infon><infon key="pub-id_pmid">15034147</infon><infon key="section_type">REF</infon><infon key="source">Nucleic acids research</infon><infon key="type">ref</infon><infon key="volume">32</infon><infon key="year">2004</infon><offset>50667</offset><text>MUSCLE: multiple sequence alignment with high accuracy and high throughput</text></passage><passage><infon key="fpage">1189</infon><infon key="issue">9</infon><infon key="lpage">91</infon><infon key="name_0">surname:Waterhouse;given-names:AM</infon><infon key="name_1">surname:Procter;given-names:JB</infon><infon key="name_2">surname:Martin;given-names:DMa</infon><infon key="name_3">surname:Clamp;given-names:M</infon><infon key="name_4">surname:Barton;given-names:GJ</infon><infon key="pub-id_doi">10.1093/bioinformatics/btp033</infon><infon key="section_type">REF</infon><infon key="source">Bioinformatics (Oxford, England)</infon><infon key="type">ref</infon><infon key="volume">25</infon><infon key="year">2009</infon><offset>50742</offset><text>Jalview Version 2—a multiple sequence alignment editor and analysis workbench</text></passage><passage><infon key="fpage">1188</infon><infon key="issue">6</infon><infon key="lpage">90</infon><infon key="name_0">surname:Crooks;given-names:GE</infon><infon key="name_1">surname:Hon;given-names:G</infon><infon key="name_2">surname:Chandonia;given-names:JM</infon><infon key="name_3">surname:Brenner;given-names:SE</infon><infon key="pub-id_doi">10.1101/gr.849004</infon><infon key="pub-id_pmid">15173120</infon><infon key="section_type">REF</infon><infon key="source">Genome Res</infon><infon key="type">ref</infon><infon key="volume">14</infon><infon key="year">2004</infon><offset>50822</offset><text>WebLogo: a sequence logo generator</text></passage><passage><infon key="fpage">125</infon><infon key="issue">Pt 2</infon><infon key="lpage">32</infon><infon key="name_0">surname:Kabsch;given-names:W</infon><infon key="pub-id_doi">10.1107/S0907444909047337</infon><infon key="pub-id_pmid">20124692</infon><infon key="section_type">REF</infon><infon key="source">Acta crystallographica Section D, Biological crystallography</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>50857</offset><text>XDS</text></passage><passage><infon key="fpage">235</infon><infon key="issue">Pt 4</infon><infon key="lpage">42</infon><infon key="name_0">surname:Winn;given-names:MD</infon><infon key="name_1">surname:Ballard;given-names:CC</infon><infon key="name_2">surname:Cowtan;given-names:KD</infon><infon key="name_3">surname:Dodson;given-names:EJ</infon><infon key="name_4">surname:Emsley;given-names:P</infon><infon key="name_5">surname:Evans;given-names:PR</infon><infon key="pub-id_doi">10.1107/S0907444910045749</infon><infon key="pub-id_pmid">21460441</infon><infon key="section_type">REF</infon><infon key="source">Acta crystallographica Section D, Biological crystallography</infon><infon key="type">ref</infon><infon key="volume">67</infon><infon key="year">2011</infon><offset>50861</offset><text>Overview of the CCP4 suite and current developments</text></passage><passage><infon key="fpage">352</infon><infon key="lpage">67</infon><infon key="name_0">surname:Afonine;given-names:PV</infon><infon key="name_1">surname:Grosse-Kunstleve;given-names:RW</infon><infon key="name_2">surname:Echols;given-names:N</infon><infon key="name_3">surname:Headd;given-names:JJ</infon><infon key="name_4">surname:Moriarty;given-names:NW</infon><infon key="name_5">surname:Mustyakimov;given-names:M</infon><infon key="pub-id_doi">10.1107/S0907444912001308</infon><infon key="pub-id_pmid">22505256</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D</infon><infon key="type">ref</infon><infon key="volume">68</infon><infon key="year">2012</infon><offset>50913</offset><text>Towards automated crystallographic structure refinement with phenix.refine</text></passage><passage><infon key="fpage">2126</infon><infon key="issue">Pt 12 Pt 1</infon><infon key="lpage">32</infon><infon key="name_0">surname:Emsley;given-names:P</infon><infon key="name_1">surname:Cowtan;given-names:K</infon><infon key="pub-id_doi">10.1107/S0907444904019158</infon><infon key="pub-id_pmid">15572765</infon><infon key="section_type">REF</infon><infon key="source">Acta crystallographica Section D, Biological crystallography</infon><infon key="type">ref</infon><infon key="volume">60</infon><infon key="year">2004</infon><offset>50988</offset><text>Coot: model-building tools for molecular graphics</text></passage><passage><infon key="fpage">505</infon><infon key="lpage">24</infon><infon key="name_0">surname:Merritt;given-names:EA</infon><infon key="name_1">surname:Bacon;given-names:DJ</infon><infon key="pub-id_pmid">18488322</infon><infon key="section_type">REF</infon><infon key="source">Methods in enzymology</infon><infon key="type">ref</infon><infon key="volume">277</infon><infon key="year">1997</infon><offset>51038</offset><text>Raster3D: photorealistic molecular graphics</text></passage><passage><infon key="fpage">291</infon><infon key="lpage">325</infon><infon key="name_0">surname:Marti-Renom;given-names:MA</infon><infon key="name_1">surname:Stuart;given-names:AC</infon><infon key="name_2">surname:Fiser;given-names:A</infon><infon key="name_3">surname:Sanchez;given-names:R</infon><infon key="name_4">surname:Melo;given-names:F</infon><infon key="name_5">surname:Sali;given-names:A</infon><infon key="pub-id_doi">10.1146/annurev.biophys.29.1.291</infon><infon key="pub-id_pmid">10940251</infon><infon key="section_type">REF</infon><infon key="source">Annu Rev Biophys Biomol Struct</infon><infon key="type">ref</infon><infon key="volume">29</infon><infon key="year">2000</infon><offset>51082</offset><text>Comparative protein structure modeling of genes and genomes</text></passage></document></collection>
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+<collection><source>PMC</source><date>20201222</date><key>pmc.key</key><document><id>4786784</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/ncomms10950</infon><infon key="article-id_pii">ncomms10950</infon><infon key="article-id_pmc">4786784</infon><infon key="article-id_pmid">26952537</infon><infon key="elocation-id">10950</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Agrawal;given-names:Anant A.</infon><infon key="name_1">surname:Salsi;given-names:Enea</infon><infon key="name_2">surname:Chatrikhi;given-names:Rakesh</infon><infon key="name_3">surname:Henderson;given-names:Steven</infon><infon key="name_4">surname:Jenkins;given-names:Jermaine L.</infon><infon key="name_5">surname:Green;given-names:Michael R.</infon><infon key="name_6">surname:Ermolenko;given-names:Dmitri N.</infon><infon key="name_7">surname:Kielkopf;given-names:Clara L.</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">7</infon><infon key="year">2016</infon><offset>0</offset><text>An extended U2AF65–RNA-binding domain recognizes the 3′ splice site signal</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>79</offset><text>How the essential pre-mRNA splicing factor U2AF65 recognizes the polypyrimidine (Py) signals of the major class of 3′ splice sites in human gene transcripts remains incompletely understood. We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3′ terminus and third nucleotide. Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF65 RRMs are complementary mechanisms for Py-tract association. Altogether, these results advance the mechanistic understanding of molecular recognition for a major class of splice site signals.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1090</offset><text> The pre-mRNA splicing factor U2AF65 recognizes 3′ splice sites in human gene transcripts, but the details are not fully understood. Here, the authors report U2AF65 structures and single molecule FRET that reveal mechanistic insights into splice site recognition.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1356</offset><text>The differential skipping or inclusion of alternatively spliced pre-mRNA regions is a major source of diversity for nearly all human gene transcripts. The splice sites are marked by relatively short consensus sequences and are regulated by additional pre-mRNA motifs (reviewed in ref.). At the 3′ splice site of the major intron class, these include a polypyrimidine (Py) tract comprising primarily Us or Cs, which is preceded by a branch point sequence (BPS) that ultimately serves as the nucleophile in the splicing reaction and an AG-dinucleotide at the 3′ splice site junction. Disease-causing mutations often compromise pre-mRNA splicing (reviewed in refs), yet a priori predictions of splice sites and the consequences of their mutations are challenged by the brevity and degeneracy of known splice site sequences. High-resolution structures of intact splicing factor–RNA complexes would offer key insights regarding the juxtaposition of the distinct splice site consensus sequences and their relationship to disease-causing point mutations.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2410</offset><text>The early-stage pre-mRNA splicing factor U2AF65 is essential for viability in vertebrates and other model organisms (for example, ref.). A tightly controlled assembly among U2AF65, the pre-mRNA, and partner proteins sequentially identifies the 3′ splice site and promotes association of the spliceosome, which ultimately accomplishes the task of splicing. Initially U2AF65 recognizes the Py-tract splice site signal. In turn, the ternary complex of U2AF65 with SF1 and U2AF35 identifies the surrounding BPS and 3′ splice site junctions. Subsequently U2AF65 recruits the U2 small nuclear ribonucleoprotein particle (snRNP) and ultimately dissociates from the active spliceosome.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3092</offset><text>Biochemical characterizations of U2AF65 demonstrated that tandem RNA recognition motifs (RRM1 and RRM2) recognize the Py tract (Fig. 1a). Milestone crystal structures of the core U2AF65 RRM1 and RRM2 connected by a shortened inter-RRM linker (dU2AF651,2) detailed a subset of nucleotide interactions with the individual U2AF65 RRMs. A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation. As such, the molecular mechanisms for Py-tract recognition by the intact U2AF65–RNA-binding domain remained unknown. Here, we use X-ray crystallography and biochemical studies to reveal new roles in Py-tract recognition for the inter-RRM linker and key residues surrounding the core U2AF65 RRMs. We use single-molecule Förster resonance energy transfer (smFRET) to characterize the conformational dynamics of this extended U2AF65–RNA-binding domain during Py-tract recognition.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>4200</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>4208</offset><text>Cognate U2AF65–Py-tract recognition requires RRM extensions</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4270</offset><text>The RNA affinity of the minimal U2AF651,2 domain comprising the core RRM1–RRM2 folds (U2AF651,2, residues 148–336) is relatively weak compared with full-length U2AF65 (Fig. 1a,b; Supplementary Fig. 1). Historically, this difference was attributed to the U2AF65 arginine–serine rich domain, which contacts pre-mRNA–U2 snRNA duplexes outside of the Py tract. We noticed that the RNA-binding affinity of the U2AF651,2 domain was greatly enhanced by the addition of seven and six residues at the respective N and C termini of the minimal RRM1 and RRM2 (U2AF651,2L, residues 141–342; Fig. 1a). In a fluorescence anisotropy assay for binding a representative Py tract derived from the well-characterized splice site of the adenovirus major late promoter (AdML), the RNA affinity of U2AF651,2L increased by 100-fold relative to U2AF651,2 to comparable levels as full-length U2AF65 (Fig. 1b; Supplementary Fig. 1a–d). Likewise, both U2AF651,2L and full-length U2AF65 showed similar sequence specificity for U-rich stretches in the 5′-region of the Py tract and promiscuity for C-rich regions in the 3′-region (Fig. 1c, Supplementary Fig. 1e–h).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>5425</offset><text>U2AF65-bound Py tract comprises nine contiguous nucleotides</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5485</offset><text>To investigate the structural basis for cognate U2AF65 recognition of a contiguous Py tract, we determined four crystal structures of U2AF651,2L bound to Py-tract oligonucleotides (Fig. 2a; Table 1). By sequential boot strapping (Methods), we optimized the oligonucleotide length, the position of a Br-dU, and the identity of the terminal nucleotide (rU, dU and rC) to achieve full views of U2AF651,2L bound to contiguous Py tracts at up to 1.5 Å resolution. The protein and oligonucleotide conformations are nearly identical among the four new U2AF651,2L structures (Supplementary Fig. 2a). The U2AF651,2L RRM1 and RRM2 associate with the Py tract in a parallel, side-by-side arrangement (shown for representative structure iv in Fig. 2b,c; Supplementary Movie 1). An extended conformation of the U2AF65 inter-RRM linker traverses across the α-helical surface of RRM1 and the central β-strands of RRM2 and is well defined in the electron density (Fig. 2b). The extensions at the N terminus of RRM1 and C terminus of RRM2 adopt well-ordered α-helices. Both RRM1/RRM2 extensions and the inter-RRM linker of U2AF651,2L directly recognize the bound oligonucleotide. We compare the global conformation of the U2AF651,2L structures with the prior dU2AF651,2 crystal structure and U2AF651,2 NMR structure in the Supplementary Discussion and Supplementary Fig. 2.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6853</offset><text>The discovery of nine U2AF65-binding sites for contiguous Py-tract nucleotides was unexpected. Based on dU2AF651,2 structures, we originally hypothesized that the U2AF65 RRMs would bind the minimal seven nucleotides observed in these structures. Surprisingly, the RRM2 extension/inter-RRM linker contribute new central nucleotide-binding sites near the RRM1/RRM2 junction and the RRM1 extension recognizes the 3′-terminal nucleotide (Fig. 2c; Supplementary Movie 1). The U2AF651,2L structures characterize ribose (r) nucleotides at all of the binding sites except the seventh and eighth deoxy-(d)U, which are likely to lack 2′-hydroxyl contacts based on the RNA-bound dU2AF651,2 structure. Qualitatively, a subset of the U2AF651,2L-nucleotide-binding sites (sites 1–3 and 7–9) share similar locations to those of the dU2AF651,2 structures (Supplementary Figs 2c,d and 3). Yet, only the U2AF651,2L interactions at sites 1 and 7 are nearly identical to those of the dU2AF651,2 structures (Supplementary Fig. 3a,f). In striking departures from prior partial views, the U2AF651,2L structures reveal three unanticipated nucleotide-binding sites at the centre of the Py tract, as well as numerous new interactions that underlie cognate recognition of the Py tract (Fig. 3a–h).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8134</offset><text>U2AF65 inter-RRM linker interacts with the Py tract</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8186</offset><text>The U2AF651,2L RRM2, the inter-RRM linker and RRM1 concomitantly recognize the three central nucleotides of the Py tract, which are likely to coordinate the conformational arrangement of these disparate portions of the protein. Residues in the C-terminal region of the U2AF65 inter-RRM linker comprise a centrally located binding site for the fifth nucleotide on the RRM2 surface and abutting the RRM1/RRM2 interface (Fig. 3d). The backbone amide of the linker V254 and the carbonyl of T252 engage in hydrogen bonds with the rU5-O4 and -N3H atoms. In the C-terminal β-strand of RRM1, the side chains of K225 and R227 donate additional hydrogen bonds to the rU5-O2 lone pair electrons. The C-terminal region of the inter-RRM linker also participates in the preceding rU4-binding site, where the V254 backbone carbonyl and D256 carboxylate position the K260 side chain to hydrogen bond with the rU4-O4 (Fig. 3c). Otherwise, the rU4 nucleotide packs against F304 in the signature ribonucleoprotein consensus motif (RNP)-2 of RRM2.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9217</offset><text>At the opposite side of the central fifth nucleotide, the sixth rU6 nucleotide is located at the inter-RRM1/RRM2 interface (Fig. 3e; Supplementary Movie 1). This nucleotide twists to face away from the U2AF65 linker and instead inserts the rU6-uracil into a sandwich between the β2/β3 loops of RRM1 and RRM2. The rU6 base edge is relatively solvent exposed; accordingly, the rU6 hydrogen bonds with U2AF65 are water mediated apart from a single direct interaction by the RRM1-N196 side chain.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9716</offset><text>We tested the contribution of the U2AF651,2L interactions with the new central nucleotide to Py-tract affinity (Fig. 3i; Supplementary Fig. 4a,b). Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1–R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively. The energetic penalties due to these mutations (ΔΔG 0.8–0.9 kcal mol−1) are consistent with the loss of each hydrogen bond with the rU5 base and support the relevance of the central nucleotide interactions observed in the U2AF651,2L structures.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10389</offset><text>U2AF65 RRM extensions interact with the Py tract</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10438</offset><text>The N- and C-terminal extensions of the U2AF65 RRM1 and RRM2 directly contact the bound Py tract. Rather than interacting with a new 5′-terminal nucleotide as we had hypothesized, the C-terminal α-helix of RRM2 instead folds across one surface of rU3 in the third binding site (Fig. 3b). There, a salt bridge between the K340 side chain and nucleotide phosphate, as well as G338-base stacking and a hydrogen bond between the backbone amide of G338 and the rU3-O4, secure the RRM2 extension. Indirectly, the additional contacts with the third nucleotide shift the rU2 nucleotide in the second binding site closer to the C-terminal β-strand of RRM2. Consequently, the U2AF651,2L-bound rU2-O4 and -N3H form dual hydrogen bonds with the K329 backbone atoms (Fig. 3a), rather than a single hydrogen bond with the K329 side chain as in the prior dU2AF651,2 structure (Supplementary Fig. 3b).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11331</offset><text>At the N terminus, the α-helical extension of U2AF65 RRM1 positions the Q147 side chain to bridge the eighth and ninth nucleotides at the 3′ terminus of the Py tract (Fig. 3f–h). The Q147 residue participates in hydrogen bonds with the -N3H of the eighth uracil and -O2 of the ninth pyrimidine. The adjacent R146 guanidinium group donates hydrogen bonds to the 3′-terminal ribose-O2′ and O3′ atoms, where it could form a salt bridge with a phospho-diester group in the context of a longer pre-mRNA. Consistent with loss of a hydrogen bond with the ninth pyrimidine-O2 (ΔΔG 1.0 kcal mol−1), mutation of the Q147 to an alanine reduced U2AF651,2L affinity for the AdML Py tract by five-fold (Fig. 3i; Supplementary Fig. 4c). We compare U2AF65 interactions with uracil relative to cytosine pyrimidines at the ninth binding site in Fig. 3g,h and the Supplementary Discussion.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>12221</offset><text>Versatile primary sequence of the U2AF65 inter-RRM linker</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12279</offset><text>The U2AF651,2L structures reveal that the inter-RRM linker mediates an extensive interface with the second α-helix of RRM1, the β2/β3 strands of RRM2 and the N-terminal α-helical extension of RRM1. Altogether, the U2AF65 inter-RRM linker residues (R228–K260) bury 2,800 Å2 of surface area in the U2AF651,2L holo-protein, suggestive of a cognate interface compared with 1,900 Å2 for a typical protein–protein complex. The path of the linker initiates at P229 following the core RRM1 β-strand, in a kink that is positioned by intra-molecular stacking among the consecutive R228, Y232 and P234 side chains (Fig. 4a, lower right). A second kink at P236, coupled with respective packing of the L235 and M238 side chains on the N-terminal α-helical RRM1 extension and the core RRM1 α2-helix, reverses the direction of the inter-RRM linker towards the RRM1/RRM2 interface and away from the RNA-binding site. In the neighbouring apical region of the linker, the V244 and V246 side chains pack in a hydrophobic pocket between two α-helices of the core RRM1. The adjacent V249 and V250 are notable for their respective interactions that connect RRM1 and RRM2 at this distal interface from the RNA-binding site (Fig. 4a, top). A third kink stacks P247 and G248 with Y245 and re-orients the C-terminal region of the linker towards the RRM2 and bound RNA. At the RNA surface, the key V254 that recognizes the fifth uracil is secured via hydrophobic contacts between its side chain and the β-sheet surface of RRM2, chiefly the consensus RNP1-F304 residue that stacks with the fourth uracil (Fig. 4a, lower left). Few direct contacts are made between the remaining residues of the linker and the U2AF65 RRM2; instead, the C-terminal conformation of the linker appears primarily RNA mediated (Fig. 3c,d).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14100</offset><text>We investigated whether the observed contacts between the RRMs and linker were critical for RNA binding by structure-guided mutagenesis (Fig. 4b). We titrated these mutant U2AF651,2L proteins into fluorescein-labelled AdML Py-tract RNA and fit the fluorescence anisotropy changes to obtain the apparent equilibrium affinities (Supplementary Fig. 4d–h). We introduced glycine substitutions to maximally reduce the buried surface area without directly interfering with its hydrogen bonds between backbone atoms and the base. First, we replaced V249 and V250 at the RRM1/RRM2 interface and V254 at the bound RNA site with glycine (3Gly). However, the resulting decrease in the AdML RNA affinity of the U2AF651,2L-3Gly mutant relative to wild-type protein was not significant (Fig. 4b). In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein. A more conservative substitution of these five residues (251–255) with an unrelated sequence capable of backbone-mediated hydrogen bonds (STVVP&gt;NLALA) confirmed the subtle impact of this versatile inter-RRM sequence on affinity for the AdML Py tract. Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). Despite 12 concurrent mutations, the AdML RNA affinity of the U2AF651,2L-12Gly variant was reduced by only three-fold relative to the unmodified protein (Fig. 4b), which is less than the penalty of the V254P mutation that disrupts the rU5 hydrogen bond (Fig. 3d,i).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15968</offset><text>To test the interplay of the U2AF65 inter-RRM linker with its N- and C-terminal RRM extensions, we constructed an internal linker deletion of 20-residues within the extended RNA-binding domain (dU2AF651,2L). We found that the affinity of dU2AF651,2L for the AdML RNA was significantly reduced relative to U2AF651,2L (four-fold, Figs 1b and 4b; Supplementary Fig. 4i). Yet, it is well known that the linker deletion in the context of the minimal RRM1–RRM2 boundaries has no detectable effect on the RNA affinities of dU2AF651,2 compared with U2AF651,2 (refs; Figs 1b and 4b; Supplementary Fig. 4j). The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. However, stretching of the truncated dU2AF651,2L linker to connect the RRM termini is expected to disrupt its nucleotide interactions. Likewise, deletion of the N-terminal RRM1 extension in the shortened constructs would remove packing interactions that position the linker in a kinked turn following P229 (Fig. 4a), consistent with the lower RNA affinities of dU2AF651,2L, dU2AF651,2 and U2AF651,2 compared with U2AF651,2L.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17374</offset><text>To further test cooperation among the U2AF65 RRM extensions and inter-RRM linker for RNA recognition, we tested the impact of a triple Q147A/V254P/R227A mutation (U2AF651,2L-3Mut) for RNA binding (Fig. 4b; Supplementary Fig. 4d). Notably, the Q147A/V254P/R227A mutation reduced the RNA affinity of the U2AF651,2L-3Mut protein by 30-fold more than would be expected based on simple addition of the ΔΔG's for the single mutations. This difference indicates that the linearly distant regions of the U2AF65 primary sequence, including Q147 in the N-terminal RRM1 extension and R227/V254 in the N-/C-terminal linker regions at the fifth nucleotide site, cooperatively recognize the Py tract. Altogether, we conclude that the conformation of the U2AF65 inter-RRM linker is key for recognizing RNA and is positioned by the RRM extension but otherwise relatively independent of the side chain composition. The non-additive effects of the Q147A/V254P/R227A triple mutation, coupled with the context-dependent penalties of an internal U2AF65 linker deletion, highlights the importance of the structural interplay among the U2AF65 linker and the N- and C-terminal extensions flanking the core RRMs.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>18570</offset><text>Importance of U2AF65–RNA contacts for pre-mRNA splicing</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18628</offset><text>We proceeded to test the importance of new U2AF65–Py-tract interactions for splicing of a model pre-mRNA substrate in a human cell line (Fig. 5; Supplementary Fig. 5). As a representative splicing substrate, we utilized a well-characterized minigene splicing reporter (called pyPY) comprising a weak (that is, degenerate, py) and strong (that is, U-rich, PY) polypyrimidine tracts preceding two alternative splice sites (Fig. 5a). When transfected into HEK293T cells containing only endogenous U2AF65, the PY splice site is used and the remaining transcript remains unspliced. When co-transfected with an expression plasmid for wild-type U2AF65, use of the py splice site significantly increases (by more than five-fold) and as documented converts a fraction of the unspliced to spliced transcript. The strong PY splice site is insensitive to added U2AF65, suggesting that endogenous U2AF65 levels are sufficient to saturate this site (Supplementary Fig. 5b). We introduced the triple mutation (V254P/R227A/Q147A) that significantly reduced U2AF651,2L association with the Py tract (Fig. 4b) in the context of full-length U2AF65 (U2AF65-3Mut). Co-transfection of the U2AF65-3Mut with the pyPY splicing substrate significantly reduced splicing of the weak ‘py' splice site relative to wild-type U2AF65 (Fig. 5b,c). We conclude that the Py-tract interactions with these residues of the U2AF65 inter-RRM linker and RRM extensions are important for splicing as well as for binding a representative of the major U2-class of splice sites.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>20165</offset><text>Sparse inter-RRM contacts underlie apo-U2AF65 dynamics</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20220</offset><text>The direct interface between U2AF651,2L RRM1 and RRM2 is minor, burying 265 Å2 of solvent accessible surface area compared with 570 Å2 on average for a crystal packing interface. A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c). This minor U2AF65 RRM1/RRM2 interface, coupled with the versatile sequence of the inter-RRM linker, highlighted the potential role for inter-RRM conformational dynamics in U2AF65-splice site recognition. Paramagnetic resonance enhancement (PRE) measurements previously had suggested a predominant back-to-back, or ‘closed' conformation of the apo-U2AF651,2 RRM1 and RRM2 in equilibrium with a minor ‘open' conformation resembling the RNA-bound inter-RRM arrangement. Yet, small-angle X-ray scattering (SAXS) data indicated that both the minimal U2AF651,2 and longer constructs comprise a highly diverse continuum of conformations in the absence of RNA that includes the ‘closed' and ‘open' conformations. To complement the static portraits of U2AF651,2L structure that we had determined by X-ray crystallography, we used smFRET to characterize the probability distribution functions and time dependence of U2AF65 inter-RRM conformational dynamics in solution.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21568</offset><text>The inter-RRM dynamics of U2AF65 were followed using FRET between fluorophores attached to RRM1 and RRM2 (Fig. 6a,b, Methods). The positions of single cysteine mutations for fluorophore attachment (A181C in RRM1 and Q324C in RRM2) were chosen based on inspection of the U2AF651,2L structures and the ‘closed' model of apo-U2AF651,2. Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50 Å for U2AF651,2L–Py tract and 30 Å for the closed apo-model) that are expected to be near the Förster radius (Ro) for the Cy3/Cy5 pair (56 Å), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the ‘closed' apo-model as opposed to the ‘open' RNA-bound structures (by ∼30%). The FRET efficiencies of either of these structurally characterized conformations also are expected to be significantly greater than elongated U2AF65 conformations that lack inter-RRM contacts.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22670</offset><text>Double-cysteine variant of U2AF651,2 was modified with equimolar amount of Cy3 and Cy5. Only traces that showed single photobleaching events for both donor and acceptor dyes and anti-correlated changes in acceptor and donor fluorescence were included in smFRET data analysis. Hence, molecules that were conjugated to two donor or two acceptor fluorophores were excluded from analysis.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23055</offset><text>We first characterized the conformational dynamics spectrum of U2AF65 in the absence of RNA (Fig. 6c,d; Supplementary Fig. 7a,b). The double-labelled U2AF651,2LFRET(Cy3/Cy5) protein was tethered to a slide via biotin-NTA/Ni+2 resin. Virtually no fluorescent molecules were detected in the absence of biotin-NTA/Ni+2, which demonstrates the absence of detectable non-specific binding of U2AF651,2LFRET to the slide. The FRET distribution histogram built from more than a thousand traces of U2AF651,2LFRET(Cy3/Cy5) in the absence of ligand showed an extremely broad distribution centred at a FRET efficiency of ∼0.4 (Fig. 6d). Approximately 40% of the smFRET traces showed apparent transitions between multiple FRET values (for example, Fig. 6c). Despite the large width of the FRET-distribution histogram, the majority (80%) of traces that showed fluctuations sampled only two distinct FRET states (for example, Supplementary Fig. 7a). Approximately 70% of observed fluctuations were interchanges between the ∼0.65 and ∼0.45 FRET values (Supplementary Fig. 7b). We cannot exclude a possibility that tethering of U2AF651,2LFRET(Cy3/Cy5) to the microscope slide introduces structural heterogeneity into the protein and, thus, contributes to the breadth of the FRET distribution histogram. However, the presence of repetitive fluctuations between particular FRET values supports the hypothesis that RNA-free U2AF65 samples several distinct conformations. This result is consistent with the broad ensembles of extended solution conformations that best fit the SAXS data collected for U2AF651,2 as well as for a longer construct (residues 136–347). We conclude that weak contacts between the U2AF65 RRM1 and RRM2 permit dissociation of these RRMs in the absence of RNA.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>24827</offset><text>U2AF65 conformational selection and induced fit by bound RNA</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24888</offset><text>We next used smFRET to probe the conformational selection of distinct inter-RRM arrangements following association of U2AF65 with the AdML Py-tract prototype. Addition of the AdML RNA to tethered U2AF651,2LFRET(Cy3/Cy5) selectively increases a fraction of molecules showing an ∼0.45 apparent FRET efficiency, suggesting that RNA binding stabilizes a single conformation, which corresponds to the 0.45 FRET state (Fig. 6e,f). To assess the possible contributions of RNA-free conformations of U2AF65 and/or structural heterogeneity introduced by tethering of U2AF651,2LFRET(Cy3/Cy5) to the slide to the observed distribution of FRET values, we reversed the immobilization scheme. We tethered the AdML RNA to the slide via a biotinylated oligonucleotide DNA handle and added U2AF651,2LFRET(Cy3/Cy5) in the absence of biotin-NTA resin (Fig. 6g,h; Supplementary Fig. 7c–g). A 0.45 FRET value was again predominant, indicating a similar RNA-bound conformation and structural dynamics for the untethered and tethered U2AF651,2LFRET(Cy3/Cy5).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>25927</offset><text>We examined the effect on U2AF651,2L conformations of purine interruptions that often occur in relatively degenerate human Py tracts. We introduced an rArA purine dinucleotide within a variant of the AdML Py tract (detailed in Methods). Insertion of adenine nucleotides decreased binding affinity of U2AF65 to RNA by approximately five-fold. Nevertheless, in the presence of saturating concentrations of rArA-interrupted RNA slide-tethered U2AF651,2LFRET(Cy3/Cy5) showed a prevalent ∼0.45 apparent FRET value (Fig. 6i,j), which was also predominant in the presence of continuous Py tract. Therefore, RRM1-to-RRM2 distance remains similar regardless of whether U2AF65 is bound to interrupted or continuous Py tract.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>26644</offset><text>The inter-fluorophore distances derived from the observed 0.45 FRET state agree with the distances between the α-carbon atoms of the respective residues in the crystal structures of U2AF651,2L bound to Py-tract oligonucleotides. It should be noted that inferring distances from FRET values is prone to significant error because of uncertainties in the determination of fluorophore orientation factor κ2 and Förster radius R0, the parameters used in distance calculations. Nevertheless, the predominant 0.45 FRET state in the presence of RNA agrees with the Py-tract-bound crystal structure of U2AF651,2L.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>27257</offset><text>Importantly, the majority of traces (∼70%) of U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA lacked FRET fluctuations and predominately exhibited a ∼0.45 FRET value (for example, Fig. 6g). The remaining ∼30% of traces for U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA showed fluctuations between distinct FRET values. The majority of traces that show fluctuations began at high (0.65–0.8) FRET value and transitioned to a ∼0.45 FRET value (Supplementary Fig. 7c–g). Hidden Markov modelling analysis of smFRET traces suggests that RNA-bound U2AF651,2L can sample at least two other conformations corresponding to ∼0.7–0.8 and ∼0.3 FRET values in addition to the predominant conformation corresponding to the 0.45 FRET state. Although a compact conformation (or multiple conformations) of U2AF651,2L corresponding to ∼0.7–0.8 FRET values can bind RNA, on RNA binding, these compact conformations of U2AF651,2L transition into a more stable structural state that corresponds to ∼0.45 FRET value and is likely similar to the side-by-side inter-RRM-arrangement of the U2AF651,2L crystal structures. Thus, the sequence of structural rearrangements of U2AF65 observed in smFRET traces (Supplementary Fig. 7c–g) suggests that a ‘conformational selection' mechanism of Py-tract recognition (that is, RNA ligand stabilization of a pre-configured U2AF65 conformation) is complemented by ‘induced fit' (that is, RNA-induced rearrangement of the U2AF65 RRMs to achieve the final ‘side-by-side' conformation), as discussed below.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>28824</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>28835</offset><text>The U2AF65 structures and analyses presented here represent a successful step towards defining a molecular map of the 3′ splice site. Several observations indicate that the numerous intramolecular contacts, here revealed among the inter-RRM linker and RRM1, RRM2, and the N-terminal RRM1 extension, synergistically coordinate U2AF65–Py-tract recognition. Truncation of U2AF65 to the core RRM1–RRM2 region reduces its RNA affinity by 100-fold. Likewise, deletion of 20 inter-RRM linker residues significantly reduces U2AF65–RNA binding only when introduced in the context of the longer U2AF651,2L construct comprising the RRM extensions, which in turn position the linker for RNA interactions. Notably, a triple mutation of three residues (V254P, Q147A and R227A) in the respective inter-RRM linker, N- and C-terminal extensions non-additively reduce RNA binding by 150-fold. Altogether, these data indicate that interactions among the U2AF65 RRM1/RRM2, inter-RRM linker, N-and C-terminal extensions are mutually inter-dependent for cognate Py-tract recognition. The implications of this finding for U2AF65 conservation and Py-tract recognition are detailed in the Supplementary Discussion.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>30033</offset><text>Recently, high-throughput sequencing studies have shown that somatic mutations in pre-mRNA splicing factors occur in the majority of patients with myelodysplastic syndrome (MDS). MDS-relevant mutations are common in the small U2AF subunit (U2AF35, or U2AF1), yet such mutations are rare in the large U2AF65 subunit (also called U2AF2)—possibly due to the selective versus nearly universal requirements of these factors for splicing. A confirmed somatic mutation of U2AF65 in patients with MDS, L187V, is located on a solvent-exposed surface of RRM1 that is distinct from the RNA interface (Fig. 7a). This L187 surface is oriented towards the N terminus of the U2AF651,2L construct, where it is expected to abut the U2AF35-binding site in the context of the full-length U2AF heterodimer. Likewise, an unconfirmed M144I mutation reported by the same group corresponds to the N-terminal residue of U2AF651,2L, which is separated by only ∼20 residues from the U2AF35-binding site. As such, we suggest that the MDS-relevant U2AF65 mutations contribute to MDS progression indirectly, by destabilizing a relevant conformation of the conjoined U2AF35 subunit rather than affecting U2AF65 functions in RNA binding or spliceosome recruitment per se.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>31277</offset><text>Our smFRET results agree with prior NMR/PRE evidence for multi-domain conformational selection as one mechanistic basis for U2AF65–RNA association (Fig. 7b). The ‘induced fit' versus ‘conformational selection' models are the prevailing views of the mechanisms underlying bio-molecular interactions (reviewed in ref.). In the former, ligand binding promotes a subsequent conformational change in the protein, whereas in the latter, the ligand selects a protein conformation from a pre-existing ensemble and thereby shifts the population towards that state. An ∼0.45 FRET value is likely to correspond to the U2AF65 conformation visualized in our U2AF651,2L crystal structures, in which the RRM1 and RRM2 bind side-by-side to the Py-tract oligonucleotide. The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. An increased prevalence of the ∼0.45 FRET value following U2AF65–RNA binding, coupled with the apparent absence of transitions in many ∼0.45-value single molecule traces (for example, Fig. 6e), suggests a population shift in which RNA binds to (and draws the equilibrium towards) a pre-configured inter-RRM proximity that most often corresponds to the ∼0.45 FRET value.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>32792</offset><text>Notably, our smFRET results reveal that U2AF65–Py-tract recognition can be characterized by an ‘extended conformational selection' model (Fig. 7b). In this recent model for macromolecular interactions, the pure ‘conformational selection' and ‘induced fit' scenarios represent the limits of a mechanistic spectrum and may compete or occur sequentially. Examples of ‘extended conformational selection' during ligand binding have been characterized for a growing number of macromolecules (for example, adenylate kinase, LAO-binding protein, poly-ubiquitin, maltose-binding protein and the preQ1 riboswitch, among others). Here, the majority of changes in smFRET traces for U2AF651,2LFRET(Cy3/Cy5) bound to slide-tethered RNA began at high (0.65–0.8) FRET value and transition to the predominant 0.45 FRET value (Supplementary Fig. 7c–g). These transitions could correspond to rearrangement from the ‘closed' NMR/PRE-based U2AF65 conformation in which the RNA-binding surface of only a single RRM is exposed and available for RNA binding, to the structural state seen in the side-by-side, RNA-bound crystal structure. As such, the smFRET approach reconciles prior inconsistencies between two major conformations that were detected by NMR/PRE experiments and a broad ensemble of diverse inter-RRM arrangements that fit the SAXS data for the apo-protein. Similar interdisciplinary structural approaches are likely to illuminate whether similar mechanistic bases for RNA binding are widespread among other members of the vast multi-RRM family.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>34345</offset><text>The finding that U2AF65 recognizes a nine base pair Py tract contributes to an elusive ‘code' for predicting splicing patterns from primary sequences in the post-genomic era (reviewed in ref.). Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. Further research will be needed to understand the roles of SF1 and U2AF35 subunits in the conformational equilibria underlying U2AF65 association with Py tracts. Moreover, structural differences among U2AF65 homologues and paralogues may regulate splice site selection. Ultimately, these guidelines will assist the identification of 3′ splice sites and the relationship of disease-causing mutations to penalties for U2AF65 association.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>35392</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>35400</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35436</offset><text>For crystallization and RNA-binding experiments, human U2AF651,2L (residues 141–342 of NCBI RefSeq NP_009210) was expressed in Escherichia coli strain BL21 Rosetta-2 as a GST-fusion protein in the vector pGEX6P-2 and purified by glutathione affinity, followed by anion exchange and gel filtration chromatography. The GST-tagged protein was bound to a GSTrap column (GE Healthcare) in 1 M NaCl, 25 mM HEPES, pH 7.4 and eluted using 150 mM NaCl, 100 mM Tris, pH 8 containing 10 mM glutathione. The GST tag was cleaved from the protein by treatment with PreScission Protease during dialysis against a buffer containing 100 mM NaCl, 25 mM HEPES, pH 8, 5% (v/v) glycerol, 5 mM DTT, 0.25 mM EDTA and 0.1 mM PMSF. Cleaved GST was separated from the U2AF651,2L by subtractive glutathione affinity chromatography in 100 mM NaCl, 25 mM Tris, pH 8, 0.2 mM TCEP followed by subtractive anion-exchange chromatography with a HiTrap Q column (GE Healthcare). The final purification step was size-exclusion chromatography on a Superdex-75 prep-grade column (GE Healthcare) that had been previously equilibrated with 100 mM NaCl, 15 mM HEPES, pH 6.8, 0.2 mM tris(2-carboxy-ethyl)phosphine (TCEP). The purified U2AF651,2L was concentrated using a Vivaspin 15 R (Sartorius) centrifugal concentrator with 10 kDa MWCO, and the protein concentration was estimated using the calculated extinction coefficient of 8,940 M−1cm−1 and absorbance at 280 nm. Shorter constructs (U2AF651,2, residues 148–336; dU2AF651,2, residues 148–237, 258–336; dU2AF651,2L, residues 141–237, 258–342) (Fig. 1a) and individual U2AF651,2L Q147A, R227A, V254P mutants used for RNA-binding experiments were purified similarly.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>37171</offset><text>For comparative RNA-binding experiments, full-length human U2AF65 (residues 1–475) and the U2AF35-UHM (U2AF homology motif; residues 43–146, NCBI RefSeq NP_006749) initially were expressed and purified separately as GST fusion proteins. Following GST cleavage and ion-exchange chromatography (SP-HiTrap and Q-HiTrap, respectively), U2AF65 was combined with slight excess U2AF35-UHM (in stoichiometric ratio of 1:1.2) and dialysed overnight. The final U2AF heterodimer was purified by size-exclusion chromatography using a Superdex-200 prep-grade column (GE Healthcare) pre-equilibrated with 150 mM NaCl, 25 mM HEPES, pH 6.8, 0.2 mM TCEP. Representative purified U2AF651,2L and U2AF65–U2AF35-UHM proteins are shown in Supplementary Fig. 1a.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>37923</offset><text>Oligonucleotide preparation</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>37951</offset><text>High-performance liquid chromatography-purified oligonucleotides (sequences shown in Supplementary Fig. 2a) were purchased for crystallization (Integrated DNA Technologies, Inc.). The lyophilized oligonucleotides were diluted in gel filtration buffer for crystallization experiments. The 5′-fluorescein (Fl)-labelled RNAs (AdML: 5′-Fl-CCCUUUUUUUUCC-3′, Py tract of the AdML splicing substrate; 5′-4rU: 5′-Fl-CCUUUUCCCCCCC-3′; 3′-4rU: 5′-Fl-CCCCCCCUUUUCC-3′) for RNA-binding experiments (Dharmacon Research, Inc., Thermo Scientific) was deprotected according to the manufacturer's protocol, vacuum dried and resuspended in nuclease-free water. RNA and RNA–DNA concentrations were calculated using the calculated molar extinction coefficients and absorbance at 260 nm.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>38740</offset><text>Fluorescence anisotropy RNA-binding experiments</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38788</offset><text>For RNA-binding experiments, purified proteins and RNA were diluted separately &gt;100-fold in binding buffer (100 mM NaCl, 15 mM HEPES, pH 6.8, 0.2 mM TCEP, 0.1 U μl−1 Superase-In (Ambion Life Technologies)). The final RNA concentration in the cuvette was 30 nM. Volume changes during addition of the protein were &lt;10% to minimize dilution effects. The fluorescence anisotropy changes during titration were measured using a FluoroMax-3 spectrophotometer temperature controlled by a circulating water bath at 23 °C. Samples were excited at 490 nm and emission intensities recorded at 520 nm with a slit width of 5 nm. The titrations were repeated three times in succession. Each titration was fit with Graphpad Prism v4.0 to obtain the apparent equilibrium dissociation constant (KD). The apparent equilibrium affinities (KA) are the reciprocal of the KD. The average KD's or KA's and s.e.m. among the three replicates were calculated using Excel and are reported in Figs 3 and 4; Supplementary Figs 1 and 4. The P values from a two-tailed unpaired t-test were calculated using Graphpad Prism v4.0.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>39908</offset><text>Transfection, immunoblotting and RT-PCR analyses</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>39957</offset><text>For transfection experiments, the full-length human U2AF65 cDNA in pCMV6-XL5 (Origene Tech. Inc., clone ID BC008740) was used (WT U2AF65) and in parallel mutated to encode the Q147A/R227A/V254P triple-mutant protein (Mut U2AF65). The pyPY minigene was a gift from M. Carmo-Fonseca (University of Lisbon, Portugal). HEK293T cells (kindly provided by Dr Lata Balakrishnan, originally purchased from ATCC, cat. no. CRL3216) were seeded into 12-well plates (2–4 × 105 cells per well) and grown as monolayers in MEM (Gibco Life Technologies) supplemented with 10% (v/v) of heat-inactivated fetal bovine serum, 1% (v/v) L-glutamine and 1% (v/v) penicillin–streptomycin. After 1 day, the cells were transiently transfected with either 0.5 μg of pyPY plasmid or a mixture of 0.5 μg of U2AF65 variant and 0.5 μg of pyPY plasmid per well using appropriately adjusted Lipofectamine 2000 (Invitrogen Life Technologies) ratio according to the manufacturer's instructions.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>40932</offset><text>For immunoblots of WT U2AF65 and Mut U2AF65 expression levels (Supplementary Fig. 5a), transfected or control cells were lysed in radioimmunoprecipitation assay buffer with proteinase and kinase inhibitors. Total protein (20 μg) was separated by SDS–PAGE, and transferred onto polyvinylidene difluoride membranes (Millipore Corp., Billerica, MA, USA) and immunoblotted using mouse monoclonal antibodies directed against U2AF65 (ref.) (MC3, cat. no. U4758 Sigma-Aldrich at 1:500 dilution) or as a control for comparison, GAPDH (glyceraldehyde-3-phosphate dehydrogenase; monoclonal clone 71.1, cat. no. G8795 Sigma-Aldrich at 1:5,000 dilution). Immunoblots were developed using anti-mouse horseradish peroxidase-conjugates (cat. no. U4758 Sigma-Aldrich, Co. at 1:2,500 or 1:10,000 dilutions for GAPDH and U2AF65, respectively) and detected using SuperSignal WestPico chemi-luminescent substrate (Pierce Thermo Scientific Inc.). Blots were imaged using a IS4000MM system (Carestream, Rochester, NY, USA). For size analysis, fluorescent images of the BioRad Precision Plus Dual Color Standards were overlaid directly.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>42051</offset><text>For reverse transcription PCR (RT-PCR), the total RNA was isolated 2 days post transfection using the Cells-to-cDNA II kit (Ambion Life Technologies). The RT-PCR reaction comprised 35 cycles (94 °C per 60 s—60 °C per 50 s—72 °C per 60 s) with forward (5′-TGAGGGGAGGTGAATGAGGAG-3′) and reverse (5′-TCCACTGGAAAGACCGCGAAG-3′) primers for the pyPY product or forward (5′-CATGTTCGTCATGGGTGTGAACCA-3′) and reverse (5′-ATGGCATGGACTGTGGTCATGAGT-3′) primers for a GAPDH control. The RT-PCR products were separated by 2% agarose gel electrophoresis and stained with ethidium bromide. The percentages of splice site use were calculated from the background corrected intensities I using the formula 100% × I(py)/[I(py)+I(PY)+I(unspliced)] for py spliced (Fig. 5b,c) or 100% × I(PY)/[I(py)+I(PY)+I(unspliced)] for PY spliced (Supplementary Fig. 5b). The band intensities of four independent biological replicates were measured using ImageQuant software.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>43029</offset><text>Crystallization, data collection and structure determination</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>43090</offset><text>Before crystallization, the purified U2AF651,2L and given oligonucleotide were mixed to achieve respective final concentrations of 1.0 and 1.1 mM and incubated on ice for 20–30 min. For each oligonucleotide, sparse matrix screens of the Jancarik and Kim Crystal Screen(in hanging drop format; Hampton Research, Corp.) and JCSG-Plus (in sitting drop format; Molecular Dimensions) were used to identify initial crystallization conditions, which were obtained from the latter screen and further optimized in hanging drop format. In optimized crystallization experiments, a mixture of sample and reservoir solution (1.2:1 μl) was equilibrated against 700 μl reservoir solution at 4 °C.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>43787</offset><text>The oligonucleotide sequences were optimized and the structures were determined as follows: in addition to the previously characterized dU2AF651,2-binding sites for seven nucleotides, the new terminal residues of the U2AF651,2L construct were presumed to contact an additional nucleotide and the crystal packing of a central nucleotide between the RRM1/RRM2 of dU2AF651,2 was presumed to represent one nucleotide. Also considering the known proclivity for deoxy(d)U to co-crystallize with dU2AF651,2 (ref.) and for 5-bromo-dU (5BrdU) to bind a given site of dU2AF651,2 (ref.), we initially designed two 9-mer oligonucleotides (5′-ribose (r)UrUrUrUrU(5BrdU)dUrUrU and 5′-rUrUrUdUdU(5BrdU)dUrUrU) and screened for co-crystallization with U2AF651,2L. The former oligonucleotide failed to produce crystals in these screens. The latter oligonucleotide comprising central dU nucleotides produced diffracting crystals, which were frozen directly from a reservoir comprising 100 mM phosphate–citrate buffer pH 4.2, 40% Peg 300. The structure determined by molecular replacement using Phenix with a data set collected at beamline (BL) 12-2 of the Stanford Synchrotron Radiation Lightsource (SSRL; Menlo Park, CA, USA) (Table 1). The search models comprising each of the individual RRMs bound to two nucleotides were derived from the dU2AF651,2 structure (PDB ID 2G4B) (translation function Z-score equivalent 12.9, log-likelihood gain 528). For comparison, searches with the NMR structure (PDB ID 2YH1) as a search model failed to find a solution. The initial structure revealed a greater number of central nucleotide-binding sites than expected. The oligonucleotide binding register had slipped to place the BrdU in the preferred site, leave the 5′ terminal-binding sites empty, and the terminal nucleotide unbound and disordered. Subsequent oligonucleotides were designed to place BrdU in the preferred site, fill the unoccupied 5′ terminal sites, capture rU at the central sites, and compare rC at the terminal site.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>45810</offset><text>The U2AF651,2L protein co-crystals with oligonucleotide 5′-phosphorylated (P)-rUrUdUdUrUdU(BrdU)dU were obtained using a reservoir of 200 mM LiCl, 100 mM sodium citrate pH 4.0, 8% (w/v) polyethylene glycol (PEG) 6,000, 10% (v/v) PEG 300, 10% (v/v) dioxane with 0.1 μl of N,N-bis[3-(D-gluconamido)propyl]deoxy-cholamide (deoxy-BigCHAP) (14 mM) added to the hanging drop and cryoprotected by sequential layering with reservoir solution supplemented with increasing PEG 300 to a final concentration of 26%. Co-crystals with either 5′-(P)rUrUdUrUrU(BrdU)dUdU or 5′-(P)rUrUrUdUrUrU(BrdU)dUrC were obtained from 1 M succinate, 100 mM HEPES, pH 7.0, 1–3% (w/v) PEG monomethylether 2,000. The former was cryoprotected by coating with a 1:1 (v/v) mixture of silicon oil and Paratone-N and the latter by sequential transfer to 21% (v/v) glycerol. Data sets for flash-cooled crystals were collected at 100 K using remote access to SSRL BL12-2. Structures were determined by molecular replacement using the initial U2AF651,2L/rUrUrUdUdU(BrdU)dUrUrU structure as a search model. Consistent sets of free-R reflections were maintained (6% of the total reflections). Models were built using COOT and refined with PHENIX. No non-glycine/non-proline residues were found in the disallowed regions of the Ramachandran plots. Clash scores and Molprobity scores calculated using the program Molprobity were above average. Structure illustrations were prepared using PYMOL. Crystallographic data and refinement statistics are given in Table 1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>47354</offset><text>Sample preparation for single-molecule FRET</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>47398</offset><text>The U2AF651,2LFRET construct used for smFRET comprises the six histidine and T7 tags from the pET28a vector (Merck), a GGGS linker and U2AF65 residues 113–343. The single cysteine of human U2AF65 was replaced by alanine (C305A), which is a natural amino-acid variation among U2AF65 homologues. Single A181C and Q324C mutations were introduced in each RRM for fluorophore attachment at residues that were carefully selected to meet experimental criteria described in the Results. The U2AF651,2LFRET was purified by the same method as described above for U2AF651,2L protein and binds RNA with similar affinity as U2AF651,2L (Supplementary Fig. 6a). Before labelling, the purified U2AF651,2LFRET protein was incubated with 10 mM DTT on ice for 30 min and then buffer exchanged into Labelling Buffer (100 mM NaCl, 25 mM HEPES pH 7.0, 5 mM EDTA, 0.5 mM tris(2-carboxy-ethyl)phosphine (TCEP)) using Zeba Spin Desalting Columns 7K MWCO (Pierce, ThermoFisher Scientific). To initiate the labelling reaction, 4 μl each of cyanine (Cy)3-Maleimide and Cy5-Maleimide (Combinix, Inc.) stock solutions (10 mM in DMSO) were pre-mixed (total volume 8 μl) and then added to 200 μl of 20 μM protein (final 20:1 molar ratio of dye:protein). The labelling reaction was incubated at room temperature in the dark for 2 h and then quenched by the addition of 10 mM DTT. The labelled protein was separated from excess dye using a Zeba Spin Desalting Column followed by size exclusion chromatography using a pre-packed Superdex-75 10/300 GL (GE Healthcare) column in Labelling Buffer. Our previous experience of conjugating cysteines with maleimide derivatives of fluorophores and suggests that nonspecific modification of aminogroups of proteins with fluorescent dyes under the employed experimental conditions is negligible. Consistent with specific labelling of A181C and Q324C, the labelling efficiencies were ∼60% each for Cy3 and Cy5 as estimated using the dye extinction coefficients (ɛCy3=150,000 M−1 cm−1 at 550 nm, ɛCy5=170,000 M−1 cm−1 at 650 nm) and the calculated extinction coefficient of the U2AF651,2LFRET protein (ɛprot=8,940 M−1 cm−1 at 280 nm), and correcting for the absorbance (A) of the dyes at 280 nm (GE Healthcare, Amersham CyDye Maleimide product booklet):</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>49725</offset><text>For smFRET experiments with a ‘strong', homogeneous Py tract, we used the prototypical AdML sequence (5′-CCUUUUUUUUCC-3′). To investigate the inter-RRM separation in the presence of a ‘weak' Py tract interrupted by purines, we compared the U2AF651,2L affinity for a purine-interrupted Py tract comprising an rUrUrUrUrU tract that is expected to bind U2AF65 RRM2/inter-RRM linker, a central rArA and an rUrUrUrCrC tract that is expected to bind RRM1. The tandem purines represent a compromise between significant inhibition of U2AF65 binding by longer A interruptions and an approximately five-fold penalty for the rArA mutation in the AdML Py tract (Supplementary Fig. 6b,c). To maintain avidity and provide flanking phosphoryl groups in case of inter-RRM adjustment, we included the 5′-C and 3′-A of parent AdML sequence, which are respective low-affinity nucleotides for binding RRM2 and RRM1 (ref.), in the final rArA-interrupted RNA oligonucleotide (5′-rCrUrUrUrUrUrArArUrUrUrCrCrA-3′).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>50732</offset><text>For the reversed immobilization of RNA via a complementary biotinyl-DNA primer experiment, the AdML Py-tract RNA was extended to include the DNA counterpart of downstream AdML intron/exon sequences that were complementary to the biotinyl-DNA primer. To increase separation from the slide surface, a hexaethylene glycol linker (18PEG) was inserted between the AdML Py-tract RNA and the tethered DNA duplex. The tethered oligonucleotide sequences included: 5′-rCrCrUrUrUrUrUrUrUrUrCrC/18PEG/dAdCdAdGdCdTdCdGdCdG-dGdTdTdGdAdGdGdAdCdAdA-3′ annealed to 5′-biotinyl-dTdTdGdTdCdCdTdCdAdA-dCdCdGdCdGdAdGdCdTdGdT-3' (purchased with high-performance liquid chromatography purification from Integrated DNA Technologies).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>51448</offset><text>Single-molecule FRET data acquisition and analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>51499</offset><text>The smFRET measurements were carried out at room temperature in 50 mM HEPES, pH 7.4, 100 mM NaCl. The imaging buffer also contained an oxygen-scavenging system (0.8 mg ml−1 glucose oxidase, 0.625% glucose, 0.02 mg ml−1 catalase), 1.5 mM Trolox (used to eliminate Cy5 blinking) and 6 mM β-mercaptoethanol. The sample chamber was assembled from quartz microscope slides and glass cover slips coated with a mixture of m-PEG and biotin-PEG and pre-treated with neutravidin (0.2 mg ml−1). Surface tethering of doubly labelled U2AF651,2LFRET(Cy3/Cy5) via its His-tag (Fig. 6c–f,i,j; Supplementary Fig. 7a,b) was achieved by pre-incubating the sample chamber with 50 nM biotinyl-NTA resin (Biotin-X NTA, Biotium), pre-loaded with three-fold excess NiSO4) for 20 min before addition of 5 nM U2AF651,2LFRET(Cy3/Cy5). After 10 min, unbound sample was removed by washing the sample chamber with imaging buffer. The AdML RNA ligand was added to the imaging buffer at a concentration of 5 μM (100-fold higher than the measured KD value), whereas the rArA-interrupted RNA was added at a concentration of 10 μM. Alternatively, to detect binding of doubly labelled U2AF651,2LFRET(Cy3/Cy5) to surface-tethered RNA ligand (Fig. 6g,h Supplementary Fig. 7c–g), 10 nM AdML RNA (pre-annealed to biotinyl-DNA primer) was incubated in the neutravidin-treated sample chamber for 20 min, and 1 nM U2AF651,2LFRET(Cy3/Cy5) was then added to the imaging buffer.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>52983</offset><text>Single-molecule FRET measurements were taken as previously described. An Olympus IX71 inverted microscope, equipped with a UPlanApo 60x/1.20w objective lens, a 532 nm laser (Spectra-Physics) for excitation of Cy3 dyes, and a 642 nm laser (Spectra-Physics) for excitation of Cy5 dyes was used. Total internal reflection (TIR) was obtained by a quartz prism (ESKMA Optics). Fluorescence emission was split into Cy3 and Cy5 fluorescence using a dual view imaging system DV2 (Photometrics) equipped with a 630 nm dichroic mirror and recorded via an Andor iXon+ EMCCD camera. Movies were recorded using the Single software (downloaded from Prof. Taekjip Ha's laboratory website at the University of Illinois at Urbana-Champaign, physics.illinois.edu/cplc/software), with the exposure time set at 100 ms. We typically took up to five 5-minute-long movies while imaging different sections of the slide for each sample. Before each measurement, we checked for non-specific binding by adding doubly-labeled U2Fret to the slide in the absence of neutravidin and imaging the slide. Non-specific binding was virtually absent.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>54105</offset><text>Collected data sets were processed with IDL and Matlab softwares, using scripts downloaded from a freely available source: physics.illinois.edu/cplc/software. Apparent FRET efficiencies (Eapp) were calculated from the emission intensities of donor (ICy3) and acceptor (ICy5) as follows: Eapp=ICy5/(ICy5+ICy3). The FRET distribution histograms were built from traces that showed single-step photobleaching in both Cy3 and Cy5 signals using a Matlab script generously provided by Prof. Peter Cornish (University of Missouri, Columbia). Anti-correlated changes in donor and acceptor intensities with constant sum of intensities indicated the presence of an energy transfer in single molecules labelled with one donor and one acceptor dye. All histograms were smoothed with a five-point window and plotted using Origin software (Origin Lab Co). Idealization of FRET trajectories was done using the hidden Markov model algorithms via HaMMy software (http://bio.physics.illinois.edu/HaMMy.asp). Transition density plots were generated from transitions detected in idealized FRET trajectories obtained by HaMMy fit of raw FRET traces via Matlab. Frequency of transitions from starting FRET efficiency value (x-axis) to ending FRET efficiency value (y-axis) was represented by a heat map. The range of FRET efficiencies from 0 to 1 was separated in 200 bins. The resulting heat map was normalized to the most populated bin in the plot; the lower- and upper-bound thresholds were set to 20% and 100% of the most populated bin, respectively.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>55637</offset><text>The surface contour plots were generated as follows: the individual single-molecule FRET traces (for example, Fig. 6g of the main text and Supplementary Fig. 7e,f) were post synchronized at the first time point showing non-zero (&gt;0.15) FRET efficiency, corresponding to binding. The time range (x-axis, 0–10 s) was separated into 100 bins. The FRET efficiency range (y-axis, 0–1 FRET) was separated into 100 bins. A heat map is used to represent the frequency of sampling of each FRET state over time; frequency in each bin was normalized to the most populated bin in the plot with lower- and upper-bound thresholds set at 10% and 80% of the most populated bin, respectively.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>56319</offset><text>Additional information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>56342</offset><text>Accession codes: Coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 5EV1, 5EV2, 5EV3 and 5EV4 for respective U2AF651,2L-oligonucleotide structures (i)–(iv).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>56550</offset><text>How to cite this article: Agrawal, A. A. et al. An extended U2AF65–RNA-binding domain recognizes the 3′ splice site signal. Nat. Commun. 7:10950 doi: 10.1038/ncomms10950 (2016).</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>56732</offset><text>Supplementary Material</text></passage><passage><infon key="fpage">470</infon><infon key="lpage">476</infon><infon key="name_0">surname:Wang;given-names:E. T.</infon><infon key="pub-id_pmid">18978772</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">456</infon><infon key="year">2008</infon><offset>56755</offset><text>Alternative isoform regulation in human tissue transcriptomes</text></passage><passage><infon key="fpage">525</infon><infon key="lpage">560</infon><infon key="name_0">surname:Burge;given-names:C. B.</infon><infon key="name_1">surname:Tuschl;given-names:T.</infon><infon key="name_2">surname:Sharp;given-names:P. A.</infon><infon key="section_type">REF</infon><infon key="source">The RNA World</infon><infon key="type">ref</infon><infon key="year">1999</infon><offset>56817</offset></passage><passage><infon key="fpage">472</infon><infon key="lpage">482</infon><infon key="name_0">surname:Singh;given-names:R. K.</infon><infon key="name_1">surname:Cooper;given-names:T. A.</infon><infon key="pub-id_pmid">22819011</infon><infon key="section_type">REF</infon><infon key="source">Trends Mol. Med.</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">2012</infon><offset>56818</offset><text>Pre-mRNA splicing in disease and therapeutics</text></passage><passage><infon key="fpage">1540</infon><infon key="lpage">1549</infon><infon key="name_0">surname:Scott;given-names:L. M.</infon><infon key="name_1">surname:Rebel;given-names:V. I.</infon><infon key="pub-id_pmid">24052622</infon><infon key="section_type">REF</infon><infon key="source">J. Natl Cancer Inst.</infon><infon key="type">ref</infon><infon key="volume">105</infon><infon key="year">2013</infon><offset>56864</offset><text>Acquired mutations that affect pre-mRNA splicing in hematologic malignancies and solid tumors</text></passage><passage><infon key="fpage">135</infon><infon key="lpage">140</infon><infon key="name_0">surname:Golling;given-names:G.</infon><infon key="pub-id_pmid">12006978</infon><infon key="section_type">REF</infon><infon key="source">Nat. Genet.</infon><infon key="type">ref</infon><infon key="volume">31</infon><infon key="year">2002</infon><offset>56958</offset><text>Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development</text></passage><passage><infon key="fpage">207</infon><infon key="lpage">219</infon><infon key="name_0">surname:Ruskin;given-names:B.</infon><infon key="name_1">surname:Zamore;given-names:P. D.</infon><infon key="name_2">surname:Green;given-names:M. R.</infon><infon key="pub-id_pmid">2963698</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">52</infon><infon key="year">1988</infon><offset>57063</offset><text>A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly</text></passage><passage><infon key="fpage">781</infon><infon key="lpage">787</infon><infon key="name_0">surname:Berglund;given-names:J. A.</infon><infon key="name_1">surname:Chua;given-names:K.</infon><infon key="name_2">surname:Abovich;given-names:N.</infon><infon key="name_3">surname:Reed;given-names:R.</infon><infon key="name_4">surname:Rosbash;given-names:M.</infon><infon key="pub-id_pmid">9182766</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">89</infon><infon key="year">1997</infon><offset>57142</offset><text>The splicing factor BBP interacts specifically with the pre-mRNA branchpoint sequence UACUAAC</text></passage><passage><infon key="fpage">858</infon><infon key="lpage">867</infon><infon key="name_0">surname:Berglund;given-names:J. A.</infon><infon key="name_1">surname:Abovich;given-names:N.</infon><infon key="name_2">surname:Rosbash;given-names:M.</infon><infon key="pub-id_pmid">9512519</infon><infon key="section_type">REF</infon><infon key="source">Genes Dev.</infon><infon key="type">ref</infon><infon key="volume">12</infon><infon key="year">1998</infon><offset>57236</offset><text>A cooperative interaction between U2AF65 and mBBP/SF1 facilitates branchpoint region recognition</text></passage><passage><infon key="fpage">832</infon><infon key="lpage">835</infon><infon key="name_0">surname:Wu;given-names:S.</infon><infon key="name_1">surname:Romfo;given-names:C. M.</infon><infon key="name_2">surname:Nilsen;given-names:T. W.</infon><infon key="name_3">surname:Green;given-names:M. R.</infon><infon key="pub-id_pmid">10617206</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">402</infon><infon key="year">1999</infon><offset>57333</offset><text>Functional recognition of the 3' splice site AG by the splicing factor U2AF35</text></passage><passage><infon key="fpage">835</infon><infon key="lpage">838</infon><infon key="name_0">surname:Zorio;given-names:D. A.</infon><infon key="name_1">surname:Blumenthal;given-names:T.</infon><infon key="pub-id_pmid">10617207</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">402</infon><infon key="year">1999</infon><offset>57411</offset><text>Both subunits of U2AF recognize the 3' splice site in Caenorhabditis elegans</text></passage><passage><infon key="fpage">838</infon><infon key="lpage">841</infon><infon key="name_0">surname:Merendino;given-names:L.</infon><infon key="name_1">surname:Guth;given-names:S.</infon><infon key="name_2">surname:Bilbao;given-names:D.</infon><infon key="name_3">surname:Martinez;given-names:C.</infon><infon key="name_4">surname:Valcarcel;given-names:J.</infon><infon key="pub-id_pmid">10617208</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">402</infon><infon key="year">1999</infon><offset>57488</offset><text>Inhibition of msl-2 splicing by Sex-lethal reveals interaction between U2AF35 and the 3' splice site AG</text></passage><passage><infon key="fpage">1173</infon><infon key="lpage">1176</infon><infon key="name_0">surname:Singh;given-names:R.</infon><infon key="name_1">surname:Valcarcel;given-names:J.</infon><infon key="name_2">surname:Green;given-names:M. R.</infon><infon key="pub-id_pmid">7761834</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">268</infon><infon key="year">1995</infon><offset>57592</offset><text>Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins</text></passage><passage><infon key="fpage">609</infon><infon key="lpage">614</infon><infon key="name_0">surname:Zamore;given-names:P. D.</infon><infon key="name_1">surname:Patton;given-names:J. G.</infon><infon key="name_2">surname:Green;given-names:M. R.</infon><infon key="pub-id_pmid">1538748</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">355</infon><infon key="year">1992</infon><offset>57696</offset><text>Cloning and domain structure of the mammalian splicing factor U2AF</text></passage><passage><infon key="fpage">3859</infon><infon key="lpage">3873</infon><infon key="name_0">surname:Jenkins;given-names:J. L.</infon><infon key="name_1">surname:Agrawal;given-names:A. A.</infon><infon key="name_2">surname:Gupta;given-names:A.</infon><infon key="name_3">surname:Green;given-names:M. R.</infon><infon key="name_4">surname:Kielkopf;given-names:C. L.</infon><infon key="pub-id_pmid">23376934</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">41</infon><infon key="year">2013</infon><offset>57763</offset><text>U2AF65 adapts to diverse pre-mRNA splice sites through conformational selection of specific and promiscuous RNA recognition motifs</text></passage><passage><infon key="fpage">49</infon><infon key="lpage">59</infon><infon key="name_0">surname:Sickmier;given-names:E. A.</infon><infon key="pub-id_pmid">16818232</infon><infon key="section_type">REF</infon><infon key="source">Mol. Cell</infon><infon key="type">ref</infon><infon key="volume">23</infon><infon key="year">2006</infon><offset>57894</offset><text>Structural basis of polypyrimidine tract recognition by the essential pre-mRNA splicing factor, U2AF65</text></passage><passage><infon key="fpage">408</infon><infon key="lpage">411</infon><infon key="name_0">surname:Mackereth;given-names:C. D.</infon><infon key="pub-id_pmid">21753750</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">475</infon><infon key="year">2011</infon><offset>57997</offset><text>Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF</text></passage><passage><infon key="fpage">50572</infon><infon key="lpage">50577</infon><infon key="name_0">surname:Kent;given-names:O. A.</infon><infon key="name_1">surname:Reayi;given-names:A.</infon><infon key="name_2">surname:Foong;given-names:L.</infon><infon key="name_3">surname:Chilibeck;given-names:K. A.</infon><infon key="name_4">surname:MacMillan;given-names:A. M.</infon><infon key="pub-id_pmid">14506271</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">278</infon><infon key="year">2003</infon><offset>58082</offset><text>Structuring of the 3' splice site by U2AF65</text></passage><passage><infon key="fpage">1706</infon><infon key="lpage">1709</infon><infon key="name_0">surname:Valcarcel;given-names:J.</infon><infon key="name_1">surname:Gaur;given-names:R. K.</infon><infon key="name_2">surname:Singh;given-names:R.</infon><infon key="name_3">surname:Green;given-names:M. R.</infon><infon key="pub-id_pmid">8781232</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">273</infon><infon key="year">1996</infon><offset>58126</offset><text>Interaction of U2AF65 RS region with pre-mRNA branch point and promotion of base pairing with U2 snRNA</text></passage><passage><infon key="fpage">363</infon><infon key="lpage">373</infon><infon key="name_0">surname:Shen;given-names:H.</infon><infon key="name_1">surname:Green;given-names:M. R.</infon><infon key="pub-id_pmid">15525510</infon><infon key="section_type">REF</infon><infon key="source">Mol. Cell</infon><infon key="type">ref</infon><infon key="volume">16</infon><infon key="year">2004</infon><offset>58229</offset><text>A pathway of sequential arginine-serine-rich domain-splicing signal interactions during mammalian spliceosome assembly</text></passage><passage><infon key="fpage">17420</infon><infon key="lpage">17425</infon><infon key="name_0">surname:Agrawal;given-names:A. A.</infon><infon key="name_1">surname:McLaughlin;given-names:K. J.</infon><infon key="name_2">surname:Jenkins;given-names:J. L.</infon><infon key="name_3">surname:Kielkopf;given-names:C. L.</infon><infon key="pub-id_pmid">25422459</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">111</infon><infon key="year">2014</infon><offset>58348</offset><text>Structure-guided U2AF65 variant improves recognition and splicing of a defective pre-mRNA</text></passage><passage><infon key="fpage">133</infon><infon key="lpage">180</infon><infon key="name_0">surname:Janin;given-names:J.</infon><infon key="name_1">surname:Bahadur;given-names:R. P.</infon><infon key="name_2">surname:Chakrabarti;given-names:P.</infon><infon key="pub-id_pmid">18812015</infon><infon key="section_type">REF</infon><infon key="source">Q. Rev. Biophys.</infon><infon key="type">ref</infon><infon key="volume">41</infon><infon key="year">2008</infon><offset>58438</offset><text>Protein-protein interaction and quaternary structure</text></passage><passage><infon key="fpage">33641</infon><infon key="lpage">33649</infon><infon key="name_0">surname:Jenkins;given-names:J. L.</infon><infon key="name_1">surname:Shen;given-names:H.</infon><infon key="name_2">surname:Green;given-names:M. R.</infon><infon key="name_3">surname:Kielkopf;given-names:C. L.</infon><infon key="pub-id_pmid">18842594</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">283</infon><infon key="year">2008</infon><offset>58491</offset><text>Solution conformation and thermodynamic characteristics of RNA binding by the splicing factor U2AF65</text></passage><passage><infon key="fpage">4523</infon><infon key="lpage">4534</infon><infon key="name_0">surname:Ito;given-names:T.</infon><infon key="name_1">surname:Muto;given-names:Y.</infon><infon key="name_2">surname:Green;given-names:M. R.</infon><infon key="name_3">surname:Yokoyama;given-names:S.</infon><infon key="pub-id_pmid">10449418</infon><infon key="section_type">REF</infon><infon key="source">EMBO J.</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">1999</infon><offset>58592</offset><text>Solution structures of the first and second RNA-binding domains of human U2 small nuclear ribonucleoprotein particle auxiliary factor (U2AF65)</text></passage><passage><infon key="fpage">8183</infon><infon key="lpage">8190</infon><infon key="name_0">surname:Pacheco;given-names:T. R.</infon><infon key="name_1">surname:Coelho;given-names:M. B.</infon><infon key="name_2">surname:Desterro;given-names:J. M.</infon><infon key="name_3">surname:Mollet;given-names:I.</infon><infon key="name_4">surname:Carmo-Fonseca;given-names:M.</infon><infon key="pub-id_pmid">16940179</infon><infon key="section_type">REF</infon><infon key="source">Mol. Cell. Biol.</infon><infon key="type">ref</infon><infon key="volume">26</infon><infon key="year">2006</infon><offset>58735</offset><text>In vivo requirement of the small subunit of U2AF for recognition of a weak 3' splice site</text></passage><passage><infon key="fpage">5223</infon><infon key="lpage">5225</infon><infon key="name_0">surname:Jenkins;given-names:J. L.</infon><infon key="name_1">surname:Laird;given-names:K. M.</infon><infon key="name_2">surname:Kielkopf;given-names:C. L.</infon><infon key="pub-id_pmid">22702716</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry</infon><infon key="type">ref</infon><infon key="volume">51</infon><infon key="year">2012</infon><offset>58825</offset><text>A broad range of conformations contribute to the solution ensemble of the essential splicing factor U2AF65</text></passage><passage><infon key="fpage">7068</infon><infon key="lpage">7076</infon><infon key="name_0">surname:Huang;given-names:J. R.</infon><infon key="pub-id_pmid">24734879</infon><infon key="section_type">REF</infon><infon key="source">J. Am. Chem. Soc.</infon><infon key="type">ref</infon><infon key="volume">136</infon><infon key="year">2014</infon><offset>58932</offset><text>Transient electrostatic interactions dominate the conformational equilibrium sampled by multidomain splicing factor U2AF65: a combined NMR and SAXS study</text></passage><passage><infon key="fpage">211</infon><infon key="lpage">231</infon><infon key="name_0">surname:Dietrich;given-names:A.</infon><infon key="name_1">surname:Buschmann;given-names:V.</infon><infon key="name_2">surname:Muller;given-names:C.</infon><infon key="name_3">surname:Sauer;given-names:M.</infon><infon key="pub-id_pmid">11999691</infon><infon key="section_type">REF</infon><infon key="source">J. Biotechnol.</infon><infon key="type">ref</infon><infon key="volume">82</infon><infon key="year">2002</infon><offset>59086</offset><text>Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single molecule data</text></passage><passage><infon key="fpage">507</infon><infon key="lpage">516</infon><infon key="name_0">surname:Roy;given-names:R.</infon><infon key="name_1">surname:Hohng;given-names:S.</infon><infon key="name_2">surname:Ha;given-names:T.</infon><infon key="pub-id_pmid">18511918</infon><infon key="section_type">REF</infon><infon key="source">Nat. Methods</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2008</infon><offset>59256</offset><text>A practical guide to single-molecule FRET</text></passage><passage><infon key="fpage">241</infon><infon key="lpage">247</infon><infon key="name_0">surname:Haferlach;given-names:T.</infon><infon key="pub-id_pmid">24220272</infon><infon key="section_type">REF</infon><infon key="source">Leukemia</infon><infon key="type">ref</infon><infon key="volume">28</infon><infon key="year">2014</infon><offset>59298</offset><text>Landscape of genetic lesions in 944 patients with myelodysplastic syndromes</text></passage><passage><infon key="fpage">64</infon><infon key="lpage">69</infon><infon key="name_0">surname:Yoshida;given-names:K.</infon><infon key="pub-id_pmid">21909114</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">478</infon><infon key="year">2011</infon><offset>59374</offset><text>Frequent pathway mutations of splicing machinery in myelodysplasia</text></passage><passage><infon key="fpage">595</infon><infon key="lpage">605</infon><infon key="name_0">surname:Kielkopf;given-names:C. L.</infon><infon key="name_1">surname:Rodionova;given-names:N. A.</infon><infon key="name_2">surname:Green;given-names:M. R.</infon><infon key="name_3">surname:Burley;given-names:S. K.</infon><infon key="pub-id_pmid">11551507</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">106</infon><infon key="year">2001</infon><offset>59441</offset><text>A novel peptide recognition mode revealed by the X-ray structure of a core U2AF35/U2AF65 heterodimer</text></passage><passage><infon key="fpage">539</infon><infon key="lpage">546</infon><infon key="name_0">surname:Csermely;given-names:P.</infon><infon key="name_1">surname:Palotai;given-names:R.</infon><infon key="name_2">surname:Nussinov;given-names:R.</infon><infon key="pub-id_pmid">20541943</infon><infon key="section_type">REF</infon><infon key="source">Trends Biochem. Sci.</infon><infon key="type">ref</infon><infon key="volume">35</infon><infon key="year">2010</infon><offset>59542</offset><text>Induced fit, conformational selection and independent dynamic segments: an extended view of binding events</text></passage><passage><infon key="fpage">13737</infon><infon key="lpage">13741</infon><infon key="name_0">surname:Hammes;given-names:G. G.</infon><infon key="name_1">surname:Chang;given-names:Y. C.</infon><infon key="name_2">surname:Oas;given-names:T. G.</infon><infon key="pub-id_pmid">19666553</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">106</infon><infon key="year">2009</infon><offset>59649</offset><text>Conformational selection or induced fit: a flux description of reaction mechanism</text></passage><passage><infon key="fpage">18055</infon><infon key="lpage">18060</infon><infon key="name_0">surname:Hanson;given-names:J. A.</infon><infon key="pub-id_pmid">17989222</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">104</infon><infon key="year">2007</infon><offset>59731</offset><text>Illuminating the mechanistic roles of enzyme conformational dynamics</text></passage><passage><infon key="fpage">838</infon><infon key="lpage">844</infon><infon key="name_0">surname:Henzler-Wildman;given-names:K. A.</infon><infon key="pub-id_pmid">18026086</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">450</infon><infon key="year">2007</infon><offset>59800</offset><text>Intrinsic motions along an enzymatic reaction trajectory</text></passage><passage><infon key="fpage">e1002054</infon><infon key="name_0">surname:Silva;given-names:D. A.</infon><infon key="name_1">surname:Bowman;given-names:G. R.</infon><infon key="name_2">surname:Sosa-Peinado;given-names:A.</infon><infon key="name_3">surname:Huang;given-names:X.</infon><infon key="pub-id_pmid">21637799</infon><infon key="section_type">REF</infon><infon key="source">PLoS Comput. Biol.</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2011</infon><offset>59857</offset><text>A role for both conformational selection and induced fit in ligand binding by the LAO protein</text></passage><passage><infon key="fpage">19346</infon><infon key="lpage">19351</infon><infon key="name_0">surname:Wlodarski;given-names:T.</infon><infon key="name_1">surname:Zagrovic;given-names:B.</infon><infon key="pub-id_pmid">19887638</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">106</infon><infon key="year">2009</infon><offset>59951</offset><text>Conformational selection and induced fit mechanism underlie specificity in noncovalent interactions with ubiquitin</text></passage><passage><infon key="fpage">313</infon><infon key="lpage">318</infon><infon key="name_0">surname:Kim;given-names:E.</infon><infon key="pub-id_pmid">23502425</infon><infon key="section_type">REF</infon><infon key="source">Nat. Chem. Biol.</infon><infon key="type">ref</infon><infon key="volume">9</infon><infon key="year">2013</infon><offset>60066</offset><text>A single-molecule dissection of ligand binding to a protein with intrinsic dynamics</text></passage><passage><infon key="fpage">14075</infon><infon key="lpage">14083</infon><infon key="name_0">surname:Suddala;given-names:K. C.</infon><infon key="name_1">surname:Wang;given-names:J.</infon><infon key="name_2">surname:Hou;given-names:Q.</infon><infon key="name_3">surname:Walter;given-names:N. G.</infon><infon key="pub-id_pmid">26471732</infon><infon key="section_type">REF</infon><infon key="source">J. Am. Chem. Soc.</infon><infon key="type">ref</infon><infon key="volume">137</infon><infon key="year">2015</infon><offset>60150</offset><text>Mg(2+) shifts ligand-mediated folding of a riboswitch from induced-fit to conformational selection</text></passage><passage><infon key="fpage">802</infon><infon key="lpage">813</infon><infon key="name_0">surname:Wang;given-names:Z.</infon><infon key="name_1">surname:Burge;given-names:C. B.</infon><infon key="pub-id_pmid">18369186</infon><infon key="section_type">REF</infon><infon key="source">RNA</infon><infon key="type">ref</infon><infon key="volume">14</infon><infon key="year">2008</infon><offset>60249</offset><text>Splicing regulation: from a parts list of regulatory elements to an integrated splicing code</text></passage><passage><infon key="fpage">e13</infon><infon key="name_0">surname:Cavaluzzi;given-names:M. J.</infon><infon key="name_1">surname:Borer;given-names:P. N.</infon><infon key="pub-id_pmid">14722228</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">32</infon><infon key="year">2004</infon><offset>60342</offset><text>Revised UV extinction coefficients for nucleoside-5'-monophosphates and unpaired DNA and RNA</text></passage><passage><infon key="fpage">975</infon><infon key="lpage">987</infon><infon key="name_0">surname:Gama-Carvalho;given-names:M.</infon><infon key="pub-id_pmid">9166400</infon><infon key="section_type">REF</infon><infon key="source">J. Cell Biol.</infon><infon key="type">ref</infon><infon key="volume">137</infon><infon key="year">1997</infon><offset>60435</offset><text>Targeting of U2AF65 to sites of active splicing in the nucleus</text></passage><passage><infon key="fpage">409</infon><infon key="lpage">411</infon><infon key="name_0">surname:Jancarik;given-names:J.</infon><infon key="name_1">surname:Kim;given-names:S.-H.</infon><infon key="section_type">REF</infon><infon key="source">J. Appl. Cryst.</infon><infon key="type">ref</infon><infon key="volume">24</infon><infon key="year">1991</infon><offset>60498</offset><text>Sparse matrix sampling: a screening method for crystallization of proteins</text></passage><passage><infon key="fpage">457</infon><infon key="lpage">459</infon><infon key="name_0">surname:Sickmier;given-names:E. A.</infon><infon key="name_1">surname:Frato;given-names:K. E.</infon><infon key="name_2">surname:Kielkopf;given-names:C. L.</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">F62</infon><infon key="year">2006</infon><offset>60573</offset><text>Crystallization and preliminary X-ray analysis of U2AF65 variant in complex with a polypyrimidine tract analogue by use of protein engineering</text></passage><passage><infon key="fpage">213</infon><infon key="lpage">221</infon><infon key="name_0">surname:Adams;given-names:P. D.</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">D66</infon><infon key="year">2010</infon><offset>60716</offset><text>PHENIX: a comprehensive Python-based system for macromolecular structure solution</text></passage><passage><infon key="fpage">2126</infon><infon key="lpage">2132</infon><infon key="name_0">surname:Emsley;given-names:P.</infon><infon key="name_1">surname:Cowtan;given-names:K.</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">D60</infon><infon key="year">2004</infon><offset>60798</offset><text>Coot: model-building tools for molecular graphics</text></passage><passage><infon key="fpage">W615</infon><infon key="lpage">W619</infon><infon key="name_0">surname:Davis;given-names:I. W.</infon><infon key="name_1">surname:Murray;given-names:L. W.</infon><infon key="name_2">surname:Richardson;given-names:J. S.</infon><infon key="name_3">surname:Richardson;given-names:D. C.</infon><infon key="pub-id_pmid">15215462</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">32</infon><infon key="year">2004</infon><offset>60848</offset><text>MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes</text></passage><passage><infon key="name_0">surname:DeLano;given-names:W. L.</infon><infon key="section_type">REF</infon><infon key="source">The PyMOL Molecular Graphics System, Version 1.8.</infon><infon key="type">ref</infon><infon key="year">2015</infon><offset>60949</offset></passage><passage><infon key="fpage">15060</infon><infon key="lpage">15065</infon><infon key="name_0">surname:Salsi;given-names:E.</infon><infon key="name_1">surname:Farah;given-names:E.</infon><infon key="name_2">surname:Dann;given-names:J.</infon><infon key="name_3">surname:Ermolenko;given-names:D. N.</infon><infon key="pub-id_pmid">25288752</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">111</infon><infon key="year">2014</infon><offset>60950</offset><text>Following movement of domain IV of elongation factor G during ribosomal translocation</text></passage><passage><infon key="fpage">1941</infon><infon key="lpage">1951</infon><infon key="name_0">surname:McKinney;given-names:S. A.</infon><infon key="name_1">surname:Joo;given-names:C.</infon><infon key="name_2">surname:Ha;given-names:T.</infon><infon key="pub-id_pmid">16766620</infon><infon key="section_type">REF</infon><infon key="source">Biophys. J.</infon><infon key="type">ref</infon><infon key="volume">91</infon><infon key="year">2006</infon><offset>61036</offset><text>Analysis of single-molecule FRET trajectories using hidden Markov modeling</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>61111</offset><text>Author contributions A.A.A. performed crystallization, refinement, molecular biology and most RNA-binding experiments. E.S. performed smFRET experiments. R.C. labelled protein and S.H. completed a subset of RNA-binding experiments. C.L.K. cryoprotected crystals, collected crystallographic data and built structures. J.L.J. performed molecular replacement and completed structure refinements. M.R.G. and C.L.K. conceived the study. C.L.K. and D.N.E. designed the experiments. C.L.K., D.N.E. and E.S. wrote the paper with input from J.L.J. and A.A.A.</text></passage><passage><infon key="file">ncomms10950-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>61661</offset><text>The intact U2AF65 RRM1/RRM2-containing domain and flanking residues are required for binding contiguous Py tracts.</text></passage><passage><infon key="file">ncomms10950-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>61776</offset><text>(a) Domain organization of full-length (fl) U2AF65 and constructs used for RNA binding and structural experiments. The N- and C-terminal residue numbers are indicated. An internal deletion (d, Δ) of residues 238–257 removes a portion of the inter-RRM linker from the dU2AF651,2 and dU2AF651,2L constructs. (b) Comparison of the apparent equilibrium affinities of various U2AF65 constructs for binding the prototypical AdML Py tract (5′-CCCUUUUUUUUCC-3′). The flU2AF65 protein includes a heterodimerization domain of the U2AF35 subunit to promote solubility and folding. The apparent equilibrium dissociation constants (KD) for binding the AdML 13mer are as follows: flU2AF65, 30±3 nM; U2AF651,2L, 35±6 nM; U2AF651,2, 3,600±300 nM. (c) Comparison of the RNA sequence specificities of flU2AF65 and U2AF651,2L constructs binding C-rich Py tracts with 4U's embedded in either the 5′- (light grey fill) or 3′- (dark grey fill) regions. The KD's for binding 5′-CCUUUUCCCCCCC-3′ are: flU2AF65, 41±2 nM; U2AF651,2L, 31±3 nM. The KD's for binding 5′-CCCCCCCUUUUCC-3′ are: flU2AF65, 414±12 nM; U2AF651,2L, 417±10 nM. Bar graphs are hatched to match the constructs shown in a. The average apparent equilibrium affinity (KA) and s.e.m. for three independent titrations are plotted. The P values from two-tailed unpaired t-tests with Welch's correction are indicated as follows: **P&lt;0.01; NS, not significant, P&gt;0.05. The purified protein and average fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 1. RRM, RNA recognition motif; RS, arginine-serine rich; UHM, U2AF homology motif; ULM, U2AF ligand motif.</text></passage><passage><infon key="file">ncomms10950-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>63443</offset><text>Structures of U2AF651,2L recognizing a contiguous Py tract.</text></passage><passage><infon key="file">ncomms10950-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>63503</offset><text>(a) Alignment of oligonucleotide sequences that were co-crystallized in the indicated U2AF651,2L structures. The regions of RRM1, RRM2 and linker contacts are indicated above by respective black and blue arrows from N- to C-terminus. For clarity, we consistently number the U2AF651,2L nucleotide-binding sites from one to nine, although in some cases the co-crystallized oligonucleotide comprises eight nucleotides and as such leaves the first binding site empty. The prior dU2AF651,2 nucleotide-binding sites are given in parentheses (site 4' interacts with dU2AF65 RRM1 and RRM2 by crystallographic symmetry). Italics, disordered in the structure. (b) Stereo views of a ‘kicked' 2|Fo|−|Fc| electron density map contoured at 1σ for the inter-RRM linker, N- and C-terminal residues (blue) or bound oligonucleotide of a representative U2AF651,2L structure (structure iv, bound to 5′-(P)rUrUrUdUrUrU(BrdU)dUrC) (magenta). (c) Cartoon diagram of this structure. Crystallographic statistics are given in Table 1 and the overall conformations of U2AF651,2L and prior dU2AF651,2/U2AF651,2 structures are compared in Supplementary Fig. 2. BrdU, 5-bromo-deoxy-uridine; d, deoxy-ribose; P-, 5′-phosphorylation; r, ribose.</text></passage><passage><infon key="file">ncomms10950-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>64725</offset><text>Representative views of the U2AF651,2L interactions with each new nucleotide of the bound Py tract.</text></passage><passage><infon key="file">ncomms10950-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>64825</offset><text>New residues of the U2AF651,2L structures are coloured a darker shade of blue, apart from residues that were tested by site-directed mutagenesis, which are coloured yellow. The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. (i) Bar graph of apparent equilibrium affinities (KA) of the wild type (blue) and the indicated mutant (yellow) U2AF651,2L proteins binding the AdML Py tract (5′-CCCUUUUUUUUCC-3′). The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; R227A, 166±2 nM; V254P, 137±10 nM; Q147A, 171±21 nM. The average KA and s.e.m. for three independent titrations are plotted. The P values from two-tailed unpaired t-tests with Welch's correction are indicated as follows: **P&lt;0.01; *P&lt;0.05; NS, not significant, P&gt;0.05. The average fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 4a–c.</text></passage><passage><infon key="file">ncomms10950-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>66426</offset><text>The U2AF65 linker/RRM and inter-RRM interactions.</text></passage><passage><infon key="file">ncomms10950-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>66476</offset><text>(a) Contacts of the U2AF65 inter-RRM linker with the RRMs. A semi-transparent space-filling surface is shown for the RRM1 (green) and RRM2 (light blue). Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. Other linker residues are coloured either dark blue for new residues in the U2AF651,2L structure or light blue for the remaining inter-RRM residues. The central panel shows an overall view with stick diagrams for mutated residues; boxed regions are expanded to show the C-terminal (bottom left) and central linker regions (top) at the inter-RRM interfaces, and N-terminal linker region contacts with RRM1 (bottom right). (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; 3Gly, 47±4 nM; 5Gly, 61±3 nM; 12Gly, 88±21 nM; NLALA, 45±3 nM; dU2AF651,2L, 123±5 nM; dU2AF651,2, 5000±100 nM; 3Mut, 5630±70 nM. The average KA and s.e.m. for three independent titrations are plotted. The P values from two-tailed unpaired t-tests with Welch's correction are indicated as follows: **P&lt;0.01; *P&lt;0.05; NS, not significant, P&gt;0.05. The fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 4d–j. (c) Close view of the U2AF65 RRM1/RRM2 interface following a two-fold rotation about the x-axis relative to a.</text></passage><passage><infon key="file">ncomms10950-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>68622</offset><text>U2AF65 inter-domain residues are important for splicing a representative pre-mRNA substrate in human cells.</text></passage><passage><infon key="file">ncomms10950-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>68730</offset><text>(a) Schematic diagram of the pyPY reporter minigene construct comprising two alternative splice sites preceded by either the weak IgM Py tract (py) or the strong AdML Py tract (PY) (sequences inset). (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). The average percentages and s.d.'s are given among four independent biological replicates. ****P&lt;0.0001 for two-tailed unpaired t-test with Welch's correction. Protein overexpression and qRT-PCR results are shown in Supplementary Fig. 5.</text></passage><passage><infon key="file">ncomms10950-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>69621</offset><text>RNA binding stabilizes the side-by-side conformation of U2AF65 RRMs.</text></passage><passage><infon key="file">ncomms10950-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>69690</offset><text>(a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of ‘side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or ‘closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2. The U2AF651,2LFRET proteins were doubly labelled at A181C/Q324C such that a mixture of Cy3/Cy5 fluorophores are expected to be present at each site. (c–f,i,j) The U2AF651,2LFRET(Cy3/Cy5) protein was immobilized on the microscope slide via biotin-NTA/Ni+2 (orange line) on a neutravidin (black X)-biotin-PEG (orange triangle)-treated surface and imaged either in the absence of ligands (c,d), in the presence of 5 μM AdML Py-tract RNA (5′-CCUUUUUUUUCC-3′) (e,f), or in the presence of 10 μM adenosine-interrupted variant RNA (5′-CUUUUUAAUUUCCA-3′) (i,j). In g and h, the immobilization protocol was reversed. The untethered U2AF651,2LFRET(Cy3/Cy5) protein (1 nM) was added to AdML RNA–polyethylene-glycol-linker–DNA oligonucleotide (10 nM), which was immobilized on the microscope slide by annealing with a complementary biotinyl-DNA oligonucleotide (black vertical line). Typical single-molecule FRET traces (c,e,g,i) show fluorescence intensities from Cy3 (green) and Cy5 (red) and the calculated apparent FRET efficiency (blue). Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). N is the number of single-molecule traces compiled for each histogram.</text></passage><passage><infon key="file">ncomms10950-f7.jpg</infon><infon key="id">f7</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>71544</offset><text>Schematic models of U2AF65 recognizing the Py tract.</text></passage><passage><infon key="file">ncomms10950-f7.jpg</infon><infon key="id">f7</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>71597</offset><text>(a) Diagram of the U2AF65, SF1 and U2AF35 splicing factors bound to the consensus elements of the 3′ splice site. A surface representation of U2AF651,2L is shown bound to nine nucleotides (nt); the relative distances and juxtaposition of the branch point sequence (BPS) and consensus AG dinucleotide at the 3′ splice site are unknown. MDS-relevant mutated residues of U2AF65 are shown as yellow spheres (L187 and M144). (b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the ‘closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to ∼0.45 FRET value, which is consistent with ‘open', side-by-side RRMs such as the U2AF651,2L crystal structures. Alternatively, a conformation of U2AF65 corresponding to ∼0.45 FRET value can directly bind to RNA; RNA binding stabilizes the ‘open', side-by-side conformation and thus shifts the U2AF65 population towards the ∼0.45 FRET value. RRM1, green; RRM2, pale blue; RRM extensions/linker, blue.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>72689</offset><text>Crystallographic data and refinement statistics*.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot; border=&quot;1&quot;&gt;&lt;colgroup&gt;&lt;col align=&quot;left&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;/colgroup&gt;&lt;thead valign=&quot;bottom&quot;&gt;&lt;tr&gt;&lt;th align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;Structure&lt;/bold&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;U2AF&lt;/bold&gt;&lt;sup&gt;&lt;bold&gt;65&lt;/bold&gt;&lt;/sup&gt;&lt;bold&gt;1,2L with rUrUrUdUdU(BrdU)dUrUrU&lt;/bold&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;U2AF&lt;/bold&gt;&lt;sup&gt;&lt;bold&gt;65&lt;/bold&gt;&lt;/sup&gt;&lt;bold&gt;1,2L with (P)rUrUdUdUrUdU(BrdU)dU&lt;/bold&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;U2AF&lt;/bold&gt;&lt;sup&gt;&lt;bold&gt;65&lt;/bold&gt;&lt;/sup&gt;&lt;bold&gt;1,2L with (P)rUrUdUrUrU(BrdU)dUdU&lt;/bold&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;U2AF&lt;/bold&gt;&lt;sup&gt;&lt;bold&gt;65&lt;/bold&gt;&lt;/sup&gt;&lt;bold&gt;1,2L with (P)rUrUrUdUrUrU(BrdU)dUrC&lt;/bold&gt;&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign=&quot;top&quot;&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;Data collection&lt;/italic&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;(i)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;(ii)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;(iii)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;(iv)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Space group&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;C222&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;C222&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;P2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;P2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Unit cell (Å) &lt;italic&gt;a,b,c&lt;/italic&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;62.1, 114.2, 59.4&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;61.9, 115.1, 59.5&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;43.4, 62.2, 77.4&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;43.5, 63.4, 77.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Resolution limits (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;32.46–2.04&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;32.57–1.86&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;38.71–1.50&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;38.83–1.57&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Completeness (%)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;95.5 (78.3)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;98.7 (95.9)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;98.2 (69.8)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;98.3 (71.7)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Redundancy&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;4.6 (4.1)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;4.3 (4.2)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;6.1 (3.0)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;6.2 (3.1)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;I&lt;/italic&gt;/&lt;italic&gt;σ&lt;/italic&gt;(&lt;italic&gt;I&lt;/italic&gt;)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;21.2 (4.2)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;24.6 (4.6)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;38.0 (6.5)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;42.9 (6.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;sym&lt;/sub&gt; (%)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;3.9 (32.1)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;3.9 (30.3)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;2.4 (14.8)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;2.2 (14.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;5&quot; align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;Refinement&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; No. reflections (work/test)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;12,124/1,055&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;17,870/1,456&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;31,802/1,996&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;28,162/2,000&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;work&lt;/sub&gt;/&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt; (%)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;17.3/22.8&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;15.1/18.8&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;15.3/18.6&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;15.4/17.6&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;5&quot; align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;No. atoms&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Protein&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;2,982&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;3,052&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;2,986&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;2,978&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Oligonucleotide&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;214&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;209&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;198&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;255&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Water&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;118&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;203&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;263&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;177&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;5&quot; align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;Bond r.m.s.d.&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Bond lengths (Å)&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;0.013&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;0.010&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;0.008&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;0.009&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Bond angles (°)&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;1.32&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;1.1&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;1.05&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;1.05&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;5&quot; align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;&amp;lt;B&amp;gt; factors (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Protein&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;46.4&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;27.4&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;26.3&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;26.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Oligonucleotide&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;61.8&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;35.2&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;24.5&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;30.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Water&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;45.2&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;35.2&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;30.7&lt;/td&gt;&lt;td align=&quot;char&quot; valign=&quot;top&quot; char=&quot;.&quot; charoff=&quot;50&quot;&gt;29.8&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>72739</offset><text>Structure	U2AF651,2L with rUrUrUdUdU(BrdU)dUrUrU	U2AF651,2L with (P)rUrUdUdUrUdU(BrdU)dU	U2AF651,2L with (P)rUrUdUrUrU(BrdU)dUdU	U2AF651,2L with (P)rUrUrUdUrUrU(BrdU)dUrC	 	Data collection	(i)	(ii)	(iii)	(iv)	 	Space group	C2221	C2221	P212121	P212121	 	Unit cell (Å) a,b,c	62.1, 114.2, 59.4	61.9, 115.1, 59.5	43.4, 62.2, 77.4	43.5, 63.4, 77.7	 	Resolution limits (Å)	32.46–2.04	32.57–1.86	38.71–1.50	38.83–1.57	 	Completeness (%)	95.5 (78.3)	98.7 (95.9)	98.2 (69.8)	98.3 (71.7)	 	Redundancy	4.6 (4.1)	4.3 (4.2)	6.1 (3.0)	6.2 (3.1)	 	I/σ(I)	21.2 (4.2)	24.6 (4.6)	38.0 (6.5)	42.9 (6.9)	 	Rsym (%)	3.9 (32.1)	3.9 (30.3)	2.4 (14.8)	2.2 (14.9)	 	Refinement	 	 No. reflections (work/test)	12,124/1,055	17,870/1,456	31,802/1,996	28,162/2,000	 	 Rwork/Rfree (%)	17.3/22.8	15.1/18.8	15.3/18.6	15.4/17.6	 	No. atoms	 	 Protein	2,982	3,052	2,986	2,978	 	 Oligonucleotide	214	209	198	255	 	 Water	118	203	263	177	 	Bond r.m.s.d.	 	 Bond lengths (Å)	0.013	0.010	0.008	0.009	 	 Bond angles (°)	1.32	1.1	1.05	1.05	 	&lt;B&gt; factors (Å2)	 	 Protein	46.4	27.4	26.3	26.7	 	 Oligonucleotide	61.8	35.2	24.5	30.5	 	 Water	45.2	35.2	30.7	29.8	 	</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>73893</offset><text>All available crystallographic data was used for refinement.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>73954</offset><text>*A single crystal was used for each structure. Values from the highest resolution shell are given in parentheses: 2.15–2.04; 1.90–1.86; 1.53–1.50; 1.61–1.57.</text></passage></document></collection>
diff --git a/raw_BioC_XML/PMC4792962_raw.xml b/raw_BioC_XML/PMC4792962_raw.xml
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+<?xml version="1.0" encoding="UTF-8"?>
+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection><source>PMC</source><date>20201221</date><key>pmc.key</key><document><id>4792962</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/ncomms10900</infon><infon key="article-id_pii">ncomms10900</infon><infon key="article-id_pmc">4792962</infon><infon key="article-id_pmid">26964885</infon><infon key="elocation-id">10900</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Huber;given-names:Eva M.</infon><infon key="name_1">surname:Heinemeyer;given-names:Wolfgang</infon><infon key="name_2">surname:Li;given-names:Xia</infon><infon key="name_3">surname:Arendt;given-names:Cassandra S.</infon><infon key="name_4">surname:Hochstrasser;given-names:Mark</infon><infon key="name_5">surname:Groll;given-names:Michael</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">7</infon><infon key="year">2016</infon><offset>0</offset><text>A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>87</offset><text>Biogenesis of the 20S proteasome is tightly regulated. The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic. Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1120</offset><text> The proteasome, an essential molecular machine, is a threonine protease, but the evolution and the components of its proteolytic centre are unclear. Here, the authors use structural biology and biochemistry to investigate the role of proteasome active site residues on maturation and activity.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1415</offset><text>The 20S proteasome core particle (CP) is the key non-lysosomal protease of eukaryotic cells. Its seven different α and seven different β subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder. While the inactive α subunits build the two outer rings, the β subunits form the inner rings. Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides. In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1). Release of the propeptides creates a functionally active CP that cleaves proteins into short peptides.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2246</offset><text>Although the chemical nature of the substrate-binding channel and hence substrate preferences are unique to each of the distinct active β subunits, all active sites employ an identical reaction mechanism to hydrolyse peptide bonds. Nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of the scissile peptide bond creates a first cleavage product and a covalent acyl-enzyme intermediate. Hydrolysis of this complex by the addition of a nucleophilic water molecule regenerates the enzyme and releases the second peptide fragment. The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage. This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed. An alternative candidate for deprotonating the Thr1 hydroxyl group is the side chain of Lys33 as it is within hydrogen-bonding distance to Thr1OH (2.7 Å). In principle it could function as the general base during both autocatalytic removal of the propeptide and protein substrate cleavage. Here we provide experimental evidences for this distinct view of the proteasome active-site mechanism. Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome. Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>4066</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>4074</offset><text>Inactivation of proteasome subunits by T1A mutations</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4127</offset><text>Proteasome-mediated degradation of cell-cycle regulators and potentially toxic misfolded proteins is required for the viability of eukaryotic cells. Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites. Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1). These results indicate that the β1 and β2 proteolytic activities are not essential for cell survival. By contrast, the T1A mutation in subunit β5 has been reported to be lethal or nearly so. Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1). Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5343</offset><text>The extremely weak growth of the β5-T1A mutant pp cis described by Chen and Hochstrasser compared with the inviability reported by Heinemeyer et al. prompted us to analyse this discrepancy. Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods). We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts. The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>6407</offset><text>Propeptide conformation and triggering of autolysis</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6459</offset><text>In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature β-subunit domains. For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2). Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.). Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1). In subunit β1, we found that Gly(-1) indeed forms a sharp turn, which relaxes on prosegment cleavage (Fig. 1a and Supplementary Fig. 2a). However, the γ-turn conformation and the associated hydrogen bond initially proposed is for geometric and chemical reasons inappropriate and would not perfectly position the carbonyl carbon atom of Gly(-1) for nucleophilic attack by Thr1. Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. Thr(-2) positions Gly(-1)O via hydrogen bonding (∼2.8 Å) in a perfect trajectory for the nucleophilic attack by Thr1Oγ (Fig. 1b and Supplementary Fig. 2b). Next, we examined the position of the β5 propeptide in the β5-T1A-K81R mutant. Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet. Nonetheless, the carbonyl carbon of Gly(-1) would be ideally placed for nucleophilic attack by Thr1Oγ (Fig. 1c and Supplementary Fig. 2c,d). As the K81R mutation is located far from the active site (Thr1Cα–Arg81Cα: 24 Å), any influence on propeptide conformation can be excluded. Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8870</offset><text>Processing of β-subunit precursors requires deprotonation of Thr1OH; however, the general base initiating autolysis is unknown. Remarkably, eukaryotic proteasomal β5 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a). As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). In agreement, the chymotrypsin-like (ChT-L) activity of H(-2)N and H(-2)F mutant yCPs was impaired in situ and in vitro (Supplementary Fig. 3c). Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO–FC electron densities. Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned. This observation indicates a mixture of processed and unprocessed β5 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10064</offset><text>Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. As expected from β5-T1A mutants, the yeasts show severe growth phenotypes, with minor variations (Supplementary Fig. 4a and Table 1). We determined crystal structures of the β5-H(-2)L-T1A, β5-H(-2)T-T1A and the β5-H(-2)A-T1A-K81R mutants (Supplementary Table 1). For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d). By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable. Nevertheless, both Leu(-2) and Thr(-2) were found to occupy the S1 specificity pocket formed by Met45 (Fig. 2a,b and Supplementary Fig. 4f–h). This result proves that the naturally occurring His(-2) of the β5 propeptide does not stably fit into the S1 site. Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12011</offset><text>Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. Although we observed fragments of 2FO–FC electron density in the β1 active site, the data were not interpretable. Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). Notably, the 2FO–FC electron-density map displays a different orientation for the β2 propeptide than has been observed for the β2-T1A proteasome. In particular, Val(-2) is displaced from the S1 site and Gly(-1) is severely shifted (movement of the carbonyl oxygen atom of 3.8 Å), thereby preventing nucleophilic attack of Thr1 (Fig. 2d and Supplementary Fig. 4j,k). These results further confirm that correct positioning of the active-site residues and Gly(-1) is decisive for the maturation of the proteasome.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13352</offset><text>The active site of the proteasome</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13386</offset><text>Proton shuttling from the proteasomal active site Thr1OH to Thr1NH2 via a nucleophilic water molecule was suggested to initiate peptide-bond hydrolysis. However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1Oγ. Instead, Lys33NH2, which is in hydrogen-bonding distance to Thr1Oγ (2.7 Å) in all catalytically active β subunits (Fig. 3a,b), was proposed to serve as the proton acceptor. Owing to its likely protonation at neutral pH, however, it was assumed not to act as the general base. A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site. Twenty years later, with a plethora of yCP X-ray structures in hand, we decided to re-analyse the active site of the proteasome and to resolve the uncertainty regarding the nature of the general base. Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1). The phenotype of the β5-K33A mutant was however less pronounced than for the β5-T1A-K81R yeast (Fig. 4a). This discrepancy in growth was traced to an additional point mutation L(-49)S in the β5-propeptide of the β5-K33A mutant (see also Supplementary Note 1). Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. This structural alteration destroys active-site integrity and abolishes catalytic activity of the β5 active site (Supplementary Fig. 5a). Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans). The Thr1 N terminus of this mutant is not blocked by the propeptide, yet its catalytic activity is reduced by ∼83% (Supplementary Fig. 6b). Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). This water hydrogen bonds also to Arg19O (∼3.0 Å) and Asp17Oδ (∼3.0 Å), and thereby presumably enables residual activity of the mutant. Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis. Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1). While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16924</offset><text>The conservative mutation of Asp17 to Asn in subunit β5 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1). Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP. As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. Autolysis and residual catalytic activity of the β5-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1. In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis. Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19094</offset><text>On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d). This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19489</offset><text>To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit. Asp166Oδ is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis. The β5-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1). X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a). Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing. The hydrogen bonds involving Ser169OH are intact and may account for residual substrate turnover. Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c). In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation. In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166. Whereas Asn can to some degree replace Asp166 due to its carbonyl group in the side chain, Ala at this position was found to prevent both autolysis and catalysis. These results suggest that Asp166 and Ser129 function as a proton shuttle and affect the protonation state of Thr1N during autolysis and catalysis.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>21052</offset><text>Substitution of the active-site Thr1 by Cys</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21096</offset><text>Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum. In yeast, this mutation causes a strong growth defect (Fig. 4a and Table 1), although the propeptide is hydrolysed, as shown here by its X-ray structure. In one of the two β5 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c). His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization. Owing to the unequal positions of the two β5 subunits within the CP in the crystal lattice, maturation and propeptide displacement may occur at different timescales in the two subunits.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22121</offset><text>Despite propeptide hydrolysis, the β5-T1C active site is catalytically inactive (Fig. 4b and Supplementary Fig. 9a). In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. Moreover, the structural data reveal that the thiol group of Cys1 is rotated by 74° with respect to the hydroxyl side chain of Thr1 (Fig. 4f and Supplementary Fig. 9b). This presumably results from the larger radius of the sulfur atom compared with oxygen. Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1. Notably, the 2FO–FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered. Together, these observations suggest that efficient peptide-bond hydrolysis requires that Lys33NH2 hydrogen bonds to the active site nucleophile.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>23124</offset><text>The benefit of Thr over Ser as the active-site nucleophile</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23183</offset><text>All proteasomes strictly employ threonine as the active-site residue instead of serine. To investigate the reason for this singularity, we analysed a β5-T1S mutant, which is viable but suffers from growth defects (Fig. 4a and Table 1). Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c). By contrast, turnover of the substrate Z-GGL-pNA, used to monitor ChT-L activity in situ but in a less quantitative fashion, is not detectably impaired (Supplementary Fig. 9a). Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24059</offset><text>However, the apo crystal structure revealed that Ser1Oγ is turned away from the substrate-binding channel (Fig. 4g). Compared with Thr1Oγ in WT CP structures, Ser1Oγ is rotated by 60°. This renders it unavailable for direct nucleophilic attack onto incoming substrates and first requires its reorientation, which is expected to delay substrate turnover. Because both conformations of Ser1Oγ are hydrogen-bonded to Lys33NH2 (Fig. 4h), the relay system is capable of hydrolysing peptide substrates, albeit at lower rates compared with Thr1. The active-site residue Thr1 is fixed in its position, as its methyl group is engaged in hydrophobic interactions with Thr3 and Ala46 (Fig. 4h). Consequently, the hydroxyl group of Thr1 requires no reorientation before substrate cleavage and is thus more catalytically efficient than Ser1. In agreement, at an elevated growing temperature of 37 °C the T1S mutant is unable to grow (Fig. 4a). In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5). Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors. Notably, the affinity of the tetrapeptide carfilzomib is less impaired, as it is better stabilized in the substrate-binding channel than the dipeptide bortezomib, which lacks a defined P3 site and has only a few interactions with the surrounding protein. Hence, the mean residence time of carfilzomib at the active site is prolonged and the probability to covalently react with Ser1 is increased. Considered together, these results provide a plausible explanation for the invariance of threonine as the active-site nucleophile in proteasomes in all three domains of life, as well as in proteasome-like proteases such as HslV (ref.).</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>25916</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>25927</offset><text>The 20S proteasome CP is the major non-lysosomal protease in eukaryotic cells, and its assembly is highly organized. The β-subunit propeptides, particularly that of β5, are key factors that help drive proper assembly of the CP complex. In addition, they prevent irreversible inactivation of the Thr1 N terminus by N-acetylation. By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli. In eukaryotes, deletion of or failure to cleave the β1 and β2 propeptides is well tolerated. However, removal of the β5 prosegment or any interference with its cleavage causes severe phenotypic defects. These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>26801</offset><text>Here we have described the atomic structures of various β5-T1A mutants, which allowed for the first time visualization of the residual β5 propeptide. Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1. From these data we conclude that only the positioning of Gly(-1) and Thr1 as well as the integrity of the proteasomal active site are required for autolysis. In this regard, inappropriate N-acetylation of the Thr1 N terminus cannot be removed by Thr1Oγ due to the rotational freedom and flexibility of the acetyl group. The propeptide needs some anchoring in the substrate-binding channel to properly position Gly(-1), but this seems to be independent of the orientation of residue (-2).</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>27707</offset><text>Autolytic activation of the CP constitutes one of the final steps of proteasome biogenesis, but the trigger for propeptide cleavage had remained enigmatic. On the basis of the numerous CP:ligand complexes solved during the past 18 years and in the current study, we provide a revised interpretation of proteasome active-site architecture. We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å). Analogously to the proteasome, a Thr–Lys–Asp triad is also found in L-asparaginase. Thus, specific protein surroundings can significantly alter the chemical properties of amino acids such as Lys to function as an acid–base catalyst.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>28792</offset><text>In this new view of the proteasomal active site, the positively charged Thr1NH3+-terminus hydrogen bonds to the amide nitrogen of incoming peptide substrates and stabilizes as well as activates them for the endoproteolytic cleavage by Thr1Oγ (Fig. 3d). Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.). Furthermore, opening of the β-lactone compound omuralide by Thr1 creates a C3-hydroxyl group, whose proton originates from Thr1NH3+. The resulting uncharged Thr1NH2 is hydrogen-bridged to the C3-OH group. In agreement, acetylation of the Thr1 N terminus irreversibly blocks hydrolytic activity, and binding of substrates is prevented for steric reasons. By acting as a proton donor during catalysis, the Thr1 N terminus may also favour cleavage of substrate peptide bonds (Fig. 3d). In all proteases, collapse of the tetrahedral transition state results in selective breakage of the substrate amide bond, while the covalent interaction between the substrate and the enzyme persists. Cleavage of the scissile peptide bond requires protonation of the emerging free amine, and in the proteasome, the Thr1 amine group is likely to assume this function. Analogously, Thr1NH3+ might promote the bivalent reaction mode of epoxyketone inhibitors by protonating the epoxide moiety to create a positively charged trivalent oxygen atom that is subsequently nucleophilically attacked by Thr1NH2.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>30357</offset><text>During autolysis the Thr1 N terminus is engaged in a hydroxyoxazolidine ring intermediate (Fig. 3d), which is unstable and short-lived. Breakdown of this tetrahedral transition state releases the Thr1 N terminus that is protonated by aspartic acid 166 via Ser129OH to yield Thr1NH3+. The residues Ser129 and Asp166 are expected to increase the pKa value of Thr1N, thereby favouring its charged state. Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. The mutation D166N lowers the pKa of Thr1N, which is thus more likely to exist in the uncharged deprotonated state (Thr1NH2). This renders the N terminus less suitable to stabilize substrates and to protonate the first cleavage product during catalysis, although it favours its ability to act as a nucleophile. This interpretation agrees with the strongly reduced catalytic activity of the β5-D166N mutant on the one hand, and the ability to react readily with carfilzomib on the other. Hence, the proteasome can be viewed as having a second triad that is essential for efficient proteolysis. While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>31717</offset><text>In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. Consequently, efficient substrate turnover or covalent modification by ligands is prevented. However, owing to Cys being a strong nucleophile, the propeptide can still be cleaved off over time. While only one single turnover is necessary for autolysis, continuous enzymatic activity is required for significant and detectable substrate hydrolysis. Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>32443</offset><text>To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally. Activity assays with the β5-T1S mutant revealed reduced turnover of Suc-LLVY-AMC. We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib. Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes. Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ). Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h). Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity. Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site. The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>34241</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>34249</offset><text>Yeast mutagenesis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34267</offset><text>Site-directed mutagenesis was performed by standard techniques using oligonucleotides listed in Supplementary Table 2. The pre2/doa3 (β5) mutant alleles in the centromeric, TRP1- or LEU2-marked shuttle vectors YCplac22 and pRS315, respectively, were verified by sequencing and subsequently introduced into the yeast strains MHY784 (ref.) or YWH20 (ref.), which express WT PRE2 from a URA3-marked plasmid. Counter-selection against the URA3 marker with 5-fluoroorotic acid yielded strains expressing only the mutant forms of β5.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34801</offset><text>The strain producing a processed β5-T1A variant and the β5 propeptide in trans is a derivative of YWH212 (ref.). It carries an additional deletion of the NAT1 gene to avoid N-acetylation of Ala1; this strain exhibits extremely slow growth rates and served for crystallographic analysis only. All strains used in this study are listed in Supplementary Table 3.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>35167</offset><text>Purification of yeast proteasomes</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35201</offset><text>Yeast strains were grown in 18-l cultures at 30 °C in YPD into early stationary phase, and the yCPs were purified according to published procedures. In brief, 120 g yeast cells were solubilized in 150 ml of 50 mM KH2PO4/K2HPO4 buffer (pH 7.5) and disrupted with a French press. Cell debris were removed by centrifugation for 30 min at 21,000 r.p.m. (4 °C). The resulting supernatant was filtered and ammonium sulfate (saturated solution) was added to a final concentration of 30% (v/v). This solution was loaded on a Phenyl Sepharose 6 Fast Flow column (GE Healthcare) pre-equilibrated with 1 M ammonium sulfate in 20 mM KH2PO4/K2HPO4 (pH 7.5). CPs were eluted by applying a linear gradient from 1 to 0 M ammonium sulfate. Proteasome-containing fractions were pooled and loaded onto a hydroxyapatite column (Bio-Rad) equilibrated with 20 mM KH2PO4/K2HPO4 (pH 7.5). Elution of the CPs was achieved by increasing the phosphate buffer concentration from 20 to 500 mM. Anion-exchange chromatogaphy (Resource Q column (GE Healthcare), elution gradient from 0 to 500 mM sodium chloride in 20 mM Tris-HCl (pH 7.5)) and subsequent size-exclusion chromatography (Superose 6 10/300 GL (GE Healthcare), 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl) resulted in pure CPs for crystallization and activity assays.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>36530</offset><text>Fluorescence-based activity assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>36564</offset><text>ChT-L (β5) activity of CPs was monitored by fluorescence spectroscopy using the model substrate Suc-LLVY-AMC. Purified yCPs (66 nM in 100 mM Tris-HCl, pH 7.5) were incubated with 300 μM substrate for 1 h at room temperature or 37 °C. The reactions were stopped by diluting samples 1:10 in 20 mM Tris-HCl, pH 7.5. AMC fluorophores released by proteasomal activity were measured in triplicate with a Varian Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies) at λexc=360 nm and λem=460 nm.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>37088</offset><text>Inhibition assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>37106</offset><text>Purified yCPs were mixed with dimethylsulfoxide as a control or serial dilutions of inhibitor and incubated for 45 min at room temperature. A final concentration of yCP of 66 nM was used. After addition of the peptide substrate Suc-LLVY-AMC to a final concentration of 200 μM and incubation for 1 h at room temperature, the reaction was stopped by diluting the samples 1:10 in 20 mM Tris-HCl, pH 7.5. AMC fluorophores released by residual proteasomal activity were measured in triplicate at λexc=360 nm and λem=460 nm. Relative fluorescence units were normalized to the dimethylsulfoxide-treated control. The calculated residual activities were plotted against the logarithm of the applied inhibitor concentration and fitted with GraphPad Prism 5. The IC50 value, the ligand concentration that leads to 50% inhibition of the enzymatic activity, was deduced from the fitted data.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>38002</offset><text>Crystallization and structure determination</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38046</offset><text>Mutant yCPs were crystallized as previously described for WT 20S proteasomes. Crystals were grown at 20 °C using the hanging drop vapour diffusion method. Drops contained a 1:1 mixture of protein (40 mg ml−1) and reservoir solution (25 mM magnesium acetate, 100 mM 2-(N-morpholino)ethanesulfonic acid (MES; pH 6.8) and 9–13% (v/v) 2-methyl-2,4-pentanediol (MPD)). Crystals were cryoprotected by addition of 5 μl cryobuffer (20 mM magnesium acetate, 100 mM MES, pH 6.8, and 30% (v/v) MPD). Inhibitor complex structures were obtained by incubating crystals in 5 μl cryobuffer supplemented with bortezomib or carfilzomib at a final concentration of 1.5 mM for at least 8 h.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38746</offset><text>Diffraction data were collected at the beamline X06SA at the Paul Scherrer Institute, SLS, Villigen, Switzerland (λ=1.0 Å). Evaluation of reflection intensities and data reduction were performed with the programme package XDS. Molecular replacement was carried out with the coordinates of the yeast 20S proteasome (PDB entry code: 5CZ4) by rigid body refinements (REFMAC5; ref.). MAIN and COOT were used to build models. TLS (Translation/Libration/Screw) refinements finally yielded excellent Rwork and Rfree, as well as root mean squared deviation bond and angle values. The coordinates, proven to have good stereochemistry from the Ramachandran plots, were deposited in the RCSB Protein Data Bank (Supplementary Table 1).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>39475</offset><text>The coordinates for the yeast 20S proteasome deposited under the entry code 1RYP do not represent the WT yCP but the double-mutant β5-K33R β1-T1A. At the time of deposition (in 1997), these data were the best available on the yCP. As yCP structure determination has become routine today, and structure refinement procedures have significantly improved, we here provide coordinates for the WT yCP at 2.3 Å resolution (PDB entry code: 5CZ4). Furthermore, the structures of most mutant yCPs described in this work were determined in their apo and ligand-bound states. For mutants with proteolytically inactive β5 subunits, the best crystallographic data obtained are given. For ligands or propeptide segments that were only partially defined in the 2FO–FC electron-density map the occupancy was reduced (for details see Supplementary Table 1).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>40325</offset><text>Additional information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>40348</offset><text>Accession codes: Coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (for PDB entry codes see Supplementary Table 1).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>40506</offset><text>How to cite this article: Huber, E. M. et al. A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome. Nat. Commun. 7:10900 doi: 10.1038/ncomms10900 (2016).</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>40694</offset><text>Supplementary Material</text></passage><passage><infon key="fpage">961</infon><infon key="lpage">972</infon><infon key="name_0">surname:Chen;given-names:P.</infon><infon key="name_1">surname:Hochstrasser;given-names:M.</infon><infon key="pub-id_pmid">8808631</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">86</infon><infon key="year">1996</infon><offset>40717</offset><text>Autocatalytic subunit processing couples active site formation in the 20S proteasome to completion of assembly</text></passage><passage><infon key="fpage">463</infon><infon key="lpage">471</infon><infon key="name_0">surname:Groll;given-names:M.</infon><infon key="pub-id_pmid">9087403</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">386</infon><infon key="year">1997</infon><offset>40828</offset><text>Structure of 20S proteasome from yeast at 2.4 Å resolution</text></passage><passage><infon key="fpage">289</infon><infon key="lpage">293</infon><infon key="name_0">surname:Krüger;given-names:E.</infon><infon key="name_1">surname:Kloetzel;given-names:P. M.</infon><infon key="name_2">surname:Enenkel;given-names:C.</infon><infon key="pub-id_pmid">11295488</infon><infon key="section_type">REF</infon><infon key="source">Biochimie</infon><infon key="type">ref</infon><infon key="volume">83</infon><infon key="year">2001</infon><offset>40890</offset><text>20S proteasome biogenesis</text></passage><passage><infon key="fpage">10976</infon><infon key="lpage">10983</infon><infon key="name_0">surname:Groll;given-names:M.</infon><infon key="pub-id_pmid">10500111</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">96</infon><infon key="year">1999</infon><offset>40916</offset><text>The catalytic sites of 20S proteasomes and their role in subunit maturation: a mutational and crystallographic study</text></passage><passage><infon key="fpage">1187</infon><infon key="lpage">1191</infon><infon key="name_0">surname:Ditzel;given-names:L.</infon><infon key="pub-id_pmid">9642094</infon><infon key="section_type">REF</infon><infon key="source">J. Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">279</infon><infon key="year">1998</infon><offset>41033</offset><text>Conformational constraints for protein self-cleavage in the proteasome</text></passage><passage><infon key="fpage">727</infon><infon key="lpage">738</infon><infon key="name_0">surname:Huber;given-names:E. M.</infon><infon key="pub-id_pmid">22341445</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">148</infon><infon key="year">2012</infon><offset>41104</offset><text>Immuno- and constitutive proteasome crystal structures reveal differences in substrate and inhibitor specificity</text></passage><passage><infon key="fpage">7835</infon><infon key="lpage">7842</infon><infon key="name_0">surname:Huber;given-names:E. M.</infon><infon key="pub-id_pmid">26020686</infon><infon key="section_type">REF</infon><infon key="source">J. Am. Chem. Soc.</infon><infon key="type">ref</infon><infon key="volume">137</infon><infon key="year">2015</infon><offset>41217</offset><text>Systematic analyses of substrate preferences of 20S proteasomes using peptidic epoxyketone inhibitors</text></passage><passage><infon key="fpage">1509</infon><infon key="lpage">1536</infon><infon key="name_0">surname:Marques;given-names:A. J.</infon><infon key="name_1">surname:Palanimurugan;given-names:R.</infon><infon key="name_2">surname:Matias;given-names:A. C.</infon><infon key="name_3">surname:Ramos;given-names:P. C.</infon><infon key="name_4">surname:Dohmen;given-names:R. J.</infon><infon key="pub-id_pmid">19265443</infon><infon key="section_type">REF</infon><infon key="source">Chem. Rev.</infon><infon key="type">ref</infon><infon key="volume">109</infon><infon key="year">2009</infon><offset>41319</offset><text>Catalytic mechanism and assembly of the proteasome</text></passage><passage><infon key="fpage">533</infon><infon key="lpage">539</infon><infon key="name_0">surname:Löwe;given-names:J.</infon><infon key="pub-id_pmid">7725097</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">268</infon><infon key="year">1995</infon><offset>41370</offset><text>Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 Å resolution</text></passage><passage><infon key="fpage">416</infon><infon key="lpage">419</infon><infon key="name_0">surname:Brannigan;given-names:J. A.</infon><infon key="pub-id_pmid">7477383</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">378</infon><infon key="year">1995</infon><offset>41466</offset><text>A protein catalytic framework with an N-terminal nucleophile is capable of self-activation</text></passage><passage><infon key="fpage">579</infon><infon key="lpage">582</infon><infon key="name_0">surname:Seemüller;given-names:E.</infon><infon key="pub-id_pmid">7725107</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">268</infon><infon key="year">1995</infon><offset>41557</offset><text>Proteasome from Thermoplasma acidophilum: a threonine protease</text></passage><passage><infon key="fpage">25637</infon><infon key="lpage">25646</infon><infon key="name_0">surname:Dick;given-names:T. P.</infon><infon key="pub-id_pmid">9748229</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">273</infon><infon key="year">1998</infon><offset>41620</offset><text>Contribution of proteasomal beta-subunits to the cleavage of peptide substrates analyzed with yeast mutants</text></passage><passage><infon key="fpage">25200</infon><infon key="lpage">25209</infon><infon key="name_0">surname:Heinemeyer;given-names:W.</infon><infon key="name_1">surname:Fischer;given-names:M.</infon><infon key="name_2">surname:Krimmer;given-names:T.</infon><infon key="name_3">surname:Stachon;given-names:U.</infon><infon key="name_4">surname:Wolf;given-names:D. H.</infon><infon key="pub-id_pmid">9312134</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">272</infon><infon key="year">1997</infon><offset>41728</offset><text>The active sites of the eukaryotic 20S proteasome and their involvement in subunit precursor processing</text></passage><passage><infon key="fpage">7156</infon><infon key="lpage">7161</infon><infon key="name_0">surname:Arendt;given-names:C. S.</infon><infon key="name_1">surname:Hochstrasser;given-names:M.</infon><infon key="pub-id_pmid">9207060</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">94</infon><infon key="year">1997</infon><offset>41832</offset><text>Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation</text></passage><passage><infon key="fpage">997</infon><infon key="lpage">1013</infon><infon key="name_0">surname:Jäger;given-names:S.</infon><infon key="name_1">surname:Groll;given-names:M.</infon><infon key="name_2">surname:Huber;given-names:R.</infon><infon key="name_3">surname:Wolf;given-names:D. H.</infon><infon key="name_4">surname:Heinemeyer;given-names:W.</infon><infon key="pub-id_pmid">10452902</infon><infon key="section_type">REF</infon><infon key="source">J. Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">291</infon><infon key="year">1999</infon><offset>41953</offset><text>Proteasome beta-type subunits: unequal roles of propeptides in core particle maturation and a hierarchy of active site function</text></passage><passage><infon key="fpage">3575</infon><infon key="lpage">3585</infon><infon key="name_0">surname:Arendt;given-names:C. S.</infon><infon key="name_1">surname:Hochstrasser;given-names:M.</infon><infon key="pub-id_pmid">10393174</infon><infon key="section_type">REF</infon><infon key="source">EMBO J.</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">1999</infon><offset>42081</offset><text>Eukaryotic 20S proteasome catalytic subunit propeptides prevent active site inactivation by N-terminal acetylation and promote particle assembly</text></passage><passage><infon key="fpage">2161</infon><infon key="lpage">2172</infon><infon key="name_0">surname:Polgar;given-names:L.</infon><infon key="pub-id_pmid">16003488</infon><infon key="section_type">REF</infon><infon key="source">Cell. Mol. Life Sci.</infon><infon key="type">ref</infon><infon key="volume">62</infon><infon key="year">2005</infon><offset>42226</offset><text>The catalytic triad of serine peptidases</text></passage><passage><infon key="fpage">1991</infon><infon key="lpage">2003</infon><infon key="name_0">surname:Li;given-names:X.</infon><infon key="name_1">surname:Li;given-names:Y.</infon><infon key="name_2">surname:Arendt;given-names:C. S.</infon><infon key="name_3">surname:Hochstrasser;given-names:M.</infon><infon key="pub-id_pmid">26627836</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">291</infon><infon key="year">2015</infon><offset>42267</offset><text>Distinct elements in the proteasomal beta5 subunit propeptide required for autocatalytic processing and proteasome assembly</text></passage><passage><infon key="fpage">6887</infon><infon key="lpage">6898</infon><infon key="name_0">surname:Schmidtke;given-names:G.</infon><infon key="pub-id_pmid">9003765</infon><infon key="section_type">REF</infon><infon key="source">EMBO J.</infon><infon key="type">ref</infon><infon key="volume">15</infon><infon key="year">1996</infon><offset>42391</offset><text>Analysis of mammalian 20S proteasome biogenesis: the maturation of beta-subunits is an ordered two-step mechanism involving autocatalysis</text></passage><passage><infon key="fpage">417</infon><infon key="lpage">420</infon><infon key="name_0">surname:Wlodawer;given-names:A.</infon><infon key="pub-id_pmid">7663937</infon><infon key="section_type">REF</infon><infon key="source">Structure</infon><infon key="type">ref</infon><infon key="volume">3</infon><infon key="year">1995</infon><offset>42529</offset><text>Proteasome: a complex protease with a new fold and a distinct mechanism</text></passage><passage><infon key="fpage">468</infon><infon key="lpage">471</infon><infon key="name_0">surname:Seemüller;given-names:E.</infon><infon key="name_1">surname:Lupas;given-names:A.</infon><infon key="name_2">surname:Baumeister;given-names:W.</infon><infon key="pub-id_pmid">8684489</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">382</infon><infon key="year">1996</infon><offset>42601</offset><text>Autocatalytic processing of the 20S proteasome</text></passage><passage><infon key="fpage">451</infon><infon key="lpage">456</infon><infon key="name_0">surname:Groll;given-names:M.</infon><infon key="name_1">surname:Berkers;given-names:C. R.</infon><infon key="name_2">surname:Ploegh;given-names:H. L.</infon><infon key="name_3">surname:Ovaa;given-names:H.</infon><infon key="pub-id_pmid">16531229</infon><infon key="section_type">REF</infon><infon key="source">Structure</infon><infon key="type">ref</infon><infon key="volume">14</infon><infon key="year">2006</infon><offset>42648</offset><text>Crystal structure of the boronic acid-based proteasome inhibitor bortezomib in complex with the yeast 20S proteasome</text></passage><passage><infon key="fpage">407</infon><infon key="lpage">417</infon><infon key="name_0">surname:Huber;given-names:E. M.</infon><infon key="name_1">surname:Heinemeyer;given-names:W.</infon><infon key="name_2">surname:Groll;given-names:M.</infon><infon key="pub-id_pmid">25599643</infon><infon key="section_type">REF</infon><infon key="source">Structure</infon><infon key="type">ref</infon><infon key="volume">23</infon><infon key="year">2015</infon><offset>42765</offset><text>Bortezomib-resistant mutant proteasomes: structural and biochemical evaluation with carfilzomib and ONX 0914</text></passage><passage><infon key="fpage">6070</infon><infon key="lpage">6074</infon><infon key="name_0">surname:Bochtler;given-names:M.</infon><infon key="name_1">surname:Ditzel;given-names:L.</infon><infon key="name_2">surname:Groll;given-names:M.</infon><infon key="name_3">surname:Huber;given-names:R.</infon><infon key="pub-id_pmid">9177170</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">94</infon><infon key="year">1997</infon><offset>42874</offset><text>Crystal structure of heat shock locus V (HslV) from Escherichia coli</text></passage><passage><infon key="fpage">765</infon><infon key="lpage">770</infon><infon key="name_0">surname:Zwickl;given-names:P.</infon><infon key="name_1">surname:Kleinz;given-names:J.</infon><infon key="name_2">surname:Baumeister;given-names:W.</infon><infon key="pub-id_pmid">7634086</infon><infon key="section_type">REF</infon><infon key="source">Nat. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">1</infon><infon key="year">1994</infon><offset>42943</offset><text>Critical elements in proteasome assembly</text></passage><passage><infon key="fpage">75</infon><infon key="lpage">83</infon><infon key="name_0">surname:Groll;given-names:M.</infon><infon key="name_1">surname:Brandstetter;given-names:H.</infon><infon key="name_2">surname:Bartunik;given-names:H.</infon><infon key="name_3">surname:Bourenkow;given-names:G.</infon><infon key="name_4">surname:Huber;given-names:R.</infon><infon key="pub-id_pmid">12614609</infon><infon key="section_type">REF</infon><infon key="source">J. Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">327</infon><infon key="year">2003</infon><offset>42984</offset><text>Investigations on the maturation and regulation of archaebacterial proteasomes</text></passage><passage><infon key="fpage">84</infon><infon key="lpage">92</infon><infon key="name_0">surname:Lubkowski;given-names:J.</infon><infon key="name_1">surname:Dauter;given-names:M.</infon><infon key="name_2">surname:Aghaiypour;given-names:K.</infon><infon key="name_3">surname:Wlodawer;given-names:A.</infon><infon key="name_4">surname:Dauter;given-names:Z.</infon><infon key="pub-id_pmid">12499544</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">59</infon><infon key="year">2003</infon><offset>43063</offset><text>Atomic resolution structure of Erwinia chrysanthemi L-asparaginase</text></passage><passage><infon key="fpage">622</infon><infon key="lpage">629</infon><infon key="name_0">surname:Gutteridge;given-names:A.</infon><infon key="name_1">surname:Thornton;given-names:J. M.</infon><infon key="pub-id_pmid">16214343</infon><infon key="section_type">REF</infon><infon key="source">Trends Biochem. Sci.</infon><infon key="type">ref</infon><infon key="volume">30</infon><infon key="year">2005</infon><offset>43130</offset><text>Understanding nature's catalytic toolkit</text></passage><passage><infon key="fpage">501</infon><infon key="lpage">512</infon><infon key="name_0">surname:Hines;given-names:J.</infon><infon key="name_1">surname:Groll;given-names:M.</infon><infon key="name_2">surname:Fahnestock;given-names:M.</infon><infon key="name_3">surname:Crews;given-names:C. M.</infon><infon key="pub-id_pmid">18482702</infon><infon key="section_type">REF</infon><infon key="source">Chem. Biol.</infon><infon key="type">ref</infon><infon key="volume">15</infon><infon key="year">2008</infon><offset>43171</offset><text>Proteasome inhibition by fellutamide B induces nerve growth factor synthesis</text></passage><passage><infon key="fpage">1679</infon><infon key="lpage">1683</infon><infon key="name_0">surname:Stein;given-names:M. L.</infon><infon key="section_type">REF</infon><infon key="source">Angew. Chem. Int. Ed.</infon><infon key="type">ref</infon><infon key="volume">53</infon><infon key="year">2014</infon><offset>43248</offset><text>Systematic comparison of peptidic proteasome inhibitors highlights the alpha-ketoamide electrophile as an auspicious reversible lead motif</text></passage><passage><infon key="fpage">4576</infon><infon key="lpage">4579</infon><infon key="name_0">surname:Groll;given-names:M.</infon><infon key="name_1">surname:Larionov;given-names:O. V.</infon><infon key="name_2">surname:Huber;given-names:R.</infon><infon key="name_3">surname:de Meijere;given-names:A.</infon><infon key="pub-id_pmid">16537370</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">103</infon><infon key="year">2006</infon><offset>43387</offset><text>Inhibitor-binding mode of homobelactosin C to proteasomes: new insights into class I MHC ligand generation</text></passage><passage><infon key="fpage">5136</infon><infon key="lpage">5141</infon><infon key="name_0">surname:Groll;given-names:M.</infon><infon key="name_1">surname:Huber;given-names:R.</infon><infon key="name_2">surname:Potts;given-names:B. C.</infon><infon key="pub-id_pmid">16608349</infon><infon key="section_type">REF</infon><infon key="source">J. Am. Chem. Soc.</infon><infon key="type">ref</infon><infon key="volume">128</infon><infon key="year">2006</infon><offset>43494</offset><text>Crystal structures of Salinosporamide A (NPI-0052) and B (NPI-0047) in complex with the 20S proteasome reveal important consequences of beta-lactone ring opening and a mechanism for irreversible binding</text></passage><passage><infon key="fpage">6270</infon><infon key="lpage">6276</infon><infon key="name_0">surname:Choi;given-names:K. S.</infon><infon key="name_1">surname:Kim;given-names:J. A.</infon><infon key="name_2">surname:Kang;given-names:H. S.</infon><infon key="pub-id_pmid">1400178</infon><infon key="section_type">REF</infon><infon key="source">J. Bacteriol.</infon><infon key="type">ref</infon><infon key="volume">174</infon><infon key="year">1992</infon><offset>43697</offset><text>Effects of site-directed mutations on processing and activities of penicillin G acylase from Escherichia coli ATCC 11105</text></passage><passage><infon key="fpage">1443</infon><infon key="lpage">1452</infon><infon key="name_0">surname:Khan;given-names:J. A.</infon><infon key="name_1">surname:Dunn;given-names:B. M.</infon><infon key="name_2">surname:Tong;given-names:L.</infon><infon key="pub-id_pmid">16216576</infon><infon key="section_type">REF</infon><infon key="source">Structure</infon><infon key="type">ref</infon><infon key="volume">13</infon><infon key="year">2005</infon><offset>43818</offset><text>Crystal structure of human Taspase1, a crucial protease regulating the function of MLL</text></passage><passage><infon key="fpage">14831</infon><infon key="lpage">14837</infon><infon key="name_0">surname:Kisselev;given-names:A. F.</infon><infon key="name_1">surname:Songyang;given-names:Z.</infon><infon key="name_2">surname:Goldberg;given-names:A. L.</infon><infon key="pub-id_pmid">10809725</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">275</infon><infon key="year">2000</infon><offset>43905</offset><text>Why does threonine, and not serine, function as the active site nucleophile in proteasomes?</text></passage><passage><infon key="fpage">373</infon><infon key="lpage">390</infon><infon key="name_0">surname:Gallastegui;given-names:N.</infon><infon key="name_1">surname:Groll;given-names:M.</infon><infon key="pub-id_pmid">22350899</infon><infon key="section_type">REF</infon><infon key="source">Methods Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">832</infon><infon key="year">2012</infon><offset>43997</offset><text>Analysing properties of proteasome inhibitors using kinetic and X-ray crystallographic studies</text></passage><passage><infon key="fpage">329</infon><infon key="lpage">336</infon><infon key="name_0">surname:Groll;given-names:M.</infon><infon key="name_1">surname:Huber;given-names:R.</infon><infon key="pub-id_pmid">16275340</infon><infon key="section_type">REF</infon><infon key="source">Methods Enzymol.</infon><infon key="type">ref</infon><infon key="volume">398</infon><infon key="year">2005</infon><offset>44092</offset><text>Purification, crystallization, and X-ray analysis of the yeast 20S proteasome</text></passage><passage><infon key="fpage">125</infon><infon key="lpage">132</infon><infon key="name_0">surname:Kabsch;given-names:W.</infon><infon key="pub-id_pmid">20124692</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>44170</offset><text>XDS</text></passage><passage><infon key="fpage">2184</infon><infon key="lpage">2195</infon><infon key="name_0">surname:Vagin;given-names:A. A.</infon><infon key="pub-id_pmid">15572771</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">60</infon><infon key="year">2004</infon><offset>44174</offset><text>REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use</text></passage><passage><infon key="fpage">1342</infon><infon key="lpage">1357</infon><infon key="name_0">surname:Turk;given-names:D.</infon><infon key="pub-id_pmid">23897458</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">69</infon><infon key="year">2013</infon><offset>44262</offset><text>MAIN software for density averaging, model building, structure refinement and validation</text></passage><passage><infon key="fpage">486</infon><infon key="lpage">501</infon><infon key="name_0">surname:Emsley;given-names:P.</infon><infon key="name_1">surname:Lohkamp;given-names:B.</infon><infon key="name_2">surname:Scott;given-names:W. G.</infon><infon key="name_3">surname:Cowtan;given-names:K.</infon><infon key="pub-id_pmid">20383002</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>44351</offset><text>Features and development of Coot</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>44384</offset><text>Author contributions E.M.H., W.H., X.L., C.S.A. and M.H. created yeast mutants; E.M.H. and W.H. performed activity and growth assays; E.M.H. and M.G. collected and analysed X-ray data; E.M.H., M.H. and M.G. wrote the manuscript.</text></passage><passage><infon key="file">ncomms10900-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>44613</offset><text>Conformation of proteasomal propeptides.</text></passage><passage><infon key="file">ncomms10900-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>44654</offset><text>(a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. Only the residues (-5) to (-1) of the β1-T1A propeptide are displayed. The major determinant of the S1 specificity pocket, residue 45, is depicted. Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow). The black arrow indicates the attack of Thr1Oγ onto the carbonyl carbon atom of Gly(-1). (b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well. Thr(-2)OH is hydrogen-bonded to Gly(-1)O (∼2.8 Å; black dashed line). The major determinant of the S1 specificity pocket, residue 45, is depicted. (c) Structural superposition of the β1-T1A, the β2-T1A and the β5-T1A-K81R propeptide remnants depict their differences in conformation. While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. Nonetheless, in all mutants the carbonyl carbon atom of Gly(-1) is ideally placed for the nucleophilic attack by Thr1Oγ. The hydrogen bond between Thr(-2)OH and Gly(-1)O (∼2.8 Å) is indicated by a black dashed line.</text></passage><passage><infon key="file">ncomms10900-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>46013</offset><text>Mutations of residue (-2) and their influence on propeptide conformation and autolysis.</text></passage><passage><infon key="file">ncomms10900-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>46101</offset><text>(a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide. The (-2) residues of both prosegments point into the S1 pocket. (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. While the residues (-2) to (-4) vary in their conformation, Gly(-1) and Ala1 are located in all structures at the same positions. (c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide. The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line). (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of Gly(-1). For all panels stereo views are provided in Supplementary Fig. 4g–j.</text></passage><passage><infon key="file">ncomms10900-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>47199</offset><text>Architecture and proposed reaction mechanism of the proteasomal active site.</text></passage><passage><infon key="file">ncomms10900-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>47276</offset><text>(a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines). Thr1OH is hydrogen-bonded to Lys33NH2 (2.7 Å), which in turn interacts with Asp17Oδ. The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. In the latter, a water molecule (red sphere) is found at the position where in the WT structure the side chain amine group of Lys33 is located. Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH. Note, the strong interaction with the water molecule causes a minor shift of Thr1, while all other active-site residues remain in place. (d) Proposed chemical reaction mechanism for autocatalytic precursor processing and proteolysis in the proteasome. The active-site Thr1 is depicted in blue, the propeptide segment and the peptide substrate are coloured in green, whereas the scissile peptide bond is highlighted in red. Autolysis (left set of structures) is initiated by deprotonation of Thr1OH via Lys33NH2 and the formation of a tetrahedral transition state. The strictly conserved oxyanion hole Gly47NH stabilizing the negatively charged intermediate is illustrated as a semicircle. Collapse of the transition state frees the Thr1 N terminus (by completing an N-to-O acyl shift of the propeptide), which is subsequently protonated by Asp166OH via Ser129OH. Next, Thr1NH2 polarizes a water molecule for the nucleophilic attack of the acyl-enzyme intermediate. On hydrolysis of the latter, the active-site Thr1 is ready for catalysis (right set of structures). Substrate processing starts with nucleophilic attack of the carbonyl carbon atom of the scissile peptide bond. The charged Thr1 N terminus may engage in the orientation of the amide moiety and donate a proton to the emerging N terminus of the C-terminal cleavage product. The resulting deprotonated Thr1NH2 finally activates a water molecule for hydrolysis of the acyl-enzyme.</text></passage><passage><infon key="file">ncomms10900-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>49537</offset><text>The proteasome favours threonine as the active-site nucleophile.</text></passage><passage><infon key="file">ncomms10900-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>49602</offset><text>(a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation. (b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (β5) using the substrate Suc-LLVY-AMC. Relative fluorescence units were measured in triplicate after 1 h of incubation at room temperature and are given as mean values. S.d.'s are indicated by error bars. (c) Illustration of the 2FO–FC electron-density map (blue mesh contoured at 1σ) for the β5-T1C propeptide fragment. The prosegment is cleaved but still bound in the substrate-binding channel. Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the β5-T1A-K81R mutant. (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). The inhibitor as well as the propeptides adopt similar conformations in the substrate-binding channel. (f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1. The SH group is rotated by 74° compared with the OH group. (g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively. The 2FO–FC electron-density map for Ser1 (blue mesh contoured at 1σ) is illustrated. (h) The methyl group of Thr1 is anchored by hydrophobic interactions with Ala46Cβ and Thr3Cγ. Ser1 lacks this stabilization and is therefore rotated by 60°.</text></passage><passage><infon key="file">ncomms10900-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>51469</offset><text>Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib.</text></passage><passage><infon key="file">ncomms10900-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>51550</offset><text>Inhibition assays (left panel). Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC. IC50 values were determined in triplicate; s.d.'s are indicated by error bars. Note that IC50 values depend on time and enzyme concentration. Proteasomes (final concentration: 66 nM) were incubated with inhibitor for 45 min before substrate addition (final concentration: 200 μM). Structures of the β5-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel). The 2FO–FC electron-density maps (blue mesh) for Ser1 (brown) and the covalently bound ligands (green; only the P1 site (Leu1) is shown) are contoured at 1σ. The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>52522</offset><text> Growth phenotypes and status of autolysis and catalysis of mutants.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot; border=&quot;1&quot;&gt;&lt;colgroup&gt;&lt;col align=&quot;left&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;/colgroup&gt;&lt;thead valign=&quot;bottom&quot;&gt;&lt;tr&gt;&lt;th align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;Mutant&lt;/bold&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;Viability&lt;/bold&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;Temperature sensitivity&lt;/bold&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;Autolysis state of the mutant subunit&lt;/bold&gt;&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn2&quot;&gt;*&lt;/xref&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;Activity of the mutant subunit&lt;/bold&gt;&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign=&quot;top&quot;&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;WT&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+++&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β1-T1A (ref.&lt;xref ref-type=&quot;bibr&quot; rid=&quot;b13&quot;&gt;13&lt;/xref&gt;)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β2-T1A (ref.&lt;xref ref-type=&quot;bibr&quot; rid=&quot;b13&quot;&gt;13&lt;/xref&gt;)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β1-T1A β2-T1A (ref.&lt;xref ref-type=&quot;bibr&quot; rid=&quot;b13&quot;&gt;13&lt;/xref&gt;)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-T1A&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+/−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-H(-2)A-T1A&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+/−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-H(-2)T-T1A&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+/−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-H(-2)L-T1A&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-T1A, pp &lt;italic&gt;trans&lt;/italic&gt;, &lt;italic&gt;nat1&lt;/italic&gt;Δ&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+/−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;pp &lt;italic&gt;trans&lt;/italic&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-T1A-K81R&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-H(-2)A-T1A-K81R&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+/−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-H(-2)T-T1A-K81R&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+/−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-H(-2)L-T1A-K81R&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-H(-2)A&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+++&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-H(-2)K&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+++&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-H(-2)F&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-H(-2)N&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5pp-β1 (ref. 18)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+/−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+/−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β2-T(-2)V&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-L(-49S)-K33A (ref.&lt;xref ref-type=&quot;bibr&quot; rid=&quot;b13&quot;&gt;13&lt;/xref&gt;)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-K33A, pp &lt;italic&gt;trans&lt;/italic&gt;&lt;xref ref-type=&quot;bibr&quot; rid=&quot;b13&quot;&gt;13&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;pp &lt;italic&gt;trans&lt;/italic&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+/−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-F(-45)S-K33R (ref.&lt;xref ref-type=&quot;bibr&quot; rid=&quot;b13&quot;&gt;13&lt;/xref&gt;)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-D17N&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+/−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn3&quot;&gt;†&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn3&quot;&gt;†&lt;/xref&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-L(-49)S-D17N&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+/−&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+/−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-D17N, pp &lt;italic&gt;trans&lt;/italic&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;pp &lt;italic&gt;trans&lt;/italic&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+/−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-D166N&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+/−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-D166N, pp &lt;italic&gt;trans&lt;/italic&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;pp &lt;italic&gt;trans&lt;/italic&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+/−&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-T1S&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β5-T1C&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;++++&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;+&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;−&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>52591</offset><text>Mutant	Viability	Temperature sensitivity	Autolysis state of the mutant subunit*	Activity of the mutant subunit	 	WT	++++	+	+	+++	 	β1-T1A (ref.)	++++	+	−	−	 	β2-T1A (ref.)	+++	++	−	−	 	β1-T1A β2-T1A (ref.)	+++	++	−	−	 	β5-T1A	+/−	++++	−	−	 	β5-H(-2)A-T1A	+/−	ND	−	−	 	β5-H(-2)T-T1A	+/−	ND	−	−	 	β5-H(-2)L-T1A	++	ND	−	−	 	β5-T1A, pp trans, nat1Δ	+/−	++++	pp trans	−	 	β5-T1A-K81R	+	++++	−	−	 	β5-H(-2)A-T1A-K81R	+/−	ND	−	−	 	β5-H(-2)T-T1A-K81R	+/−	ND	−	−	 	β5-H(-2)L-T1A-K81R	++	ND	−	−	 	β5-H(-2)A	++++	++	+	+++	 	β5-H(-2)K	++++	++	+	+++	 	β5-H(-2)F	++++	+++	+	++	 	β5-H(-2)N	++++	+++	+	++	 	β5pp-β1 (ref. 18)	++++	+	+/−	+/−	 	β2-T(-2)V	++++	+	−	−	 	β5-L(-49S)-K33A (ref.)	+	++++	−	−	 	β5-K33A, pp trans	+	++++	pp trans	+/−	 	β5-F(-45)S-K33R (ref.)	++	++++	+	−	 	β5-D17N	+/−	++++	ND†	ND†	 	β5-L(-49)S-D17N	+	++++	+/−	+/−	 	β5-D17N, pp trans	+	++++	pp trans	+/−	 	β5-D166N	++	++++	+	+/−	 	β5-D166N, pp trans	+++	++++	pp trans	+/−	 	β5-T1S	+++	++++	+	++	 	β5-T1C	++	++++	+	−	 	</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>53689</offset><text>ND, not determined.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>53709</offset><text>*The autolysis state was assessed by purification and crystallization of the mutant proteasomes.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>53806</offset><text>†Purification of this mutant proteasome was not possible.</text></passage></document></collection>
diff --git a/raw_BioC_XML/PMC4832331_raw.xml b/raw_BioC_XML/PMC4832331_raw.xml
new file mode 100644
index 0000000000000000000000000000000000000000..f97cdd3eb931eb25cf1b8bd4a114d40d88e7509c
--- /dev/null
+++ b/raw_BioC_XML/PMC4832331_raw.xml
@@ -0,0 +1,99 @@
+<?xml version="1.0" encoding="UTF-8"?>
+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection><source>PMC</source><date>20201215</date><key>pmc.key</key><document><id>4832331</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/srep24601</infon><infon key="article-id_pii">srep24601</infon><infon key="article-id_pmc">4832331</infon><infon key="article-id_pmid">27080013</infon><infon key="elocation-id">24601</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0
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+reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Kandiah;given-names:Eaazhisai</infon><infon key="name_1">surname:Carriel;given-names:Diego</infon><infon key="name_10">surname:Gutsche;given-names:Irina</infon><infon key="name_2">surname:Perard;given-names:Julien</infon><infon key="name_3">surname:Malet;given-names:Hélène</infon><infon key="name_4">surname:Bacia;given-names:Maria</infon><infon key="name_5">surname:Liu;given-names:Kaiyin</infon><infon key="name_6">surname:Chan;given-names:Sze W. S.</infon><infon key="name_7">surname:Houry;given-names:Walid A.</infon><infon key="name_8">surname:Ollagnier de Choudens;given-names:Sandrine</infon><infon key="name_9">surname:Elsen;given-names:Sylvie</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">6</infon><infon key="year">2016</infon><offset>0</offset><text>Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>136</offset><text>The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1452</offset><text>Enterobacterial inducible decarboxylases of basic amino acids lysine, arginine and ornithine have a common evolutionary origin and belong to the α-family of pyridoxal-5′-phosphate (PLP)-dependent enzymes. They counteract acid stress experienced by the bacterium in the host digestive and urinary tract, and in particular in the extremely acidic stomach. Each decarboxylase is induced by an excess of the target amino acid and a specific range of extracellular pH, and works in conjunction with a cognate inner membrane antiporter. Decarboxylation of the amino acid into a polyamine is catalysed by a PLP cofactor in a multistep reaction that consumes a cytoplasmic proton and produces a CO2 molecule passively diffusing out of the cell, while the polyamine is excreted by the antiporter in exchange for a new amino acid substrate. Consequently, these enzymes buffer both the bacterial cytoplasm and the local extracellular environment. These amino acid decarboxylases are therefore called acid stress inducible or biodegradative to distinguish them from their biosynthetic lysine and ornithine decarboxylase paralogs catalysing the same reaction but responsible for the polyamine production at neutral pH.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2662</offset><text>Inducible enterobacterial amino acid decarboxylases have been intensively studied since the early 1940 because the ability of bacteria to withstand acid stress can be linked to their pathogenicity in humans. In particular, the inducible lysine decarboxylase LdcI (or CadA) attracts attention due to its broad pH range of activity and its capacity to promote survival and growth of pathogenic enterobacteria such as Salmonella enterica serovar Typhimurium, Vibrio cholerae and Vibrio vulnificus under acidic conditions. Furthermore, both LdcI and the biosynthetic lysine decarboxylase LdcC of uropathogenic Escherichia coli (UPEC) appear to play an important role in increased resistance of this pathogen to nitrosative stress produced by nitric oxide and other damaging reactive nitrogen intermediates accumulating during the course of urinary tract infections (UTI). This effect is attributed to cadaverine, the diamine produced by decarboxylation of lysine by LdcI and LdcC, that was shown to enhance UPEC colonisation of the bladder. In addition, the biosynthetic E. coli lysine decarboxylase LdcC, long thought to be constitutively expressed in low amounts, was demonstrated to be strongly upregulated by fluoroquinolones via their induction of RpoS. A direct correlation between the level of cadaverine and the resistance of E. coli to these antibiotics commonly used as a first-line treatment of UTI could be established. Both acid pH and cadaverine induce closure of outer membrane porins thereby contributing to bacterial protection from acid stress, but also from certain antibiotics, by reduction in membrane permeability.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4295</offset><text>The crystal structure of the E. coli LdcI as well as its low resolution characterisation by electron microscopy (EM) showed that it is a decamer made of two pentameric rings. Each monomer is composed of three domains – an N-terminal wing domain (residues 1–129), a PLP-binding core domain (residues 130–563), and a C-terminal domain (CTD, residues 564–715). Monomers tightly associate via their core domains into 2-fold symmetrical dimers with two complete active sites, and further build a toroidal D5-symmetrical structure held by the wing and core domain interactions around the central pore, with the CTDs at the periphery.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4931</offset><text>Ten years ago we showed that the E. coli AAA+ ATPase RavA, involved in multiple stress response pathways, tightly interacted with LdcI but was not capable of binding to LdcC. We described how two double pentameric rings of the LdcI tightly associate with five hexameric rings of RavA to form a unique cage-like architecture that enables the bacterium to withstand acid stress even under conditions of nutrient deprivation eliciting stringent response. Furthermore, we recently solved the structure of the E. coli LdcI-RavA complex by cryo-electron microscopy (cryoEM) and combined it with the crystal structures of the individual proteins. This allowed us to make a pseudoatomic model of the whole assembly, underpinned by a cryoEM map of the LdcI-LARA complex (with LARA standing for LdcI associating domain of RavA), and to identify conformational rearrangements and specific elements essential for complex formation. The main determinants of the LdcI-RavA cage assembly appeared to be the N-terminal loop of the LARA domain of RavA and the C-terminal β-sheet of LdcI.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6005</offset><text>In spite of this wealth of structural information, the fact that LdcC does not interact with RavA, although the two lysine decarboxylases are 69% identical and 84% similar, and the physiological significance of the absence of this interaction remained unexplored. To solve this discrepancy, in the present work we provided a three-dimensional (3D) cryoEM reconstruction of LdcC and compared it with the available LdcI and LdcI-RavA structures. Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). This comparison pinpointed differences between the biodegradative and the biosynthetic lysine decarboxylases and brought to light interdomain movements associated to pH-dependent enzyme activation and RavA binding, notably at the predicted RavA binding site at the level of the C-terminal β-sheet of LdcI. Consequently, we tested the capacity of cage formation by LdcI-LdcC chimeras where we interchanged the C-terminal β-sheets in question. Finally, we performed multiple sequence alignment of 22 lysine decarboxylases from Enterobacteriaceae containing the ravA-viaA operon in their genome. Remarkably, this analysis revealed that several specific residues in the above-mentioned β-sheet, independently of the rest of the protein sequence, are sufficient to define if a particular lysine decarboxylase should be classified as an “LdcC-like” or an “LdcI-like”. Moreover, this classification perfectly agrees with the genetic environment of the lysine decarboxylase genes. This fascinating parallelism between the propensity for RavA binding and the genetic environment of an enterobacterial lysine decarboxylase, as well as the high degree of conservation of this small structural motif, emphasize the functional importance of the interaction between biodegradative enterobacterial lysine decarboxylases and the AAA+ ATPase RavA.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>8130</offset><text>Results and Discussion</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8153</offset><text>CryoEM 3D reconstructions of LdcC, LdcIa and LdcI-LARA</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8208</offset><text>In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity – a 5.5 Å resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1 Å resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2). In addition, we improved our earlier cryoEM map of the LdcI-LARA complex from 7.5 Å to 6.2 Å resolution (Figs 1E,F and S3). Based on these reconstructions, reliable pseudoatomic models of the three assemblies were obtained by flexible fitting of either the crystal structure of LdcIi or a derived structural homology model of LdcC (Table S1). Significant differences between these pseudoatomic models can be interpreted as movements between specific biological states of the proteins as described below.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>9121</offset><text>The wing domains as a stable anchor at the center of the double-ring</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9190</offset><text>As a first step of a comparative analysis, we superimposed the three cryoEM reconstructions (LdcIa, LdcI-LARA and LdcC) and the crystal structure of the LdcIi decamer (Fig. 2 and Movie S1). This superposition reveals that the densities lining the central hole of the toroid are roughly at the same location, while the rest of the structure exhibits noticeable changes. Specifically, at the center of the double-ring the wing domains of the subunits provide the conserved basis for the assembly with the lowest root mean square deviation (RMSD) (between 1.4 and 2 Å for the Cα atoms only), whereas the peripheral CTDs containing the RavA binding interface manifest the highest RMSD (up to 4.2 Å) (Table S2). In addition, the wing domains of all structures are very similar, with the RMSD after optimal rigid body alignment (RMSDmin) less than 1.1 Å. Thus, taking the limited resolution of the cryoEM maps into account, we consider that the wing domains of all the four structures are essentially identical and that in the present study the RMSD of less than 2 Å can serve as a baseline below which differences may be assumed as insignificant. This preservation of the central part of the double-ring assembly may help the enzymes to maintain their decameric state upon activation and incorporation into the LdcI-RavA cage.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10525</offset><text>The core domain and the active site rearrangements upon pH-dependent enzyme activation and LARA binding</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10629</offset><text>Both visual inspection (Fig. 2) and RMSD calculations (Table S2) show that globally the three structures at active pH (LdcIa, LdcI-LARA and LdcC) are more similar to each other than to the structure determined at high pH conditions (LdcIi). The decameric enzyme is built of five dimers associating into a 5-fold symmetrical double-ring (two monomers making a dimer are delineated in Fig. 1). As common for the α family of the PLP-dependent decarboxylases, dimerization is required for the enzymatic activity because the active site is buried in the dimer interface (Fig. 3A,B). This interface is formed essentially by the core domains with some contribution of the CTDs. The core domain is built by the PLP-binding subdomain (PLP-SD, residues 184–417) flanked by two smaller subdomains rich in partly disordered loops – the linker region (residues 130–183) and the subdomain 4 (residues 418–563). Zooming in the variations in the PLP-SD shows that most of the structural changes concern displacements in the active site (Fig. 3C–F). The most conspicuous differences between the PLP-SDs can be linked to the pH-dependent activation of the enzymes. The resolution of the cryoEM maps does not allow modeling the position of the PLP moiety and calls for caution in detailed mechanistic interpretations in terms of individual amino acids. Therefore we restrict our analysis to secondary structure elements. In particular, transition from LdcIi to LdcI-LARA involves ~3.5 Å and ~4.5 Å shifts away from the 5-fold axis in the active site α-helices spanning residues 218–232 and 246–254 respectively (Fig. 3C–E). Between these two extremes, the PLP-SDs of LdcIa and LdcC are similar both in the context of the decamer (Fig. 3F) and in terms of RMSDmin = 0.9 Å, which probably reflects the fact that, at the optimal pH, these lysine decarboxylases have a similar enzymatic activity. In addition, our earlier biochemical observation that the enzymatic activity of LdcIa is unaffected by RavA binding is consistent with the relatively small changes undergone by the active site upon transition from LdcIa to LdcI-LARA. Worthy of note, our previous comparison of the crystal structure of LdcIi with that of the inducible arginine decarboxylase AdiA revealed high conservation of the PLP-coordinating residues and identified a patch of negatively charged residues lining the active site channel as a potential binding site for the target amino acid substrate (Figs S3 and S4 in ref.).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13132</offset><text>Rearrangements of the ppGpp binding pocket upon pH-dependent enzyme activation and LARA binding</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13228</offset><text>An inhibitor of the LdcI and LdcC activity, the stringent response alarmone ppGpp, is known to bind at the interface between neighboring monomers within each ring (Fig. S4). The ppGpp binding pocket is made up by residues from all domains and is located approximately 30 Å away from the PLP moiety. Whereas the crystal structure of the ppGpp-LdcIi was solved to 2 Å resolution, only a 4.1 Å resolution structure of the ppGpp-free LdcIi could be obtained. At this resolution, the apo-LdcIi and ppGpp-LdcIi structures (both solved at pH 8.5) appeared indistinguishable except for the presence of ppGpp (Fig. S11 in ref. ). Thus, we speculated that inhibition of LdcI by ppGpp would be accompanied by a transduction of subtle structural changes at the level of individual amino acid side chains between the ppGpp binding pocket and the active site of the enzyme. All our current cryoEM reconstructions of the lysine decarboxylases were obtained in the absence of ppGpp in order to be closer to the active state of the enzymes under study. While differences in the ppGpp binding site could indeed be visualized (Fig. S4), the level of resolution warns against speculations about their significance. The fact that interaction with RavA reduces the ppGpp affinity for LdcI despite the long distance of ~30 Å between the LARA domain binding site and the closest ppGpp binding pocket (Fig. S5) seems to favor an allosteric regulation mechanism. Interestingly, although a number of ppGpp binding residues are strictly conserved between LdcI and AdiA that also forms decamers at low pH optimal for its arginine decarboxylase activity, no ppGpp regulation of AdiA could be demonstrated.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>14916</offset><text>Swinging and stretching of the CTDs upon pH-dependent LdcI activation and LARA binding</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15003</offset><text>Inspection of the superimposed decameric structures (Figs 2 and S6) suggests a depiction of the wing domains as an anchor around which the peripheral CTDs swing. This swinging movement seems to be mediated by the core domains and is accompanied by a stretching of the whole LdcI subunits attracted by the RavA magnets. Indeed, all CTDs have very similar structures (RMSDmin &lt;1 Å). Yet the superposition of the decamers lays bare a progressive movement of the CTD as a whole upon enzyme activation by pH and the binding of LARA. The LdcIi monomer is the most compact, whereas LdcIa and especially LdcI-LARA gradually extend their CTDs towards the LARA domain of RavA (Figs 2 and 4). These small but noticeable swinging and stretching (up to ~4 Å) may be related to the incorporation of the LdcI decamer into the LdcI-RavA cage.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>15836</offset><text>The C-terminal β-sheet of a lysine decarboxylase as a major determinant of the interaction with RavA</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15940</offset><text>In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. However, at the level of this structural element the two proteins are actually surpisingly similar. Therefore, we wanted to check the influence of the primary sequence of the two proteins in this region on their ability to interact with RavA. To this end, we swapped the relevant β-sheets of the two proteins and produced their chimeras, namely LdcIC (i.e. LdcI with the C-terminal β-sheet of LdcC) and LdcCI (i.e. LdcC with the C-terminal β-sheet of LdcI) (Fig. 5A–C). Both constructs could be purified and could form decamers visually indistinguishable from the wild-type proteins. As expected, binding of LdcI to RavA was completely abolished by this procedure and no LdcIC-RavA complex could be detected. On the contrary, introduction of the C-terminal β-sheet of LdcI into LdcC led to an assembly of the LdcCI-RavA complex. On the negative stain EM grid, the chimeric cages appeared less rigid than the native LdcI-RavA, which probably means that the environment of the β-sheet contributes to the efficiency of the interaction and the stability of the entire architecture (Fig. 5D–F).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>17457</offset><text>The C-terminal β-sheet of a lysine decarboxylase is a highly conserved signature allowing to distinguish between LdcI and LdcC</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17587</offset><text>Alignment of the primary sequences of the E. coli LdcI and LdcC shows that some amino acid residues of the C-terminal β-sheet are the same in the two proteins, whereas others are notably different in chemical nature. Importantly, most of the amino acid differences between the two enzymes are located in this very region. Thus, to advance beyond our experimental confirmation of the C-terminal β-sheet as a major determinant of the capacity of a particular lysine decarboxylase to form a cage with RavA, we set out to investigate whether certain residues in this β-sheet are conserved in lysine decarboxylases of different enterobacteria that have the ravA-viaA operon in their genome. We inspected the genetic environment of lysine decarboxylases from 22 enterobacterial species referenced in the NCBI database, corrected the gene annotation where necessary (Tables S3 and S4), and performed multiple sequence alignment coupled to a phylogenetic analysis (see Methods). This procedure yielded several unexpected and exciting results. First of all, consensus sequence for the entire lysine decarboxylase family was derived. Second, the phylogenetic analysis clearly split the lysine decarboxylases into two groups (Fig. 6A). All lysine decarboxylases predicted to be “LdcI-like” or biodegradable based on their genetic environment, as for example their organization in an operon with a gene encoding the CadB antiporter (see Methods), were found in one group, whereas all enzymes predicted as “LdcC-like” or biosynthetic partitioned into another group. Thus, consensus sequences could also be determined for each of the two groups (Figs 6B,C and S7). Inspection of these consensus sequences revealed important differences between the groups regarding charge, size and hydrophobicity of several residues precisely at the level of the C-terminal β-sheet that is responsible for the interaction with RavA (Fig. 6B–D). For example, in our previous study, site-directed mutations identified Y697 as critically required for the RavA binding. Our current analysis shows that Y697 is strictly conserved in the “LdcI-like” group whereas the “LdcC-like” enzymes always have a lysine in this position; it also uncovers several other residues potentially essential for the interaction with RavA which can now be addressed by site-directed mutagenesis. The third and most remarkable finding was that exactly the same separation into “LdcI-like” and “LdcC”-like groups can be obtained based on a comparison of the C-terminal β-sheets only, without taking the rest of the primary sequence into account. Therefore the C-terminal β-sheet emerges as being a highly conserved signature sequence, sufficient to unambiguously discriminate between the “LdcI-like” and “LdcC-like” enterobacterial lysine decarboxylases independently of any other information (Figs 6 and S7). Our structures show that this motif is not involved in the enzymatic activity or the oligomeric state of the proteins. Thus, enterobacteria identified here (Fig. 6, Table S4) appear to exert evolutionary pressure on the biodegradative lysine decarboxylase towards the RavA binding. One of the elucidated roles of the LdcI-RavA cage is to maintain LdcI activity under conditions of enterobacterial starvation by preventing LdcI inhibition by the stringent response alarmone ppGpp. Furthermore, the recently documented interaction of both LdcI and RavA with specific subunits of the respiratory complex I, together with the unanticipated link between RavA and maturation of numerous iron-sulfur proteins, tend to suggest an additional intriguing function for this 3.5 MDa assembly. The conformational rearrangements of LdcI upon enzyme activation and RavA binding revealed in this work, and our amazing finding that the molecular determinant of the LdcI-RavA interaction is the one that straightforwardly determines if a particular enterobacterial lysine decarboxylase belongs to “LdcI-like” or “LdcC-like” proteins, should give a new impetus to functional studies of the unique LdcI-RavA cage. Besides, the structures and the pseudoatomic models of the active ppGpp-free states of both the biodegradative and the biosynthetic E. coli lysine decarboxylases offer an additional tool for analysis of their role in UPEC infectivity. Together with the apo-LdcI and ppGpp-LdcIi crystal structures, our cryoEM reconstructions provide a structural framework for future studies of structure-function relationships of lysine decarboxylases from other enterobacteria and even of their homologues outside Enterobacteriaceae. For example, the lysine decarboxylase of Eikenella corrodens is thought to play a major role in the periodontal disease and its inhibitors were shown to retard gingivitis development. Finally, cadaverine being an important platform chemical for the production of industrial polymers such as nylon, structural information is valuable for optimisation of bacterial lysine decarboxylases used for its production in biotechnology.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>22628</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>22636</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>22672</offset><text>LdcI and LdcC were expressed and purified as described from an E. coli strain that cannot produce ppGpp (MG1655 ΔrelA ΔspoT strain). LdcI was stored in 20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, 0.1 mM PLP, pH 6.8 (buffer A) and LdcC in 20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, 0.1 mM PLP, pH 7.5 (buffer B).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>22993</offset><text>Chimeric LdcIC and LdcCI were constructed, expressed and purified as follows. The chimeras were designed by exchange, between LdcI and LdcC, of residues from 631 to 640 and from 697 to the C-terminus, corresponding to the regions around the two strands of the C-terminal β-sheet (Fig. 5B,C), while leaving the rest of the sequence unaltered. The synthetic ldcIC and ldcCI genes (2148 bp and 2154 bp respectively), provided within a pUC57 vector (GenScript) were subcloned into pET-TEV vector based on pET-28a (Invitrogen) containing an N-terminal TEV-cleavable 6x-His-Tag. Proteins were expressed in Rosetta 2 (DE3) cells (Novagen) in LB medium supplemented with kanamycin and chloramphenicol at 37 °C, upon induction with 0.5 mM IPTG at 18 °C. Cells were harvested by centrifugation, the pellet resuspended in a 50 mM Tris-HCl, 150 mM NaCl, pH 8 buffer supplemented with Complete EDTA free (Roche) and 0.1 mM PMSF (Sigma), and disrupted by sonication at 4 °C. After centrifugation at 75000 g at 4 °C for 20 min, the supernatant was loaded on a Ni-NTA column. The eluted protein-containing fractions were pooled and the His-Tag removed by incubation with the TEV protease at 1/100 ratio and an extensive dialysis in a 50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 5 mM EDTA, pH 8 buffer. After a second dialysis in a 50 mM Tris-HCl, 150 mM NaCl, pH 8 buffer supplemented with 10 mM imidazole, the sample was loaded on a Ni-NTA column in the same buffer, which allowed to separate the TEV protease and LdcCI/LdcIC. Finally, the pure proteins were obtained by size exclusion chromatography on a Superdex-S200 column in buffer A.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24652</offset><text>LdcIa -cryoEM data collection and 3D reconstruction</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24704</offset><text>LdcI was prepared at 2 mg/ml in a buffer containing 25 mM MES, 100 mM NaCl, 0.2 mM PLP, 1 mM DTT, pH 6.2. 3 μl of sample were applied to glow-discharged quantifoil grids 300 mesh 2/1 (Quantifoil Micro Tools GmbH, Germany), excess solution was blotted during 2.5 s with a Vitrobot (FEI) and the grid frozen in liquid ethane. Data collection was performed on a FEI Polara microscope operated at 300 kV under low dose conditions. Micrographs were recorded on Kodak SO-163 film at 59,000 magnification, with defocus ranging from 0.6 to 4.9 μm. Films were digitized on a Zeiss scanner (Photoscan) at a step size of 7 μm giving a pixel size of 1.186 Å. The contrast transfer function (CTF) for each micrograph was determined with CTFFIND3.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>25462</offset><text>Initially ~2500 particles of 256 × 256 pixels were extracted manually, binned 4 times and subjected to one round of multivariate statistical analysis and classification using IMAGIC. Representative class averages corresponding to projections in different orientations were used as input for an ab-initio 3D reconstruction by RICOserver (rico.ibs.fr/). The resulting 3D reconstruction was refined using RELION, which yielded an 18 Å resolution map. Using projections of this model as a template, particles of size 256 × 256 pixels were semi-automatically selected from all the micrographs using the Fast Projection Matching (FPM) algorithm. The resulting dataset of ~46000 particles was processed in RELION with the previous map as an initial model and with a full CTF correction after the first peak. The final map comprised 44207 particles with a resolution of 7.4 Å as per the gold-standard FSC = 0.143 criterion. It was sharpened with EMBfactor using calculated B-factor of −350 Å2 and masked with a soft mask to obtain a final map with a resolution of 6.1 Å (Fig. S3, Table S1).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>26571</offset><text>LdcC - cryoEM data collection and 3D reconstruction</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26623</offset><text>LdcC was prepared at 2 mg/ml in a buffer containing 25 mM HEPES, 100 mM NaCl, 0.2 mM PLP, 1 mM DTT, pH 7.2. Grids were prepared and sample imaged as LdcIa. Data were processed essentially as LdcIa described above. Briefly, an initial ~20 Å resolution model was generated by angular reconstitution after manual picking of circa 3000 particles from the first micrographs, filtered to 60 Å resolution, refined with RELION and used for a semi-automatic selection with FPM. The dataset was processed in RELION with a full CTF correction to yield a final reconstruction comprising 61000 particles. The map was sharpened with Relion postprocessing, using a soft mask and automated B-factor estimation (−690 Å2), yielding a map at 5.5 Å resolution (Fig. S1, Table S1).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>27400</offset><text>LdcI-LARA - 3D reconstruction</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>27430</offset><text>In our first study, the dataset was processed in SPIDER and the CTF correction involved a simple phase-flipping. Therefore, for consistency with the present work, we revisited the dataset and processed it in RELION with a full CTF correction after the first peak. It was sharpened with EMBfactor using calculated B-factor of −350 Å2 and masked with a soft mask to obtain a final map with a resolution of 6.2 Å (Fig. S2). This reconstruction is of a slightly better quality in terms of the continuity of the internal density. Therefore we used this improved map for fitting of the atomic model and further analysis (Fig. S2, Table S1).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>28073</offset><text>Additional image processing</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28101</offset><text>As a crosscheck, each data set was also refined either from other initial models: a “featureless donut” with approximate dimensions of the decamer, and low pass-filtered reconstructions from the two other data sets (i.e. the LdcC reconstruction was used as a model for the LdcIa and LdcI-LARA data sets, etc). All refinements converged to the same solutions independently of the starting model. Local resolution of all maps was determined with ResMap.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>28557</offset><text>LdcCI and LdcIC chimeras —negative stain EM and 2D image analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28625</offset><text>0.4 mg/ml of RavA (in a 20 mM Tris-HCl, 500 mM NaCl, 10 mM MgCl2, 1 mM DTT, 5% glycerol, pH 6.8 buffer) was mixed with 0.3 mg/ml of either LdcI, LdcC, LdcCI or LdcIC in the presence of 2 mM ADP and 10 mM MgCl2 in a buffer containing 20 mM Hepes and 150 mM NaCl at pH 7.4. After 10 minutes incubation at room temperature, 3 μl of mixture were applied to the clear side of the carbon on a carbon-mica interface and negatively stained with 2% uranyl acetate. Images were recorded with a JEOL 1200 EX II microscope at 100 kV at a nominal magnification of 15000 on a CCD camera yielding a pixel size of 4.667 Å. No complexes between RavA and LdcC or LdcIC could be observed, whereas the LdcCI-RavA preparation manifested cage-like particles similar to the previously published LdcI-RavA, but also unbound RavA and LdcCI, which implies that the affinity of RavA to the LdcCI chimera is lower than its affinity to the native LdcI. 1260 particles of 96 × 96 pixels were extracted interactively from several micrographs. 2D centering, multivariate statistical analysis and classification were performed using IMAGIC. Class-averages similar to the cage-like LdcI-RavA complex were used as references for multi-reference alignment followed by multivariate statistical analysis and classification.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>29946</offset><text>Fitting of atomic models into cryoEM maps</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>29988</offset><text>A homology model of LdcC was obtained using the atomic coordinates of the LdcI monomer (3N75) as the template in SWISS-MODEL server. The RMSD between the template and the resulting model was 0.26 Å. The LdcC model was then fitted as a rigid body into the LdcC cryoEM map using the fit-in-map module of UCSF Chimera. This rigid fit indicated movements of several parts of the protein. Therefore, the density corresponding to one LdcC monomer was extracted and flexible fitting was performed using IMODFIT at 8 Å resolution. This monomeric model was then docked into the decameric cryoEM map with URO and its graphical version VEDA that use symmetry information for fitting in Fourier space. The Cα RMSDmin between the initial model of the LdcC monomer and the final IMODFIT LdcC model is 1.2 Å. In the case of LdcIa, the density corresponding to an individual monomer was extracted and the fit performed similarly to the one described above, with the final model of the decameric particle obtained with URO and VEDA. The Cα RMSDmin between the LdcIi monomer and the final IMODFIT model is 1.4 Å. For LdcI-LARA, the density accounting for one LdcI monomer bound to a LARA domain was extracted and further separated into individual densities corresponding to LdcI and to LARA. The fit of LdcI was performed as for LdcC and LdcIa, while the crystal structure of LARA was docked into the monomeric LdcI-LARA map as a rigid body using SITUS. The resulting pseudoatomic models were used to create the final model of the LdcI-LARA decamer with URO and VEDA. The Cα RMSDmin between the LdcIi monomer and the final IMODFIT model is 1.4 Å. A brief summary of relevant parameters is provided in Table S1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>31699</offset><text>Sequence analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31717</offset><text>Out of a non-exhaustive list of 50 species of Enterobacteriaceae (Table S3), 22 were found to contain genes annotated as ldcI or ldcC and containing the ravA-viaA operon (Table S4). An analysis using MUSCLE with default parameters showed that these genes share more than 65% identity. To verify annotation of these genes, we compared their genetic environment with that of E. coli ldcI and ldcC. Indeed, in E. coli the ldcI gene is in operon with the cadB gene encoding a lysine-cadaverine antiporter, whereas the ldcC gene is present between the accA gene (encoding an acetyl-CoA carboxylase alpha subunit carboxyltransferase) and the yaeR gene (coding for an unknown protein belonging to the family of Glyoxalase/Dioxygenase/Bleomycin resistance proteins). Compared with this genetic environment, the annotation of several ldcI and ldcC genes in enterobacteria was found to be inconsistent (Table S4). For example, several strains contain genes annotated as ldcC in the genetic background of ldcI and vice versa, as in the case of Salmonella enterica and Trabulsiella guamensi. Furthermore, the gene with an “ldcC-like” environment was found to be annotated as cadA in particular strains of Citrobacter freundii, Cronobacter sakazakii, Enterobacter cloacae subsp. Cloaca, Erwinia amylovora, Pantoea agglomerans, Rahnella aquatilis, Shigella dysenteriae, and Yersinia enterocolitica subsp. enterocolitica, whereas in Hafnia alvei, Kluyvera ascorbata, and Serratia marcescens subsp. marcescens, the gene with an “ldcI-like” environment was found to be annotated as ldcC. In addition, as far as the genetic environment is concerned, Plesiomonas appears to have two ldc genes with the organization of the E. coli ldcI (operon cadA-cadB). Consequently, we corrected for gene annotation consistent with the genetic environment and made multiple sequence alignments using version 8.0.1 of the CLC Genomics Workbench software. A phylogenetic tree was generated based on Maximum Likelihood and following the Neighbor-Joining method with the WAG protein substitution model. The reliability of the generated phylogenetic tree was assessed by bootstrap analysis. The presented unrooted phylogenetic tree shows the nodes that are reliable over 95% (Fig. 6A). Remarkably, the multiple sequence alignment and the resulting phylogenetic tree clearly grouped together all sequences annotated as ldcI on the one side, and all sequences annotated as ldcC on the other side. Thus, we conclude that all modifications in gene annotations that we introduced for the sake of consistency with the genetic environment are perfectly corroborated by the multiple sequence alignment and the phylogenetic analysis. Since the regulation of the lysine decarboxylase gene (i.e. inducible or constitutive) cannot be assessed by this analysis, we call the resulting groups “ldcI-like” and “ldcC-like” as referred to the E. coli enzymes, and make a parallel between the membership in a given group and the ability of the protein to form a cage complex with RavA.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>34762</offset><text>Additional Information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34785</offset><text>Accession codes: CryoEM maps and corresponding fitted atomic structures (main chain atoms) have been deposited in the Electron Microscopy Data Bank and Protein Data Bank, respectively, with accession codes EMD-3205 and 5FKZ for LdcC, EMD-3204 and 5FKX for LdcIa and EMD-3206 and 5FL2 for LdcI-LARA.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35084</offset><text>How to cite this article: Kandiah, E. et al. Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA. Sci. Rep. 6, 24601; doi: 10.1038/srep24601 (2016).</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>35317</offset><text>Supplementary Material</text></passage><passage><infon key="fpage">436</infon><infon key="lpage">447</infon><infon key="name_0">surname:Christen;given-names:P.</infon><infon key="name_1">surname:Mehta;given-names:P. K.</infon><infon key="section_type">REF</infon><infon key="source">Chem. Rec.
+N. Y. N</infon><infon key="type">ref</infon><infon key="volume">1</infon><infon key="year">2001</infon><offset>35340</offset><text>From cofactor to enzymes. The molecular evolution of pyridoxal-5′-phosphate-dependent enzymes</text></passage><passage><infon key="fpage">383</infon><infon key="lpage">415</infon><infon key="name_0">surname:Eliot;given-names:A. C.</infon><infon key="name_1">surname:Kirsch;given-names:J. F.</infon><infon key="pub-id_pmid">15189147</infon><infon key="section_type">REF</infon><infon key="source">Annu. Rev. Biochem.</infon><infon key="type">ref</infon><infon key="volume">73</infon><infon key="year">2004</infon><offset>35436</offset><text>Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations</text></passage><passage><infon key="fpage">301</infon><infon key="lpage">314</infon><infon key="name_0">surname:Zhao;given-names:B.</infon><infon key="name_1">surname:Houry;given-names:W. A.</infon><infon key="section_type">REF</infon><infon key="source">Biochem. Cell Biol. Biochim. Biol. Cell.</infon><infon key="type">ref</infon><infon key="volume">88</infon><infon key="year">2010</infon><offset>35522</offset><text>Acid stress response in enteropathogenic gammaproteobacteria: an aptitude for survival</text></passage><passage><infon key="fpage">65</infon><infon key="lpage">81</infon><infon key="name_0">surname:Kanjee;given-names:U.</infon><infon key="name_1">surname:Houry;given-names:W. A.</infon><infon key="pub-id_pmid">23701194</infon><infon key="section_type">REF</infon><infon key="source">Annu. Rev.
+Microbiol.</infon><infon key="type">ref</infon><infon key="volume">67</infon><infon key="year">2013</infon><offset>35609</offset><text>Mechanisms of acid resistance in Escherichia coli</text></passage><passage><infon key="fpage">e22397</infon><infon key="name_0">surname:Viala;given-names:J. P. M.</infon><infon key="pub-id_pmid">21799843</infon><infon key="section_type">REF</infon><infon key="source">PloS One</infon><infon key="type">ref</infon><infon key="volume">6</infon><infon key="year">2011</infon><offset>35659</offset><text>Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella</text></passage><passage><infon key="fpage">600</infon><infon key="lpage">618</infon><infon key="name_0">surname:Gale;given-names:E. F.</infon><infon key="name_1">surname:Epps;given-names:H. M.</infon><infon key="pub-id_pmid">16747564</infon><infon key="section_type">REF</infon><infon key="source">Biochem.
+J.</infon><infon key="type">ref</infon><infon key="volume">36</infon><infon key="year">1942</infon><offset>35754</offset><text>The effect of the pH of the medium during growth on the enzymic activities of bacteria (Escherichia coli and Micrococcus lysodeikticus) and the biological significance of the changes produced</text></passage><passage><infon key="fpage">232</infon><infon key="lpage">242</infon><infon key="name_0">surname:Gale;given-names:E. F.</infon><infon key="name_1">surname:Epps;given-names:H. M.</infon><infon key="pub-id_pmid">16747785</infon><infon key="section_type">REF</infon><infon key="source">Biochem. J.</infon><infon key="type">ref</infon><infon key="volume">38</infon><infon key="year">1944</infon><offset>35946</offset><text>Studies on bacterial amino-acid decarboxylases: 1. l(+)-lysine decarboxylase</text></passage><passage><infon key="fpage">836</infon><infon key="lpage">849</infon><infon key="name_0">surname:Merrell;given-names:D. S.</infon><infon key="name_1">surname:Camilli;given-names:A.</infon><infon key="pub-id_pmid">10564522</infon><infon key="section_type">REF</infon><infon key="source">Mol. Microbiol.</infon><infon key="type">ref</infon><infon key="volume">34</infon><infon key="year">1999</infon><offset>36023</offset><text>The cadA gene of Vibrio cholerae is induced during infection and plays a role in acid tolerance</text></passage><passage><infon key="fpage">8586</infon><infon key="lpage">8592</infon><infon key="name_0">surname:Kim;given-names:J.-S.</infon><infon key="name_1">surname:Choi;given-names:S. H.</infon><infon key="name_2">surname:Lee;given-names:J.
+K.</infon><infon key="pub-id_pmid">17012399</infon><infon key="section_type">REF</infon><infon key="source">J. Bacteriol.</infon><infon key="type">ref</infon><infon key="volume">188</infon><infon key="year">2006</infon><offset>36119</offset><text>Lysine decarboxylase expression by Vibrio vulnificus is induced by SoxR in response to superoxide stress</text></passage><passage><infon key="fpage">928</infon><infon key="lpage">933</infon><infon key="name_0">surname:Bower;given-names:J. M.</infon><infon key="name_1">surname:Mulvey;given-names:M. A.</infon><infon key="pub-id_pmid">16428396</infon><infon key="section_type">REF</infon><infon key="source">J. Bacteriol.</infon><infon key="type">ref</infon><infon key="volume">188</infon><infon key="year">2006</infon><offset>36224</offset><text>Polyamine-mediated resistance of uropathogenic Escherichia coli to nitrosative stress</text></passage><passage><infon key="fpage">2104</infon><infon key="lpage">2112</infon><infon key="name_0">surname:Bower;given-names:J. M.</infon><infon key="name_1">surname:Gordon-Raagas;given-names:H. B.</infon><infon key="name_2">surname:Mulvey;given-names:M. A.</infon><infon key="pub-id_pmid">19255192</infon><infon key="section_type">REF</infon><infon key="source">Infect. Immun.</infon><infon key="type">ref</infon><infon key="volume">77</infon><infon key="year">2009</infon><offset>36310</offset><text>Conditioning of uropathogenic Escherichia coli for enhanced colonization of host</text></passage><passage><infon key="fpage">575</infon><infon key="lpage">579</infon><infon key="name_0">surname:Akhova;given-names:A. V.</infon><infon key="name_1">surname:Tkachenko;given-names:A. G.</infon><infon key="section_type">REF</infon><infon key="source">Microbiology</infon><infon key="type">ref</infon><infon key="volume">78</infon><infon key="year">2009</infon><offset>36391</offset><text>Lysine Decarboxylase Activity as a Factor of Fluoroquinolone Resistance in Escherichia coli</text></passage><passage><infon key="fpage">1267</infon><infon key="lpage">1270</infon><infon key="name_0">surname:Kikuchi;given-names:Y.</infon><infon key="name_1">surname:Kurahashi;given-names:O.</infon><infon key="name_2">surname:Nagano;given-names:T.</infon><infon key="name_3">surname:Kamio;given-names:Y.</infon><infon key="pub-id_pmid">9692215</infon><infon key="section_type">REF</infon><infon key="source">Biosci. Biotechnol. Biochem.</infon><infon key="type">ref</infon><infon key="volume">62</infon><infon key="year">1998</infon><offset>36483</offset><text>RpoS-dependent expression of the second lysine decarboxylase gene in Escherichia coli</text></passage><passage><infon key="fpage">13</infon><infon key="lpage">19</infon><infon key="name_0">surname:Samartzidou;given-names:H.</infon><infon key="name_1">surname:Mehrazin;given-names:M.</infon><infon key="name_2">surname:Xu;given-names:Z.</infon><infon key="name_3">surname:Benedik;given-names:M.
+J.</infon><infon key="name_4">surname:Delcour;given-names:A.
+H.</infon><infon key="pub-id_pmid">12486035</infon><infon key="section_type">REF</infon><infon key="source">J. Bacteriol.</infon><infon key="type">ref</infon><infon key="volume">185</infon><infon key="year">2003</infon><offset>36569</offset><text>Cadaverine inhibition of porin plays a role in cell survival at acidic pH</text></passage><passage><infon key="fpage">330</infon><infon key="lpage">334</infon><infon key="name_0">surname:Bekhit;given-names:A.</infon><infon key="name_1">surname:Fukamachi;given-names:T.</infon><infon key="name_2">surname:Saito;given-names:H.</infon><infon key="name_3">surname:Kobayashi;given-names:H.</infon><infon key="pub-id_pmid">21372380</infon><infon key="section_type">REF</infon><infon key="source">Biol. Pharm. Bull.</infon><infon key="type">ref</infon><infon key="volume">34</infon><infon key="year">2011</infon><offset>36643</offset><text>The role of OmpC and OmpF in acidic resistance in Escherichia coli</text></passage><passage><infon key="fpage">1042</infon><infon key="lpage">1049</infon><infon key="name_0">surname:Tkachenko;given-names:A. G.</infon><infon key="name_1">surname:Pozhidaeva;given-names:O. N.</infon><infon key="name_2">surname:Shumkov;given-names:M. S.</infon><infon key="section_type">REF</infon><infon key="source">Biochem.
+Biokhimiia</infon><infon key="type">ref</infon><infon key="volume">71</infon><infon key="year">2006</infon><offset>36710</offset><text>Role of polyamines in formation of multiple antibiotic resistance of Escherichia coli under stress conditions</text></passage><passage><infon key="fpage">931</infon><infon key="lpage">944</infon><infon key="name_0">surname:Kanjee;given-names:U.</infon><infon key="pub-id_pmid">21278708</infon><infon key="section_type">REF</infon><infon key="source">EMBO J.</infon><infon key="type">ref</infon><infon key="volume">30</infon><infon key="year">2011</infon><offset>36820</offset><text>Linkage between the bacterial acid stress and stringent responses: the structure of the inducible lysine decarboxylase</text></passage><passage><infon key="fpage">662</infon><infon key="lpage">670</infon><infon key="name_0">surname:Sabo;given-names:D. L.</infon><infon key="name_1">surname:Boeker;given-names:E. A.</infon><infon key="name_2">surname:Byers;given-names:B.</infon><infon key="name_3">surname:Waron;given-names:H.</infon><infon key="name_4">surname:Fischer;given-names:E. H.</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry (Mosc.)</infon><infon key="type">ref</infon><infon key="volume">13</infon><infon key="year">1974</infon><offset>36939</offset><text>Purification and physical properties of inducible Escherichia coli lysine decarboxylase</text></passage><passage><infon key="fpage">1532</infon><infon key="lpage">1546</infon><infon key="name_0">surname:Snider;given-names:J.</infon><infon key="pub-id_pmid">16301313</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">281</infon><infon key="year">2006</infon><offset>37027</offset><text>Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase</text></passage><passage><infon key="fpage">211</infon><infon key="lpage">221</infon><infon key="name_0">surname:Wong;given-names:K. S.</infon><infon key="name_1">surname:Houry;given-names:W. A.</infon><infon key="pub-id_pmid">22491058</infon><infon key="section_type">REF</infon><infon key="source">J. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">179</infon><infon key="year">2012</infon><offset>37139</offset><text>Novel structural and functional insights into the MoxR family of AAA+ ATPases</text></passage><passage><infon key="fpage">22499</infon><infon key="lpage">22504</infon><infon key="name_0">surname:El Bakkouri;given-names:M.</infon><infon key="pub-id_pmid">21148420</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl. Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">107</infon><infon key="year">2010</infon><offset>37217</offset><text>Structure of RavA MoxR AAA+ protein reveals the design principles of a molecular cage modulating the inducible lysine decarboxylase activity</text></passage><passage><infon key="fpage">e85529</infon><infon key="name_0">surname:Wong;given-names:K. S.</infon><infon key="pub-id_pmid">24454883</infon><infon key="section_type">REF</infon><infon key="source">PloS
+One</infon><infon key="type">ref</infon><infon key="volume">9</infon><infon key="year">2014</infon><offset>37358</offset><text>The MoxR ATPase RavA and its cofactor ViaA interact with the NADH:ubiquinone oxidoreductase I in Escherichia coli</text></passage><passage><infon key="fpage">e03653</infon><infon key="name_0">surname:Malet;given-names:H.</infon><infon key="pub-id_pmid">25097238</infon><infon key="section_type">REF</infon><infon key="source">eLife</infon><infon key="type">ref</infon><infon key="volume">3</infon><infon key="year">2014</infon><offset>37472</offset><text>Assembly principles of a unique cage formed by hexameric and decameric E. coli proteins</text></passage><passage><infon key="fpage">167</infon><infon key="lpage">172</infon><infon key="name_0">surname:Yamamoto;given-names:Y.</infon><infon key="name_1">surname:Miwa;given-names:Y.</infon><infon key="name_2">surname:Miyoshi;given-names:K.</infon><infon key="name_3">surname:Furuyama;given-names:J.</infon><infon key="name_4">surname:Ohmori;given-names:H.</infon><infon key="pub-id_pmid">9339543</infon><infon key="section_type">REF</infon><infon key="source">Genes Genet. Syst.</infon><infon key="type">ref</infon><infon key="volume">72</infon><infon key="year">1997</infon><offset>37560</offset><text>The Escherichia coli ldcC gene encodes another lysine decarboxylase, probably a constitutive enzyme</text></passage><passage><infon key="fpage">857</infon><infon key="lpage">876</infon><infon key="name_0">surname:Käck;given-names:H.</infon><infon key="name_1">surname:Sandmark;given-names:J.</infon><infon key="name_2">surname:Gibson;given-names:K.</infon><infon key="name_3">surname:Schneider;given-names:G.</infon><infon key="name_4">surname:Lindqvist;given-names:Y.</infon><infon key="pub-id_pmid">10452893</infon><infon key="section_type">REF</infon><infon key="source">J. Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">291</infon><infon key="year">1999</infon><offset>37660</offset><text>Crystal structure of diaminopelargonic acid synthase: evolutionary relationships between pyridoxal-5′-phosphate-dependent enzymes</text></passage><passage><infon key="fpage">9388</infon><infon key="lpage">9398</infon><infon key="name_0">surname:Kanjee;given-names:U.</infon><infon key="name_1">surname:Gutsche;given-names:I.</infon><infon key="name_2">surname:Ramachandran;given-names:S.</infon><infon key="name_3">surname:Houry;given-names:W.
+A.</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry
+(Mosc.)</infon><infon key="type">ref</infon><infon key="volume">50</infon><infon key="year">2011</infon><offset>37792</offset><text>The enzymatic activities of the Escherichia coli basic aliphatic amino acid decarboxylases exhibit a pH zone of inhibition</text></passage><passage><infon key="fpage">863</infon><infon key="lpage">871</infon><infon key="name_0">surname:Erhardt;given-names:H.</infon><infon key="pub-id_pmid">22063474</infon><infon key="section_type">REF</infon><infon key="source">Biochim. Biophys. Acta</infon><infon key="type">ref</infon><infon key="volume">1817</infon><infon key="year">2012</infon><offset>37915</offset><text>Disruption of individual nuo-genes leads to the formation of partially assembled NADH:ubiquinone oxidoreductase (complex I) in Escherichia coli</text></passage><passage><infon key="fpage">1176</infon><infon key="lpage">1184</infon><infon key="name_0">surname:Lohinai;given-names:Z.</infon><infon key="pub-id_pmid">26110450</infon><infon key="section_type">REF</infon><infon key="source">J. Periodontol.</infon><infon key="type">ref</infon><infon key="volume">86</infon><infon key="year">2015</infon><offset>38059</offset><text>Biofilm Lysine Decarboxylase, a New Therapeutic Target for Periodontal Inflammation</text></passage><passage><infon key="fpage">6706</infon><infon key="lpage">6712</infon><infon key="name_0">surname:Peters;given-names:J. L.</infon><infon key="pub-id_pmid">22975025</infon><infon key="section_type">REF</infon><infon key="source">Vaccine</infon><infon key="type">ref</infon><infon key="volume">30</infon><infon key="year">2012</infon><offset>38143</offset><text>Effects of immunization with natural and recombinant lysine decarboxylase on canine gingivitis development</text></passage><passage><infon key="fpage">1048</infon><infon key="lpage">1056</infon><infon key="name_0">surname:Lohinai;given-names:Z.</infon><infon key="pub-id_pmid">22141361</infon><infon key="section_type">REF</infon><infon key="source">J. Periodontol.</infon><infon key="type">ref</infon><infon key="volume">83</infon><infon key="year">2012</infon><offset>38250</offset><text>Bacterial lysine decarboxylase influences human dental biofilm lysine content, biofilm accumulation, and subclinical gingival inflammation</text></passage><passage><infon key="fpage">1108</infon><infon key="lpage">1113</infon><infon key="name_0">surname:Kim;given-names:H. J.</infon><infon key="pub-id_pmid">25674800</infon><infon key="section_type">REF</infon><infon key="source">J. Microbiol. Biotechnol</infon><infon key="type">ref</infon><infon key="volume">25</infon><infon key="year">2015</infon><offset>38389</offset><text>Optimization of Direct Lysine Decarboxylase Biotransformation for Cadaverine Production with Whole-Cell Biocatalysts at High Lysine Concentration</text></passage><passage><infon key="fpage">799</infon><infon key="lpage">806</infon><infon key="name_0">surname:Ma;given-names:W.</infon><infon key="pub-id_pmid">25515797</infon><infon key="section_type">REF</infon><infon key="source">Biotechnol. Lett.</infon><infon key="type">ref</infon><infon key="volume">37</infon><infon key="year">2015</infon><offset>38535</offset><text>Enhanced cadaverine production from L-lysine using recombinant Escherichia coli co-overexpressing CadA and CadB</text></passage><passage><infon key="fpage">965</infon><infon key="lpage">972</infon><infon key="name_0">surname:Li;given-names:N.</infon><infon key="name_1">surname:Chou;given-names:H.</infon><infon key="name_2">surname:Yu;given-names:L.</infon><infon key="name_3">surname:Xu;given-names:Y.</infon><infon key="section_type">REF</infon><infon key="source">Biotechnol. Bioprocess Eng.</infon><infon key="type">ref</infon><infon key="volume">19</infon><infon key="year">2014</infon><offset>38647</offset><text>Cadaverine production by heterologous expression of Klebsiella oxytoca lysine decarboxylase</text></passage><passage><infon key="fpage">129</infon><infon key="lpage">228</infon><infon key="name_0">surname:Dubochet;given-names:J.</infon><infon key="pub-id_pmid">3043536</infon><infon key="section_type">REF</infon><infon key="source">Q. Rev.
+Biophys.</infon><infon key="type">ref</infon><infon key="volume">21</infon><infon key="year">1988</infon><offset>38739</offset><text>Cryo-electron microscopy of vitrified specimens</text></passage><passage><infon key="fpage">334</infon><infon key="lpage">347</infon><infon key="name_0">surname:Mindell;given-names:J. A.</infon><infon key="name_1">surname:Grigorieff;given-names:N.</infon><infon key="pub-id_pmid">12781660</infon><infon key="section_type">REF</infon><infon key="source">J. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">142</infon><infon key="year">2003</infon><offset>38787</offset><text>Accurate determination of local defocus and specimen tilt in electron microscopy</text></passage><passage><infon key="fpage">17</infon><infon key="lpage">24</infon><infon key="name_0">surname:van Heel;given-names:M.</infon><infon key="name_1">surname:Harauz;given-names:G.</infon><infon key="name_2">surname:Orlova;given-names:E.
+V.</infon><infon key="name_3">surname:Schmidt;given-names:R.</infon><infon key="name_4">surname:Schatz;given-names:M.</infon><infon key="pub-id_pmid">8742718</infon><infon key="section_type">REF</infon><infon key="source">J.
+Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">116</infon><infon key="year">1996</infon><offset>38868</offset><text>A new generation of the IMAGIC image processing system</text></passage><passage><infon key="fpage">13</infon><infon key="lpage">23</infon><infon key="name_0">surname:Navaza;given-names:J.</infon><infon key="pub-id_pmid">14643206</infon><infon key="section_type">REF</infon><infon key="source">J. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">144</infon><infon key="year">2003</infon><offset>38923</offset><text>On the three-dimensional reconstruction of icosahedral particles</text></passage><passage><infon key="fpage">519</infon><infon key="lpage">530</infon><infon key="name_0">surname:Scheres;given-names:S. H. W.</infon><infon key="pub-id_pmid">23000701</infon><infon key="section_type">REF</infon><infon key="source">J. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">180</infon><infon key="year">2012</infon><offset>38988</offset><text>RELION: implementation of a Bayesian approach to cryo-EM structure determination</text></passage><passage><infon key="fpage">253</infon><infon key="lpage">260</infon><infon key="name_0">surname:Estrozi;given-names:L. F.</infon><infon key="name_1">surname:Navaza;given-names:J.</infon><infon key="pub-id_pmid">20599509</infon><infon key="section_type">REF</infon><infon key="source">J. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">172</infon><infon key="year">2010</infon><offset>39069</offset><text>Ab initio high-resolution single-particle 3D reconstructions: the symmetry adapted functions way</text></passage><passage><infon key="fpage">853</infon><infon key="lpage">854</infon><infon key="name_0">surname:Scheres;given-names:S. H. W.</infon><infon key="name_1">surname:Chen;given-names:S.</infon><infon key="pub-id_pmid">22842542</infon><infon key="section_type">REF</infon><infon key="source">Nat. Methods</infon><infon key="type">ref</infon><infon key="volume">9</infon><infon key="year">2012</infon><offset>39166</offset><text>Prevention of overfitting in cryo-EM structure determination</text></passage><passage><infon key="fpage">170</infon><infon key="lpage">175</infon><infon key="name_0">surname:Fernández;given-names:J. J.</infon><infon key="name_1">surname:Luque;given-names:D.</infon><infon key="name_2">surname:Castón;given-names:J. R.</infon><infon key="name_3">surname:Carrascosa;given-names:J. L.</infon><infon key="pub-id_pmid">18614378</infon><infon key="section_type">REF</infon><infon key="source">J. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">164</infon><infon key="year">2008</infon><offset>39227</offset><text>Sharpening high resolution information in single particle electron cryomicroscopy</text></passage><passage><infon key="fpage">63</infon><infon key="lpage">65</infon><infon key="name_0">surname:Kucukelbir;given-names:A.</infon><infon key="name_1">surname:Sigworth;given-names:F. J.</infon><infon key="name_2">surname:Tagare;given-names:H. D.</infon><infon key="pub-id_pmid">24213166</infon><infon key="section_type">REF</infon><infon key="source">Nat.
+Methods</infon><infon key="type">ref</infon><infon key="volume">11</infon><infon key="year">2014</infon><offset>39309</offset><text>Quantifying the local resolution of cryo-EM density maps</text></passage><passage><infon key="fpage">3381</infon><infon key="lpage">3385</infon><infon key="name_0">surname:Schwede;given-names:T.</infon><infon key="name_1">surname:Kopp;given-names:J.</infon><infon key="name_2">surname:Guex;given-names:N.</infon><infon key="name_3">surname:Peitsch;given-names:M. C.</infon><infon key="pub-id_pmid">12824332</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">31</infon><infon key="year">2003</infon><offset>39366</offset><text>SWISS-MODEL: An automated protein homology-modeling server</text></passage><passage><infon key="fpage">1605</infon><infon key="lpage">1612</infon><infon key="name_0">surname:Pettersen;given-names:E. F.</infon><infon key="pub-id_pmid">15264254</infon><infon key="section_type">REF</infon><infon key="source">J. Comput. Chem.</infon><infon key="type">ref</infon><infon key="volume">25</infon><infon key="year">2004</infon><offset>39425</offset><text>UCSF Chimera–a visualization system for exploratory research and analysis</text></passage><passage><infon key="fpage">261</infon><infon key="lpage">270</infon><infon key="name_0">surname:Lopéz-Blanco;given-names:J. R.</infon><infon key="name_1">surname:Chacón;given-names:P.</infon><infon key="pub-id_pmid">23999189</infon><infon key="section_type">REF</infon><infon key="source">J. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">184</infon><infon key="year">2013</infon><offset>39501</offset><text>iMODFIT: efficient and robust flexible fitting based on vibrational analysis in internal coordinates</text></passage><passage><infon key="fpage">1820</infon><infon key="lpage">1825</infon><infon key="name_0">surname:Navaza;given-names:J.</infon><infon key="name_1">surname:Lepault;given-names:J.</infon><infon key="name_2">surname:Rey;given-names:F. A.</infon><infon key="name_3">surname:Alvarez-Rúa;given-names:C.</infon><infon key="name_4">surname:Borge;given-names:J.</infon><infon key="pub-id_pmid">12351826</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol.
+Crystallogr</infon><infon key="type">ref</infon><infon key="volume">58</infon><infon key="year">2002</infon><offset>39602</offset><text>On the fitting of model electron densities into EM reconstructions: a reciprocal-space formulation</text></passage><passage><infon key="fpage">651</infon><infon key="lpage">658</infon><infon key="name_0">surname:Siebert;given-names:X.</infon><infon key="name_1">surname:Navaza;given-names:J.</infon><infon key="pub-id_pmid">19564685</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol.
+Crystallogr</infon><infon key="type">ref</infon><infon key="volume">65</infon><infon key="year">2009</infon><offset>39701</offset><text>UROX 2.0: an interactive tool for fitting atomic models into electron-microscopy reconstructions</text></passage><passage><infon key="fpage">1792</infon><infon key="lpage">1797</infon><infon key="name_0">surname:Edgar;given-names:R. C.</infon><infon key="pub-id_pmid">15034147</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">32</infon><infon key="year">2004</infon><offset>39798</offset><text>MUSCLE: multiple sequence alignment with high accuracy and high throughput</text></passage><passage><infon key="fpage">691</infon><infon key="lpage">699</infon><infon key="name_0">surname:Whelan;given-names:S.</infon><infon key="name_1">surname:Goldman;given-names:N.</infon><infon key="pub-id_pmid">11319253</infon><infon key="section_type">REF</infon><infon key="source">Mol. Biol.
+Evol.</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">2001</infon><offset>39873</offset><text>A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>39995</offset><text>Author Contributions E.K., H.M. and I.G. carried out EM data collection with assistance of M.B. and analyzed the data. D.C. performed cloning, multiple sequence alignment and phylogenetic analysis under the direction of S.E. and I.G., J.P. cloned and purified chimeric proteins under the direction of S.O.C., K.L. and S.W.S.C. purified LdcI, LdcC and LARA under the direction of W.A.H., I.G. conceived and directed the studies and wrote the manuscript with input from E.K.</text></passage><passage><infon key="file">srep24601-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>40468</offset><text>3D cryoEM reconstructions of LdcC, LdcI-LARA and LdcIa.</text></passage><passage><infon key="file">srep24601-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>40524</offset><text>(A,C,E) cryoEM map of the LdcC (A), LdcIa
+(C) and LdcI-LARA (E) decamers with one protomer in light
+grey. In the rest of the protomers, the wing, core and C-terminal domains
+are colored from light to dark in shades of green for LdcC (A), pink
+for LdcIa (C) and blue for LdcI in LdcI-LARA (E).
+In (E), the LARA domain density is shown in dark grey. Two monomers
+making a dimer are delineated. Scale bar 50 Å.
+(B,D,F) One protomer from the cryoEM map of the LdcC (B),
+LdcIa (D) and LdcI-LARA (F) in light grey with
+the pseudoatomic model represented as cartoons and colored as the densities
+in (A,C,E). Each domain is indicated for clarity. Scale bar
+50 Å. See also Figs S1 and S3.</text></passage><passage><infon key="file">srep24601-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>41210</offset><text>Analysis of conformational rearrangements.</text></passage><passage><infon key="file">srep24601-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>41253</offset><text>Superposition of the pseudoatomic models of LdcC, LdcI from LdcI-LARA and
+LdcIa colored as in Fig. 1, and the
+crystal structure of LdcIi in shades of yellow. Only one of the
+two rings of the double toroid is shown for clarity. The dashed circle
+indicates the central region that remains virtually unchanged between all
+the structures, while the periphery undergoes visible movements. Scale bar
+50 Å.</text></passage><passage><infon key="file">srep24601-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>41656</offset><text>Conformational rearrangements in the enzyme active site.</text></passage><passage><infon key="file">srep24601-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>41713</offset><text>(A) LdcIi crystal structure, with one ring represented as a
+grey surface and the second as a cartoon. A monomer with its PLP cofactor is
+delineated. The PLP moieties of the cartoon ring are shown in red.
+(B) The LdcIi dimer extracted from the crystal structure
+of the decamer. One monomer is colored in shades of yellow as in Figs 1 and 2, while the monomer
+related by C2 symmetry is grey. The PLP is red. The active site is boxed.
+(C–F) Close-up views of the active site. The PLP
+moiety in red is from the LdcIi crystal structure. We did not
+attempt to model it in the cryoEM maps. The dimer interface is shown as a
+dashed line and the active site α-helices mentioned in the text
+are highlighted. (C) Compares LdcIi (yellow) and
+LdcIa (pink), (D) compares LdcIa (pink) and
+LdcI-LARA (blue), and (E) compares LdcIi (yellow),
+LdcIa (pink) and LdcI-LARA (blue) simultaneously in order to
+show the progressive shift described in the text. (F) Shows the
+similarity between LdcIa and LdcC at the level of the secondary
+structure elements composing the active site. Colors are as in the other
+figures.</text></passage><passage><infon key="file">srep24601-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>42813</offset><text>Stretching of the LdcI monomer upon pH-dependent enzyme activation and LARA
+binding.</text></passage><passage><infon key="file">srep24601-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>42898</offset><text>(A–C) A slice through the pseudoatomic models of the LdcI
+monomers extracted from the superimposed decamers (Fig.
+2) The rectangle indicates the regions enlarged in
+(D–F). (A) compares LdcIi (yellow)
+and LdcIa (pink), (B) compares LdcIa (pink) and
+LdcI-LARA (blue), and (C) compares LdcIi (yellow),
+LdcIa (pink) and LdcI-LARA (blue) simultaneously in order to
+show the progressive stretching described in the text. The cryoEM density of
+the LARA domain is represented as a grey surface to show the position of the
+binding site and the direction of the movement. (D–F)
+Inserts zooming at the CTD part in proximity of the LARA binding site. Loop
+regions are removed for a clearer visual comparison. An arrow indicates a
+swinging movement.</text></passage><passage><infon key="file">srep24601-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>43641</offset><text>Analysis of the LdcIC and LdcCI chimeras.</text></passage><passage><infon key="file">srep24601-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>43683</offset><text>(A) A slice through the pseudoatomic models of the LdcIa
+(purple) and LdcC (green) monomers extracted from the superimposed decamers
+(Fig. 2). (B) The C-terminal
+β-sheet in LdcIa and LdcC enlarged from
+(A,C) Exchanged primary sequences (capital letters) and
+their immediate vicinity (lower case letters) colored as in
+(A,B), with the corresponding secondary structure elements
+and the amino acid numbering shown. (D,E) A gallery of negative stain
+EM images of (D) the wild type LdcI-RavA cage and (E) the
+LdcCI-RavA cage-like particles. (F) Some representative class
+averages of the LdcCI-RavA cage-like particles. Scale bar
+20 nm.</text></passage><passage><infon key="file">srep24601-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>44318</offset><text>Sequence analysis of enterobacterial lysine decarboxylases.</text></passage><passage><infon key="file">srep24601-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>44378</offset><text>(A) Maximum likelihood tree with the
+“LdcC-like” and the
+“LdcI-like” groups highlighted in green and pink,
+respectively. Only nodes with higher values than 95% are shown (500
+replicates of the original dataset, see Methods for details). Scale bar
+indicates the average number of substitutions per site. (B) Analysis
+of consensus “LdcI-like” and
+“LdcC-like” sequences around the first and second
+C-terminal β-strands. The height of the bars and the letters
+representing the amino acids reflects the degree of conservation of each
+particular position is in the alignment. Amino acids are colored according
+to a polarity color scheme with hydrophobic residues in black, hydrophilic
+in green, acidic in red and basic in blue. Numbering as in E. coli.
+(C) Signature sequences of LdcI and LdcC in the C-terminal
+β-sheet. Polarity differences are highlighted. (D)
+Position and nature of these differences at the surface of the respective
+cryoEM maps with the color code as in B. See also Fig. S7 and Tables S3 and S4.</text></passage></document></collection>
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+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection><source>PMC</source><date>20201216</date><key>pmc.key</key><document><id>4833862</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/ncomms11196</infon><infon key="article-id_pii">ncomms11196</infon><infon key="article-id_pmc">4833862</infon><infon key="article-id_pmid">27073141</infon><infon key="elocation-id">11196</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Hunkeler;given-names:Moritz</infon><infon key="name_1">surname:Stuttfeld;given-names:Edward</infon><infon key="name_2">surname:Hagmann;given-names:Anna</infon><infon key="name_3">surname:Imseng;given-names:Stefan</infon><infon key="name_4">surname:Maier;given-names:Timm</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">7</infon><infon key="year">2016</infon><offset>0</offset><text>The dynamic organization of fungal acetyl-CoA carboxylase</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>58</offset><text>Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1127</offset><text> Acetyl-CoA carboxylases are central regulatory hubs of fatty acid metabolism and are important targets for drug development in obesity and cancer. Here, the authors demonstrate that the regulation of these highly dynamic enzymes in fungi is governed by a mechanism based on phosphorylation-dependent conformational variability.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1456</offset><text>Biotin-dependent acetyl-CoA carboxylases (ACCs) are essential enzymes that catalyse the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. This reaction provides the committed activated substrate for the biosynthesis of fatty acids via fatty-acid synthase. By catalysing this rate-limiting step in fatty-acid biosynthesis, ACC plays a key role in anabolic metabolism. ACC inhibition and knock-out studies show the potential of targeting ACC for treatment of the metabolic syndrome. Furthermore, elevated ACC activity is observed in malignant tumours. A direct link between ACC and cancer is provided by cancer-associated mutations in the breast cancer susceptibility gene 1 (BRCA1), which relieve inhibitory interactions of BRCA1 with ACC. Thus, ACC is a relevant drug target for type 2 diabetes and cancer. Microbial ACCs are also the principal target of antifungal and antibiotic compounds, such as Soraphen A.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2376</offset><text>The principal functional protein components of ACCs have been described already in the late 1960s for Escherichia coli (E. coli) ACC: Biotin carboxylase (BC) catalyses the ATP-dependent carboxylation of a biotin moiety, which is covalently linked to the biotin carboxyl carrier protein (BCCP). Carboxyltransferase (CT) transfers the activated carboxyl group from carboxybiotin to acetyl-CoA to yield malonyl-CoA. Prokaryotic ACCs are transient assemblies of individual BC, CT and BCCP subunits. Eukaryotic ACCs, instead, are multienzymes, which integrate all functional components into a single polypeptide chain of ∼2,300 amino acids. Human ACC occurs in two closely related isoforms, ACC1 and 2, located in the cytosol and at the outer mitochondrial membrane, respectively. In addition to the canonical ACC components, eukaryotic ACCs contain two non-catalytic regions, the large central domain (CD) and the BC–CT interaction domain (BT). The CD comprises one-third of the protein and is a unique feature of eukaryotic ACCs without homologues in other proteins. The function of this domain remains poorly characterized, although phosphorylation of several serine residues in the CD regulates ACC activity. The BT domain has been visualized in bacterial carboxylases, where it mediates contacts between α- and β-subunits.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3706</offset><text>Structural studies on the functional architecture of intact ACCs have been hindered by their huge size and pronounced dynamics, as well as the transient assembly mode of bacterial ACCs. However, crystal structures of individual components or domains from prokaryotic and eukaryotic ACCs, respectively, have been solved. The structure determination of the holoenzymes of bacterial biotin-dependent carboxylases, which lack the characteristic CD, such as the pyruvate carboxylase (PC), propionyl-CoA carboxylase, 3-methyl-crotonyl-CoA carboxylase and a long-chain acyl-CoA carboxylase revealed strikingly divergent architectures despite a general conservation of all functional components. In these structures, the BC and CT active sites are at distances between 40 and 80 Å, such that substrate transfer could be mediated solely by the mobility of the flexibly tethered BCCP.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4584</offset><text>Human ACC1 is regulated allosterically, via specific protein–protein interactions, and by reversible phosphorylation. Dynamic polymerization of human ACC1 is linked to increased activity and is regulated allosterically by the activator citrate and the inhibitor palmitate, or by binding of the small protein MIG-12 (ref.). Human ACC1 is further regulated by specific phosphorylation-dependent binding of BRCA1 to Ser1263 in the CD. BRCA1 binds only to the phosphorylated form of ACC1 and prevents ACC activation by phosphatase-mediated dephosphorylation. Furthermore, phosphorylation by AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKA) leads to a decrease in ACC1 activity. AMPK phosphorylates ACC1 in vitro at Ser80, Ser1201 and Ser1216 and PKA at Ser78 and Ser1201. However, regulatory effects on ACC1 activity are mainly mediated by phosphorylation of Ser80 and Ser1201 (refs). Phosphorylated Ser80, which is highly conserved only in higher eukaryotes, presumably binds into the Soraphen A-binding pocket. The regulatory Ser1201 shows only moderate conservation across higher eukaryotes, while the phosphorylated Ser1216 is highly conserved across all eukaryotes. However, no effect of Ser1216 phosphorylation on ACC activity has been reported in higher eukaryotes.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5882</offset><text>For fungal ACC, neither spontaneous nor inducible polymerization has been detected despite considerable sequence conservation to human ACC1. The BRCA1-interacting phosphoserine position is not conserved in fungal ACC, and no other phospho-dependent protein–protein interactions of fungal ACC have been described. In yeast ACC, phosphorylation sites have been identified at Ser2, Ser735, Ser1148, Ser1157 and Ser1162 (ref.). Of these, only Ser1157 is highly conserved in fungal ACC and aligns to Ser1216 in human ACC1. Its phosphorylation by the AMPK homologue SNF1 results in strongly reduced ACC activity.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6491</offset><text>Despite the outstanding relevance of ACC in primary metabolism and disease, the dynamic organization and regulation of the giant eukaryotic, and in particular fungal ACC, remain poorly characterized. Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a). Integrating these data with small-angle X-ray scattering (SAXS) and electron microscopy (EM) observations yield a comprehensive representation of the dynamic structure and regulation of fungal ACC.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>7107</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>7115</offset><text>The organization of the yeast ACC CD</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7152</offset><text>First, we focused on structure determination of the 82-kDa CD. The crystal structure of the CD of SceACC (SceCD) was determined at 3.0 Å resolution by experimental phasing and refined to Rwork/Rfree=0.20/0.24 (Table 1). The overall extent of the SceCD is 70 by 75 Å (Fig. 1b and Supplementary Fig. 1a,b), and the attachment points of the N-terminal 26-residue linker to the BCCP domain and the C-terminal CT domain are separated by 46 Å (the N- and C termini are indicated with spheres in Fig. 1b). SceCD comprises four distinct domains, an N-terminal α-helical domain (CDN), and a central four-helix bundle linker domain (CDL), followed by two α–β-fold C-terminal domains (CDC1/CDC2). CDN adopts a letter C shape, where one of the ends is a regular four-helix bundle (Nα3-6), the other end is a helical hairpin (Nα8,9) and the bridging region comprises six helices (Nα1,2,7,10–12). CDL is composed of a small, irregular four-helix bundle (Lα1–4) and tightly interacts with the open face of CDC1 via an interface of 1,300 Å2 involving helices Lα3 and Lα4. CDL does not interact with CDN apart from the covalent linkage and forms only a small contact to CDC2 via a loop between Lα2/α3 and the N-terminal end of Lα1, with an interface area of 400 Å2. CDC1/CDC2 share a common fold; they are composed of six-stranded β-sheets flanked on one side by two long, bent helices inserted between strands β3/β4 and β4/β5. CDC2 is extended at its C terminus by an additional β-strand and an irregular β-hairpin. On the basis of a root mean square deviation of main chain atom positions of 2.2 Å, CDC1/CDC2 are structurally more closely related to each other than to any other protein (Fig. 1c); they may thus have evolved by duplication. Close structural homologues could not be found for the CDN or the CDC domains.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>9025</offset><text>A regulatory loop mediates interdomain interactions</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9077</offset><text>To define the functional state of insect-cell-expressed ACC variants, we employed mass spectrometry (MS) for phosphorylation site detection. In insect-cell-expressed full-length SceACC, the highly conserved Ser1157 is the only fully occupied phosphorylation site with functional relevance in S. cerevisiae. Additional phosphorylation was detected for Ser2101 and Tyr2179; however, these sites are neither conserved across fungal ACC nor natively phosphorylated in yeast. MS analysis of dissolved crystals confirmed the phosphorylated state of Ser1157 also in SceCD crystals. The SceCD structure thus authentically represents the state of SceACC, where the enzyme is inhibited by SNF1-dependent phosphorylation.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9788</offset><text>In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands β2 and β3 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here. This regulatory loop wedges between the CDC1 and CDC2 domains and provides the largest contribution to the interdomain interface. The N-terminal region of the regulatory loop also directly contacts the C-terminal region of CDC2 leading into CT. Phosphoserine 1157 is tightly bound by two highly conserved arginines (Arg1173 and Arg1260) of CDC1 (Fig. 1d). Already the binding of phosphorylated Ser1157 apparently stabilizes the regulatory loop conformation; the accessory phosphorylation sites Ser1148 and Ser1162 in the same loop may further modulate the strength of interaction between the regulatory loop and the CDC1 and CDC2 domains. Phosphorylation of the regulatory loop thus determines interdomain interactions of CDC1 and CDC2, suggesting that it may exert its regulatory function by modifying the overall structure and dynamics of the CD.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10924</offset><text>The functional role of Ser1157 was confirmed by an activity assay based on the incorporation of radioactive carbonate into acid non-volatile material. Phosphorylated SceACC shows only residual activity (kcat=0.4±0.2 s−1, s.d. based on five replicate measurements), which increases 16-fold (kcat=6.5±0.3 s−1) after dephosphorylation with λ protein phosphatase. The values obtained for dephosphorylated SceACC are comparable to earlier measurements of non-phosphorylated yeast ACC expressed in E. coli.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>11436</offset><text>The variable CD is conserved between yeast and human</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11489</offset><text>To compare the organization of fungal and human ACC CD, we determined the structure of a human ACC1 fragment that comprises the BT and CD domains (HsaBT-CD), but lacks the mobile BCCP in between (Fig. 1a). An experimentally phased map was obtained at 3.7 Å resolution for a cadmium-derivatized crystal and was interpreted by a poly-alanine model (Fig. 1e and Table 1). Each of the four CD domains in HsaBT-CD individually resembles the corresponding SceCD domain; however, human and yeast CDs exhibit distinct overall structures. In agreement with their tight interaction in SceCD, the relative spatial arrangement of CDL and CDC1 is preserved in HsaBT-CD, but the human CDL/CDC1 didomain is tilted by 30° based on a superposition of human and yeast CDC2 (Supplementary Fig. 1c). As a result, the N terminus of CDL at helix Lα1, which connects to CDN, is shifted by 12 Å. Remarkably, CDN of HsaBT-CD adopts a completely different orientation compared with SceCD. With CDL/CDC1 superposed, CDN in HsaBT-CD is rotated by 160° around a hinge at the connection of CDN/CDL (Supplementary Fig. 1d). This rotation displaces the N terminus of CDN in HsaBT-CD by 51 Å compared with SceCD, resulting in a separation of the attachment points of the N-terminal linker to the BCCP domain and the C-terminal CT domain by 67 Å (the attachment points are indicated with spheres in Fig. 1e). The BT domain of HsaBT-CD consists of a helix that is surrounded at its N terminus by an antiparallel eight-stranded β-barrel. It resembles the BT of propionyl-CoA carboxylase; only the four C-terminal strands of the β-barrel are slightly tilted.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13128</offset><text>On the basis of MS analysis of insect-cell-expressed human full-length ACC, Ser80 shows the highest degree of phosphorylation (90%). Ser29 and Ser1263, implicated in insulin-dependent phosphorylation and BRCA1 binding, respectively, are phosphorylated at intermediate levels (40%). The highly conserved Ser1216 (corresponding to S. cerevisiae Ser1157), as well as Ser1201, both in the regulatory loop discussed above, are not phosphorylated. However, residual phosphorylation levels were detected for Ser1204 (7%) and Ser1218 (7%) in the same loop. MS analysis of the HsaBT-CD crystallization sample reveals partial proteolytic digestion of the regulatory loop. Accordingly, most of this loop is not represented in the HsaBT-CD crystal structure. The absence of the regulatory loop might be linked to the less-restrained interface of CDL/CDC1 and CDC2 and altered relative orientations of these domains. Besides the regulatory loop, also the phosphopeptide target region for BRCA1 interaction is not resolved presumably because of pronounced flexibility.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14183</offset><text>At the level of isolated yeast and human CD, the structural analysis indicates the presence of at least two hinges, one with large-scale flexibility at the CDN/CDL connection, and one with tunable plasticity between CDL/CDC1 and CDC2, plausibly affected by phosphorylation in the regulatory loop region.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>14487</offset><text>The integration of CD into the fungal ACC multienzyme</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14541</offset><text>To further obtain insights into the functional architecture of fungal ACC, we characterized larger multidomain fragments up to the intact enzymes. Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). No crystals diffracting to sufficient resolution were obtained for larger BC-containing fragments, or for full-length Cth or SceACC. To improve crystallizability, we generated ΔBCCP variants of full-length ACC, which, based on SAXS analysis, preserve properties of intact ACC (Supplementary Table 1 and Supplementary Fig. 2a–c). For CthΔBCCP, crystals diffracting to 8.4 Å resolution were obtained. However, molecular replacement did not reveal a unique positioning of the BC domain. Owing to the limited resolution the discussion of structures of CthCD-CT and CthΔBCCP is restricted to the analysis of domain localization. Still, these structures contribute considerably to the visualization of an intrinsically dynamic fungal ACC.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15756</offset><text>In all these crystal structures, the CT domains build a canonical head-to-tail dimer, with active sites formed by contributions from both protomers (Fig. 2 and Supplementary Fig. 3a). The connection of CD and CT is provided by a 10-residue peptide stretch, which links the N terminus of CT to the irregular β-hairpin/β-strand extension of CDC2 (Supplementary Fig. 3b). The connecting region is remarkably similar in isolated CD and CthCD-CTCter structures, indicating inherent conformational stability. CD/CT contacts are only formed in direct vicinity of the covalent linkage and involve the β-hairpin extension of CDC2 as well as the loop between strands β2/β3 of the CT N-lobe, which contains a conserved RxxGxN motif. The neighbouring loop on the CT side (between CT β1/β2) is displaced by 2.5 Å compared to isolated CT structures (Supplementary Fig. 3c). On the basis of an interface area of ∼600 Å2 and its edge-to-edge connection characteristics, the interface between CT and CD might be classified as conformationally variable. Indeed, the comparison of the positioning of eight instances of the C-terminal part of CD relative to CT in crystal structures determined here, reveals flexible interdomain linking (Fig. 3a). The CDC2/CT interface acts as a true hinge with observed rotation up to 16°, which results in a translocation of the distal end of CDC2 by 8 Å.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17149</offset><text>The interface between CDC2 and CDL/CDC1, which is mediated by the phosphorylated regulatory loop in the SceCD structure, is less variable than the CD–CT junction, and permits only limited rotation and tilting (Fig. 3b). Analysis of the impact of phosphorylation on the interface between CDC2 and CDL/CDC1 in CthACC variant structures is precluded by the limited crystallographic resolution. However, MS analysis of CthCD-CT and CthΔBCCP constructs revealed between 60 and 70% phosphorylation of Ser1170 (corresponding to SceACC Ser1157).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17691</offset><text>The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23 Å (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of CthΔBCCP, denoted as CthCD-CT1/2 and CthΔBCCP1, respectively). In addition, CDN can rotate around hinges in the connection between CDN/CDL by 70° (Fig. 4b, observed in the second protomer of CthΔBCCP, denoted as CthΔBCCP2) and 160° (Fig. 4c, observed in SceCD) leading to displacement of the anchor site for the BCCP linker by up to 33 and 40 Å, respectively.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18366</offset><text>Conformational variability in the CD thus contributes considerably to variations in the spacing between the BC and CT domains, and may extend to distance variations beyond the mobility range of the flexibly tethered BCCP. On the basis of the occurrence of related conformational changes between fungal and human ACC fragments, the observed set of conformations may well represent general states present in all eukaryotic ACCs.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>18793</offset><text>Large-scale conformational variability of fungal ACC</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18846</offset><text>To obtain a comprehensive view of fungal ACC dynamics in solution, we employed SAXS and EM. SAXS analysis of CthACC agrees with a dimeric state and an elongated shape with a maximum extent of 350 Å (Supplementary Table 1). The smooth appearance of scattering curves and derived distance distributions might indicate substantial interdomain flexibility (Supplementary Fig. 2a–c). Direct observation of individual full-length CthACC particles, according to MS results predominantly in a phosphorylated low-activity state, in negative stain EM reveals a large set of conformations from rod-like extended to U-shaped particles. Class averages, obtained by maximum-likelihood-based two-dimensional (2D) classification, are focused on the dimeric CT domain and the full BC–BCCP–CD domain of only one protomer, due to the non-coordinated motions of the lateral BC/CD regions relative to the CT dimer. They identify the connections between CDN/CDL and between CDC2/CT as major contributors to conformational heterogeneity (Supplementary Fig. 4a,b). The flexibility in the CDC2/CT hinge appears substantially larger than the variations observed in the set of crystal structures. The BC domain is not completely disordered, but laterally attached to BT/CDN in a generally conserved position, albeit with increased flexibility. Surprisingly, in both the linear and U-shaped conformations, the approximate distances between the BC and CT active sites would remain larger than 110 Å. These observed distances are considerably larger than in static structures of any other related biotin-dependent carboxylase. Furthermore, based on an average length of the BCCP–CD linker in fungal ACC of 26 amino acids, mobility of the BCCP alone would not be sufficient to bridge the active sites of BC and CT. Consequently, increased flexibility or additional modes of conformational changes may be required for productive catalysis. The most relevant candidate site for mediating such additional flexibility and permitting an extended set of conformations is the CDC1/CDC2 interface, which is rigidified by the Ser1157-phosphorylated regulatory loop, as depicted in the SceCD crystal structure.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>21030</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>21041</offset><text>Altogether, the architecture of fungal ACC is based on the central dimeric CT domain (Fig. 4d). The CD consists of four distinct subdomains and acts as a tether from the CT to the mobile BCCP and an oriented BC domain. The CD has no direct role in substrate recognition or catalysis but contributes to the regulation of all eukaryotic ACCs. In higher eukaryotic ACCs, regulation via phosphorylation is achieved by combining the effects of phosphorylation at Ser80, Ser1201 and Ser1263. In fungal ACC, however, Ser1157 in the regulatory loop of the CD is the only phosphorylation site that has been demonstrated to be both phosphorylated in vivo and involved in the regulation of ACC activity. In its phosphorylated state, the regulatory loop containing Ser1157 wedges between CDC1/CDC2 and presumably limits the conformational freedom at this interdomain interface. However, flexibility at this hinge may be required for full ACC activity, as the distances between the BCCP anchor points and the active sites of BC and CT observed here are such large that mobility of the BCCP alone is not sufficient for substrate transfer. The current data thus suggest that regulation of fungal ACC is mediated by controlling the dynamics of the unique CD, rather than directly affecting catalytic turnover at the active sites of BC and CT. A comparison between fungal and human ACC will help to further discriminate mechanistic differences that contribute to the extended control and polymerization of human ACC.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>22541</offset><text>Most recently, a crystal structure of near full-length non-phosphorylated ACC from S. cerevisae (lacking only 21 N-terminal amino acids, here denoted as flACC) was published by Wei and Tong. In flACC, the ACC dimer obeys twofold symmetry and assembles in a triangular architecture with dimeric BC domains (Supplementary Fig. 5a). In their study, mutational data indicate a requirement for BC dimerization for catalytic activity. The transition from the elongated open shape, observed in our experiments, towards a compact triangular shape is based on an intricate interplay of several hinge-bending motions in the CD (Fig. 4d). Comparison of flACC with our CthΔBCCP structure reveals the CDC2/CT hinge as a major contributor to conformational flexibility (Supplementary Fig. 5b,c). In flACC, CDC2 rotates ∼120° with respect to the CT domain. A second hinge can be identified between CDC1/CDC2. On the basis of a superposition of CDC2, CDC1 of the phosphorylated SceCD is rotated by 30° relative to CDC1 of the non-phosphorylated flACC (Supplementary Fig. 5d), similar to what we have observed for the non-phosphorylated HsaBT-CD (Supplementary Fig. 1d). When inspecting all individual protomer and fragment structures in their study, Wei and Tong also identify the CDN/CDC1 connection as a highly flexible hinge, in agreement with our observations.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>23895</offset><text>The only bona fide regulatory phophorylation site of fungal ACC in the regulatory loop is directly participating in CDC1/CDC2 domain interactions and thus stabilizes the hinge conformation. In flACC, the regulatory loop is mostly disordered, illustrating the increased flexibility due to the absence of the phosphoryl group. Only in three out of eight observed protomers a short peptide stretch (including Ser1157) was modelled. In those instances the Ser1157 residue is located at a distance of 14–20 Å away from the location of the phosphorylated serine observed here, based on superposition of either CDC1 or CDC2. Applying the conformation of the CDC1/CDC2 hinge observed in SceCD on flACC leads to CDN sterically clashing with CDC2 and BT/CDN clashing with CT (Supplementary Fig. 6a,b). Thus, in accordance with the results presented here, phosphorylation of Ser1157 in SceACC most likely limits flexibility in the CDC1/CDC2 hinge such that activation through BC dimerization is not possible (Fig. 4d), which however does not exclude intermolecular dimerization. In addition, EM micrographs of phosphorylated and dephosphorylated SceACC display for both samples mainly elongated and U-shaped conformations and reveal no apparent differences in particle shape distributions (Supplementary Fig. 7). This implicates that the triangular shape with dimeric BC domains has a low population also in the active form, even though a biasing influence of grid preparation cannot be excluded completely.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>25397</offset><text>Large-scale conformational variability has also been observed in most other carrier protein-based multienzymes, including polyketide and fatty-acid synthases (with the exception of fungal-type fatty-acid synthases), non-ribosomal peptide synthetases and the pyruvate dehydrogenase complexes, although based on completely different architectures. Together, this structural information suggests that variable carrier protein tethering is not sufficient for efficient substrate transfer and catalysis in any of these systems. The determination of a set of crystal structures of SceACC in two states, unphosphorylated and phosphorylated at the major regulatory site Ser1157, provides a unique depiction of multienzyme regulation by post-translational modification (Fig. 4d). The phosphorylated regulatory loop binds to an allosteric site at the interface of two non-catalytic domains and restricts conformational freedom at several hinges in the dynamic ACC. It disfavours the adoption of a rare, compact conformation, in which intramolecular dimerization of the BC domains results in catalytic turnover. The regulation of activity thus results from restrained large-scale conformational dynamics rather than a direct or indirect influence on active site structure. To our best knowledge, ACC is the first multienzyme for which such a phosphorylation-dependent mechanical control mechanism has been visualized. However, the example of ACC now demonstrates the possibility of regulating activity by controlled dynamics of non-enzymatic linker regions also in other families of carrier-dependent multienzymes. Understanding such structural and dynamic constraints imposed by scaffolding and linking in carrier protein-based multienzyme systems is a critical prerequisite for engineering of efficient biosynthetic assembly lines.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>27220</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>27228</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>27264</offset><text>All proteins were expressed in the Baculovirus Expression Vector System. The MultiBac insect cell expression plasmid pACEBACI (Geneva Biotech) was modified to host a GATEWAY (LifeTechnologies) cassette with an N-terminal 10xHis-tag, named pAB1GW-NH10 hereafter. Full-length HsaACC (Genebank accession #Q13085), SceACC (#Q00955) and CthACC (#G0S3L5) were cloned into pAB1GW-NH10 using GATEWAY according to the manufacturer's manual. Truncated variants were constructed by PCR amplification, digestion of the template DNA with DpnI, phosphorylation of the PCR product and religation of the linear fragment to a circular plasmid. The following constructs were used for this study: SceACC (1–2,233), CthACC (1–2,297), CthΔBCCP (1–2,297, Δ700–765), CthCD-CT (788–2,297), CthCD-CTCter (1,114–2,297), SceCD (768–1,494) and HsaBT-CD (622–1,584, Δ753–818). Bacmid and virus production was carried out according to MultiBac instructions. Baculovirus generation and amplification as well as protein expression were performed in Sf21 cells (Expression Systems) in Insect-Xpress medium (Lonza). The cells were harvested 68–96 h post infection by centrifugation and stored at −80 °C until being processed.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28486</offset><text>Cells were lysed by sonication and the lysate was cleared by ultracentrifugation. Soluble protein was purified using Ni-NTA (Genscript) and size exclusion chromatography (Superose 6, GE Healthcare). The affinity tag was removed by tobacco etch virus (TEV) protease cleavage overnight at 4 °C. TEV protease and uncleaved protein were removed by orthogonal Ni-NTA purification before size exclusion chromatography. SceACC, CthACC and CthΔBCCP were further purified by high-resolution anion exchange chromatography before size exclusion chromatography. Purified SceCD, CthCD-CTCter, CthCD-CT, CthΔBCCP, CthACC and SceACC were concentrated to 10 mg ml−1 in 30 mM 3-(N-morpholino) propanesulfonic acid (MOPS) pH 7, 200 mM ammonium sulfate, 5% glycerol and 10 mM dithiothreitol. Purified HsaBT-CD was concentrated to 20 mg ml−1 in 20 mM bicine pH 8.0, 200 mM NaCl, 5% glycerol and 5 mM tris(2-carboxyethyl) phosphine (TCEP). Proteins were used directly or were stored at −80 °C after flash-freezing in liquid nitrogen.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>29533</offset><text>Protein crystallization</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>29557</offset><text>All crystallization experiments were conducted using sitting drop vapour diffusion. SceCD crystals were grown at 19 °C by mixing protein and reservoir solution (0.1 M BisTrisPropane pH 6.5, 0.05–0.2 M di-sodium malonate, 20–30% polyethylene glycol (PEG) 3350, 10 mM trimethylamine or 2% benzamidine) in a 1:1 or 2:1 ratio. Crystals appeared after several days and continued to grow for 20–200 days. Crystals were cryoprotected by short incubation in mother liquor supplemented with 22% ethylene glycol and flash-cooled in liquid nitrogen. For heavy metal derivatization the crystals were incubated in stabilization solution supplemented with 1 mM Thimerosal or 10 mM EuCl2, and then backsoaked for 15 s in stabilization solution without heavy metal.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>30328</offset><text>Initial crystals of HsaBT-CD grew in 0.1 M Tris pH 8.5, 0.35 M tri-potassium citrate and 2–3.5% PEG10000 at 19 °C. After several rounds of optimization, good-quality diffraction crystals were obtained at 19 °C in 0.1 M MES pH 6, 0.25–0.35 M tri-potassium citrate, 2–5% PEG10000 and 0.01–0.04 M cadmium chloride. The protein drop contained a 1:1 ratio of protein and reservoir solution. Crystals grew immediately and stopped growing after 3 days. They were dehydrated and cryoprotected in several steps in artificial mother liquor containing incrementally increasing concentrations of tri-potassium citrate, PEG10000 and ethylene glycol and then flash-cooled in liquid nitrogen. The final solution was composed of 0.1 M MES pH 6, 0.5 M tri-potassium citrate, 6.75% PEG10000, 0.01 M cadmium chloride and 22% ethylene glycol.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31179</offset><text>CthCD-CTCter crystals were grown at 19 °C by mixing protein and reservoir solution (0.1 M HEPES pH 7.5, 2–7% Tacsimate pH 7, 7.5–15% PEG monomethyl ether 5000) in a 1:1 ratio. Crystals appeared after several days and continued to grow for up to 2 weeks. Crystals were cryoprotected by short incubation in mother liquor supplemented with 22% ethylene glycol.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31546</offset><text>CthCD-CT ACC crystals were grown at 19 °C by mixing protein and reservoir solution (0.1 M Bicine pH 8.5–9.5, 4–8% PEG8000) in a 1:1 or 1:2 ratio. Crystals grew 8– 10 days and were cryoprotected by short incubation in mother liquor supplemented with 22% ethylene glycol before flash-cooling in liquid nitrogen.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31867</offset><text>CthΔBCCP ACC crystals were grown at 19 °C by mixing protein and reservoir solution (0.1 M Morpheus buffer 3 (Molecular Dimensions, MD2-100-102), 7–12% Morpheus ethylene glycols mix (MD2-100-74), 8–12% PEG4000, 17–23% glycerol) in a 1:1 or 1:2 ratio. Crystals grew up to 3 weeks and were cryoprotected in reservoir solution before flash-cooling in liquid nitrogen.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>32243</offset><text>Structure determination and analysis of phosphorylation</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>32299</offset><text>All X-ray diffraction data were collected at beamlines X06SA (PXI) or X06DA (PXIII) at the Swiss Light Source (SLS, Paul Scherrer Institute, Villigen, Switzerland) equipped with PILATUS detectors. The wavelength of data collection was 1.000 Å for native crystals, and 1.527 and 1.907 Å for crystals derivatized with europium and cadmium, respectively. Raw data were processed using XDS. Molecular replacement was carried out using Phaser 2.5.7 and 2.6.0, density modification was performed using Parrot and resolve, multicrystal averaging was carried out using phenix. All model building procedures were conducted using Coot and figures were prepared using PyMOL (Schrödinger LLC).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>32988</offset><text>Diffraction of initial SceCD crystals in space group P43212 with unit cell dimensions of a=b=110.3 Å and c=131.7 Å was limited to 3.5 Å. The resolution was improved to 3 Å by addition of trimethylamine or benzamidine to the reservoir solution without significant changes in unit cell dimensions. Crystals derivatized with thimerosal and europium were used for initial SAD phase determination using the SHELXC/D package. Two mercury and four europium sites were located, and an initial model was placed in the resulting maps. Since crystals derivatized with europium were slightly non-isomorphous with a c axis length of 127 Å, multicrystal averaging was used for density modification and provided directly interpretable maps. Iterative cycles of model building and refinement in Buster (version 2.10.2; Global Phasing Ltd) converged at Rwork/Rfree of 0.20/0.24. The final model lacks the disordered N terminus (amino acids 768–789), an extended loop in the CDC1 domain (1,203–1,215), a short stretch (1,147–1,149) preceding the regulatory loop and the two very C-terminal residues (1,493–1,494). On the basis of temperature factor analysis, the start and end of the regulatory loop show higher disorder than the region around the interacting phosphoserine 1157. MS analysis of dissolved crystals detected quantitative phosphorylation of the regulatory Ser1157, as also found for full-length SceACC, and additionally albeit with much lower occurrence, phosphorylation of Ser790, Ser1137, Ser1148 and Ser1159. A modelled phosphoryl position for Ser1159 could overlap with the one of Ser1157, and might be represented in the crystal. For all other phosphorylation sites no difference density could be observed, probably because of very low occupancy. PDBeFold was used to search for structural homologues. The thresholds for lowest acceptable percentage of matched secondary structure elements were 70% for the search query and 20% for the result.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34956</offset><text>Initial HsaBT-CD crystals were obtained in space group I4122 with a=b=240.1 Å and c=768.9 Å and diffracted to 7.5 Å. Optimized and dehydrated crystals also belonged to space group I4122 but with unit cell parameters a=b=267.3 Å and c=210.6 Å and diffracted to a resolution of 3.7 Å. Phase information was obtained from SAD based on bound cadmium ions from the crystallization condition. Six cadmium positions were located in a 4.0-Å resolution data set at 1.9 Å wavelength using SHELXC/D via the HKL2MAP interface. Density modification and phasing based on this anomalous data set, a 3.7-Å resolution data set at 1.0 Å wavelength and additional non-isomorphous lower-resolution data sets led to a high-quality electron density map. At the intermediate resolution obtained, the map was interpreted by a poly-alanine model, which was guided by predicted secondary structure as well as sequence and structural alignment with SceCD. The final model contains five cadmium ions and refines using phenix against experimental data with Rwork/Rfree of 0.35/0.38, as expected for a poly-alanine model. Two HsaBT-CD monomers are packed in the asymmetric unit via the CDN and BT domains. Density on top of the β-barrel of one BT most likely representing parts of the BT–CD linker guided the assignment of this BT to its linked CD partner domain. This BT-to-CD assignment was further supported by the analysis of an additional lower-resolution crystal form. Cadmium ions were found to participate in crystal packing.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>36489</offset><text>In HsaACC, phosphorylation at regulatory sites was detected as provided in the main text. No phosphorylation was detected for other phosphosites previously identified in large-scale phosphoproteomics studies, namely serines 5, 23, 25, 48, 53, 78, 488, 786, 1273 (refs).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>36759</offset><text>Two different crystal forms were obtained for CthCD-CTCter (denoted as CthCD-CTCter1 and CthCD-CTCter2), diffracting to 3.6 and 4.5 Å. Both forms packed in space group P212121 with unit cell constants of a=97.7 Å, b=165.3 Å and c=219.2 Å or a=100.2 Å, b=153.5 Å and c=249.2 Å, respectively. Phases were determined by molecular replacement using a homology model based on SceCT (pdb 1od2) as search model in Phaser; multicrystal averaging was applied in density modification. The CT domain was rebuilt and an initial homology model based on the SceCD structure was fitted into difference density for CthCD-CTCter1. Iterative cycles of rebuilding and refinement in Buster converged at Rwork/Rfree of 0.20/0.24. The refined CD fragment served as a starting model for rebuilding CthCD-CTCter2 at lower resolution. Coordinate refinement in Buster was additionally guided by reference model restraints and converged at Rwork/Rfree of 0.24/0.24. Residues 1,114–1,185, 1,213–1,252, 1,380–1,385, 1,465–1,468 and 2,188–2,195 were disordered in both crystal forms and are not included in the models. Helical regions C terminal to Glu2264 of both protomers of CthCD-CTCter1 and C terminal to Leu2259 and Arg2261 of the two protomers of CthCD-CTCter2, respectively, could not be built unambiguously and were therefore interpreted by placing poly-alanine stretches. Conservation was mapped on the CthCD-CTCter1 crystal structure using al2co based on a sequence alignment of 367 fungal ACC sequences calculated by Clustal Omega. MS analysis of purified protein detected 7% phosphorylation at Ser1170 (corresponding to Ser1157 in SceCD).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38412</offset><text>CthCD-CT crystallized in space group P31212 with unit cell constants of a=b=195.0 Å and c=189.5 Å and crystals diffracted to a resolution of 7.2 Å. The structure was solved by molecular replacement using a model composed of CthCT and CDC2 as search model in Phaser. CDC1 and CDN were placed manually into the resulting maps, and the model was refined using rigid-body, domain-wise TLS and B-factor refinement and NCS- and reference model-restrained coordinate refinement in Buster to Rwork/Rfree of 0.23/0.25. Owing to the low resolution, the maximum allowed B-factor in Buster refinement was increased from the default value of 300–500 Å2, minimizing B-factor clipping to 5% of all atoms. Residues 1,033–1,035, 1,134–1,152, 1,213–1,252, 1,380–1,385, 1,465–1,468 and 2,188–2,195 were not included in the models. Helical regions C terminal to Leu2259 and Arg2261 on the two protomers, respectively, were interpreted as described for CthCD-CTCter. Loop conformations, including the regulatory loop, were modelled as observed in SceCD. MS analysis of purified protein detected 60% phosphorylation at Ser1170 (corresponding to Ser1157 in SceCD). Conservation was mapped on the CthCD-CT crystal structure as for CthCD-CTCter.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>39659</offset><text>CthΔBCCP ACC crystallized in space group P6422 with unit cell constants of a=b=462.2 Å and c=204.6 Å, resolution was limited to 8.4 Å. Structure determination and refinement was performed as for CthCD-CT, with a maximum allowed B-factor of 500 Å2, minimizing B-factor clipping to 3% of all atoms. Although substantial difference density is observed, no defined positions of the BT and BC domains could be derived because of disorder or partial in situ proteolysis or combinations thereof. In addition, residues 1,032–1,039, 1,134–1,152, 1,213–1,252, 1,380–1,385, 1,465–1,468 and 2,188–2,195 were not included in the model. The MissingAtom macro implemented in Buster was employed to account for missing atoms, the final Rwork/Rfree were 0.30/0.32. A region C terminal to Leu2259 on one protomer was interpreted as poly-alanine. Loop conformations, including the regulatory loop, were modelled as observed in SceCD. MS analysis of purified protein detected 70% phosphorylation at Ser1170 (corresponding to Ser1157 in SceCD).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>40708</offset><text>Small-angle X-ray scattering</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>40737</offset><text>Proteins were thawed on ice and dialysed overnight against 30 mM MOPS pH 7, 200 mM ammonium sulfate, 5% glycerol and 10 mM dithiothreitol. Raw scattering data were measured at SAXS beamline B21 at Diamond Light Source. The samples were measured at concentrations of 2.5, 5 and 10 mg ml−1. Data were processed using the ATSAS package according to standard procedures. A slight increase in scattering in the very low-resolution range was observed with increasing protein concentrations, which may be because of interparticle attraction or minor aggregation. Scattering intensities were thus extrapolated to zero concentration using point-wise extrapolation implemented in Primus. Direct comparison of raw scattering curves demonstrates the similarity of CthACC and CthΔBCCP, and the derived values such as Rg and Porod Volume match within expected error margins. Molecular mass estimations based on the SAXS–MOW method derive values of 534.7 and 534.0 kDa for CthACC and CthΔBCCP, respectively. The relative discrepancies to the theoretical weights of 516.8 kDa (CthACC) and 503.0 kDa (CthΔBCCP) are 3.5% and 6.2%, respectively, which is in a typical range for this method.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>41932</offset><text>Electron microscopy</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>41952</offset><text>Full-length CthACC was diluted to 0.01 mg ml−1 in 30 mM MOPS pH 7.0, 200 mM ammonium sulfate, 5% glycerol and 10 mM dithiothreitol. Protein sample was adsorbed to a 200-μm copper grid and stained with 2% uranyl acetate. Grids of CthACC were imaged on a CM-200 microscope (Philips) equipped with a TVIPS F416 4k CMOS camera (Tietz Video and Image Processing Systems). The voltage used was 200 kV, and a magnification of × 50,000 results in a pixel size of 2.14 Å. Initial image processing and particle picking was carried out using Xmipp. Overall, 22,309 particles were picked semi-automatically from 236 micrographs with a box size of 300 × 300 pixels. After extraction, particles with a z-score of more than three were discarded and 22,257 particles were aligned and classified into 48 2D class averages using the maximum-likelihood target function in Fourier space (MLF2D). After 72 iterations, 4,226 additional particles were discarded and the remaining 18,031 particles were re-aligned and classified into 36 classes using MLF2D with a high-resolution cutoff of 30 Å. After 44 iterations the alignment converged and class averages were extracted.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>43128</offset><text>In vitro biotinylation and activity assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>43170</offset><text>To ensure full functionality, SceACC was biotinylated in vitro using the E. coli biotin ligase BirA. The reaction mixture contained 10 μM ACC, 3.7 μM BirA, 50 mM Tris-HCl, pH 8, 5.5 mM MgCl2, 0.5 mM biotin, 60 mM NaCl, 3 mM ATP and 10% glycerol, and the reaction was allowed to proceed for 7 h at 30 °C.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>43495</offset><text>The catalytic activity of phosphorylated and dephosphorylated SceACC was measured by following the incorporation of radioactive 14C into acid-stable non-volatile material. Dephosphorylated ACC was prepared by overnight treatment with λ protein phosphatase (New England Biolabs) of partially purified ACC before the final gel filtration step. The removal of the phosphoryl group from Ser1157 was confirmed by MS. The reaction mixture contained 0.5 μg recombinant ACC in 100 mM potassium phosphate, pH 8, 3 mM ATP, 5 mM MgCl2, 50 mM NaH14CO3 (specific activity 7.4 MBq mmol−1) and 1 mM acetyl-CoA in a total reaction volume of 100 μl. The reaction mixture was incubated for 15 min at 30 °C, stopped by addition of 200 μl 6 M HCl and subsequently evaporated to dryness at 85 °C. The non-volatile residue was redissolved in 100 μl of water, 1 ml Ultima Gold XR scintillation medium (Perkin Elmer) was added and the 14C radioactivity was measured in a Packard Tricarb 2000CA liquid scintillation analyser. Measurements were carried out in five replicates and catalytic activities were calculated using a standard curve derived from measurements of varying concentrations of NaH14CO3 in reaction buffer.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>44734</offset><text>Additional information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>44757</offset><text>Accession codes: Atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 5I6E (SceCD), 5I87 (HsaBT-CD), 5I6F/5I6G (CthCD-CTCter1/2), 5I6H (CthCD-CT) and 5I6I (CthΔBCCP).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>44982</offset><text>How to cite this article: Hunkeler, M. et al. The dynamic organization of fungal acetyl-CoA carboxylase. Nat. Commun. 7:11196 doi: 10.1038/ncomms11196 (2016).</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>45141</offset><text>Supplementary Material</text></passage><passage><infon key="fpage">225</infon><infon key="lpage">226</infon><infon key="name_0">surname:Wakil;given-names:S. J.</infon><infon key="name_1">surname:Titchener;given-names:E. B.</infon><infon key="name_2">surname:Gibson;given-names:D. M.</infon><infon key="pub-id_pmid">13560478</infon><infon key="section_type">REF</infon><infon key="source">Biochim. Biophys. Acta</infon><infon key="type">ref</infon><infon key="volume">29</infon><infon key="year">1958</infon><offset>45164</offset><text>Evidence for the participation of biotin in the enzymic synthesis of fatty acids</text></passage><passage><infon key="fpage">537</infon><infon key="lpage">579</infon><infon key="name_0">surname:Wakil;given-names:S. J.</infon><infon key="name_1">surname:Stoops;given-names:J. K.</infon><infon key="name_2">surname:Joshi;given-names:V. C.</infon><infon key="pub-id_pmid">6137188</infon><infon key="section_type">REF</infon><infon key="source">Annu. Rev. Biochem.</infon><infon key="type">ref</infon><infon key="volume">52</infon><infon key="year">1983</infon><offset>45245</offset><text>Fatty acid synthesis and its regulation</text></passage><passage><infon key="fpage">2613</infon><infon key="lpage">2616</infon><infon key="name_0">surname:Abu-Elheiga;given-names:L.</infon><infon key="name_1">surname:Matzuk;given-names:M. M.</infon><infon key="name_2">surname:Abo-Hashema;given-names:K. A.</infon><infon key="name_3">surname:Wakil;given-names:S. J.</infon><infon key="pub-id_pmid">11283375</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">291</infon><infon key="year">2001</infon><offset>45285</offset><text>Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl-CoA carboxylase 2</text></passage><passage><infon key="fpage">10207</infon><infon key="lpage">10212</infon><infon key="name_0">surname:Abu-Elheiga;given-names:L.</infon><infon key="name_1">surname:Oh;given-names:W.</infon><infon key="name_2">surname:Kordari;given-names:P.</infon><infon key="name_3">surname:Wakil;given-names:S. J.</infon><infon key="pub-id_pmid">12920182</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">100</infon><infon key="year">2003</infon><offset>45382</offset><text>Acetyl-CoA carboxylase 2 mutant mice are protected against obesity and diabetes induced by high-fat/high-carbohydrate diets</text></passage><passage><infon key="fpage">283</infon><infon key="lpage">289</infon><infon key="name_0">surname:Harwood;given-names:H. J. J.</infon><infon key="section_type">REF</infon><infon key="source">Curr. Opin. Invest. Drugs</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2004</infon><offset>45506</offset><text>Acetyl-CoA carboxylase inhibition for the treatment of metabolic syndrome</text></passage><passage><infon key="fpage">2115</infon><infon key="lpage">2120</infon><infon key="name_0">surname:Milgraum;given-names:L. Z.</infon><infon key="name_1">surname:Witters;given-names:L. A.</infon><infon key="name_2">surname:Pasternack;given-names:G. R.</infon><infon key="name_3">surname:Kuhajda;given-names:F. P.</infon><infon key="pub-id_pmid">9815604</infon><infon key="section_type">REF</infon><infon key="source">Clin. Cancer Res.</infon><infon key="type">ref</infon><infon key="volume">3</infon><infon key="year">1997</infon><offset>45580</offset><text>Enzymes of the fatty acid synthesis pathway are highly expressed in in situ breast carcinoma</text></passage><passage><infon key="fpage">358</infon><infon key="lpage">365</infon><infon key="name_0">surname:Swinnen;given-names:J. V.</infon><infon key="name_1">surname:Brusselmans;given-names:K.</infon><infon key="name_2">surname:Verhoeven;given-names:G.</infon><infon key="pub-id_pmid">16778563</infon><infon key="section_type">REF</infon><infon key="source">Curr. Opin. Clin. Nutr. Metab. Care</infon><infon key="type">ref</infon><infon key="volume">9</infon><infon key="year">2006</infon><offset>45673</offset><text>Increased lipogenesis in cancer cells: new players, novel targets</text></passage><passage><infon key="fpage">519</infon><infon key="lpage">525</infon><infon key="name_0">surname:Williams;given-names:R. S.</infon><infon key="name_1">surname:Lee;given-names:M. S.</infon><infon key="name_2">surname:Hau;given-names:D. D.</infon><infon key="name_3">surname:Glover;given-names:J. N.</infon><infon key="pub-id_pmid">15133503</infon><infon key="section_type">REF</infon><infon key="source">Nat. Struct. Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">11</infon><infon key="year">2004</infon><offset>45739</offset><text>Structural basis of phosphopeptide recognition by the BRCT domain of BRCA1</text></passage><passage><infon key="fpage">5767</infon><infon key="lpage">5773</infon><infon key="name_0">surname:Shen;given-names:Y.</infon><infon key="name_1">surname:Tong;given-names:L.</infon><infon key="pub-id_pmid">18452305</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry</infon><infon key="type">ref</infon><infon key="volume">47</infon><infon key="year">2008</infon><offset>45814</offset><text>Structural evidence for direct interactions between the BRCT domains of human BRCA1 and a phospho-peptide from human ACC1</text></passage><passage><infon key="fpage">1784</infon><infon key="lpage">1803</infon><infon key="name_0">surname:Tong;given-names:L.</infon><infon key="pub-id_pmid">15968460</infon><infon key="section_type">REF</infon><infon key="source">Cell Mol. Life Sci.</infon><infon key="type">ref</infon><infon key="volume">62</infon><infon key="year">2005</infon><offset>45936</offset><text>Acetyl-coenzyme A carboxylase: crucial metabolic enzyme and attractive target for drug discovery</text></passage><passage><infon key="fpage">S138</infon><infon key="lpage">S143</infon><infon key="name_0">surname:Wakil;given-names:S. J.</infon><infon key="name_1">surname:Abu-Elheiga;given-names:L. A.</infon><infon key="pub-id_pmid">19047759</infon><infon key="section_type">REF</infon><infon key="source">J. Lipid Res.</infon><infon key="type">ref</infon><infon key="volume">50</infon><infon key="year">2009</infon><offset>46033</offset><text>Fatty acid metabolism: target for metabolic syndrome</text></passage><passage><infon key="fpage">881</infon><infon key="lpage">891</infon><infon key="name_0">surname:Shen;given-names:Y.</infon><infon key="name_1">surname:Volrath;given-names:S. L.</infon><infon key="name_2">surname:Weatherly;given-names:S. C.</infon><infon key="name_3">surname:Elich;given-names:T. D.</infon><infon key="name_4">surname:Tong;given-names:L.</infon><infon key="pub-id_pmid">15610732</infon><infon key="section_type">REF</infon><infon key="source">Mol. Cell</infon><infon key="type">ref</infon><infon key="volume">16</infon><infon key="year">2004</infon><offset>46086</offset><text>A mechanism for the potent inhibition of eukaryotic acetyl-coenzyme A carboxylase by soraphen A, a macrocyclic polyketide natural product</text></passage><passage><infon key="fpage">305</infon><infon key="lpage">332</infon><infon key="name_0">surname:Campbell;given-names:J. W.</infon><infon key="name_1">surname:Cronan;given-names:J. E.;suffix:Jr.</infon><infon key="pub-id_pmid">11544358</infon><infon key="section_type">REF</infon><infon key="source">Annu. Rev. Microbiol.</infon><infon key="type">ref</infon><infon key="volume">55</infon><infon key="year">2001</infon><offset>46224</offset><text>Bacterial fatty acid biosynthesis: targets for antibacterial drug discovery</text></passage><passage><infon key="fpage">105</infon><infon key="lpage">110</infon><infon key="name_0">surname:Weatherly;given-names:S. C.</infon><infon key="name_1">surname:Volrath;given-names:S. L.</infon><infon key="name_2">surname:Elich;given-names:T. D.</infon><infon key="pub-id_pmid">14766011</infon><infon key="section_type">REF</infon><infon key="source">Biochem. J.</infon><infon key="type">ref</infon><infon key="volume">380</infon><infon key="year">2004</infon><offset>46300</offset><text>Expression and characterization of recombinant fungal acetyl-CoA carboxylase and isolation of a soraphen-binding domain</text></passage><passage><infon key="fpage">561</infon><infon key="lpage">568</infon><infon key="name_0">surname:Alberts;given-names:A. W.</infon><infon key="name_1">surname:Vagelos;given-names:P. R.</infon><infon key="pub-id_pmid">4868901</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">59</infon><infon key="year">1968</infon><offset>46420</offset><text>Acetyl CoA carboxylase. I. Requirement for two protein fractions</text></passage><passage><infon key="fpage">1319</infon><infon key="lpage">1326</infon><infon key="name_0">surname:Alberts;given-names:A. W.</infon><infon key="name_1">surname:Nervi;given-names:A. M.</infon><infon key="name_2">surname:Vagelos;given-names:P. R.</infon><infon key="pub-id_pmid">4901473</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">63</infon><infon key="year">1969</infon><offset>46485</offset><text>Acetyl CoA carboxylase, II. Deomonstration of biotin-protein and biotin carboxylase subunits</text></passage><passage><infon key="fpage">407</infon><infon key="lpage">435</infon><infon key="name_0">surname:Cronan;given-names:J. E.;suffix:Jr.</infon><infon key="name_1">surname:Waldrop;given-names:G. L.</infon><infon key="pub-id_pmid">12121720</infon><infon key="section_type">REF</infon><infon key="source">Prog. Lipid Res.</infon><infon key="type">ref</infon><infon key="volume">41</infon><infon key="year">2002</infon><offset>46578</offset><text>Multi-subunit acetyl-CoA carboxylases</text></passage><passage><infon key="fpage">1502</infon><infon key="lpage">1509</infon><infon key="name_0">surname:Bianchi;given-names:A.</infon><infon key="pub-id_pmid">1967254</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">265</infon><infon key="year">1990</infon><offset>46616</offset><text>Identification of an isozymic form of acetyl-CoA carboxylase</text></passage><passage><infon key="fpage">1444</infon><infon key="lpage">1449</infon><infon key="name_0">surname:Abu-Elheiga;given-names:L.</infon><infon key="pub-id_pmid">10677481</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">97</infon><infon key="year">2000</infon><offset>46677</offset><text>The subcellular localization of acetyl-CoA carboxylase 2</text></passage><passage><infon key="fpage">223</infon><infon key="lpage">227</infon><infon key="name_0">surname:Brownsey;given-names:R. W.</infon><infon key="name_1">surname:Boone;given-names:A. N.</infon><infon key="name_2">surname:Elliott;given-names:J. E.</infon><infon key="name_3">surname:Kulpa;given-names:J. E.</infon><infon key="name_4">surname:Lee;given-names:W. M.</infon><infon key="pub-id_pmid">16545081</infon><infon key="section_type">REF</infon><infon key="source">Biochem. Soc. Trans.</infon><infon key="type">ref</infon><infon key="volume">34</infon><infon key="year">2006</infon><offset>46734</offset><text>Regulation of acetyl-CoA carboxylase</text></passage><passage><infon key="fpage">1059</infon><infon key="lpage">1064</infon><infon key="name_0">surname:Munday;given-names:M. R.</infon><infon key="pub-id_pmid">12440972</infon><infon key="section_type">REF</infon><infon key="source">Biochem. Soc. Trans.</infon><infon key="type">ref</infon><infon key="volume">30</infon><infon key="year">2002</infon><offset>46771</offset><text>Regulation of mammalian acetyl-CoA carboxylase</text></passage><passage><infon key="fpage">e01130</infon><infon key="lpage">01114</infon><infon key="name_0">surname:Shi;given-names:S.</infon><infon key="name_1">surname:Chen;given-names:Y.</infon><infon key="name_2">surname:Siewers;given-names:V.</infon><infon key="name_3">surname:Nielsen;given-names:J.</infon><infon key="pub-id_pmid">24803522</infon><infon key="section_type">REF</infon><infon key="source">MBio</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2014</infon><offset>46818</offset><text>Improving production of malonyl coenzyme A-derived metabolites by abolishing Snf1-dependent regulation of Acc1</text></passage><passage><infon key="fpage">1001</infon><infon key="lpage">1005</infon><infon key="name_0">surname:Huang;given-names:C. S.</infon><infon key="pub-id_pmid">20725044</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">466</infon><infon key="year">2010</infon><offset>46929</offset><text>Crystal structure of the alpha(6)beta(6) holoenzyme of propionyl-coenzyme A carboxylase</text></passage><passage><infon key="fpage">219</infon><infon key="lpage">223</infon><infon key="name_0">surname:Huang;given-names:C. S.</infon><infon key="name_1">surname:Ge;given-names:P.</infon><infon key="name_2">surname:Zhou;given-names:Z. H.</infon><infon key="name_3">surname:Tong;given-names:L.</infon><infon key="pub-id_pmid">22158123</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">481</infon><infon key="year">2012</infon><offset>47017</offset><text>An unanticipated architecture of the 750-kDa alpha6beta6 holoenzyme of 3-methylcrotonyl-CoA carboxylase</text></passage><passage><infon key="fpage">10249</infon><infon key="lpage">10256</infon><infon key="name_0">surname:Waldrop;given-names:G. L.</infon><infon key="name_1">surname:Rayment;given-names:I.</infon><infon key="name_2">surname:Holden;given-names:H. M.</infon><infon key="pub-id_pmid">7915138</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry</infon><infon key="type">ref</infon><infon key="volume">33</infon><infon key="year">1994</infon><offset>47121</offset><text>Three-dimensional structure of the biotin carboxylase subunit of acetyl-CoA carboxylase</text></passage><passage><infon key="fpage">1407</infon><infon key="lpage">1419</infon><infon key="name_0">surname:Athappilly;given-names:F. K.</infon><infon key="name_1">surname:Hendrickson;given-names:W. A.</infon><infon key="pub-id_pmid">8747466</infon><infon key="section_type">REF</infon><infon key="source">Structure</infon><infon key="type">ref</infon><infon key="volume">3</infon><infon key="year">1995</infon><offset>47209</offset><text>Structure of the biotinyl domain of acetyl-coenzyme A carboxylase determined by MAD phasing</text></passage><passage><infon key="fpage">2064</infon><infon key="lpage">2067</infon><infon key="name_0">surname:Zhang;given-names:H.</infon><infon key="name_1">surname:Yang;given-names:Z.</infon><infon key="name_2">surname:Shen;given-names:Y.</infon><infon key="name_3">surname:Tong;given-names:L.</infon><infon key="pub-id_pmid">12663926</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">299</infon><infon key="year">2003</infon><offset>47301</offset><text>Crystal structure of the carboxyltransferase domain of acetyl-coenzyme A carboxylase</text></passage><passage><infon key="fpage">1712</infon><infon key="lpage">1722</infon><infon key="name_0">surname:Bilder;given-names:P.</infon><infon key="pub-id_pmid">16460018</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry</infon><infon key="type">ref</infon><infon key="volume">45</infon><infon key="year">2006</infon><offset>47386</offset><text>The structure of the carboxyltransferase component of acetyl-coA carboxylase reveals a zinc-binding motif unique to the bacterial enzyme</text></passage><passage><infon key="fpage">863</infon><infon key="lpage">891</infon><infon key="name_0">surname:Tong;given-names:L.</infon><infon key="pub-id_pmid">22869039</infon><infon key="section_type">REF</infon><infon key="source">Cell Mol. Life Sci.</infon><infon key="type">ref</infon><infon key="volume">70</infon><infon key="year">2013</infon><offset>47523</offset><text>Structure and function of biotin-dependent carboxylases</text></passage><passage><infon key="fpage">1076</infon><infon key="lpage">1079</infon><infon key="name_0">surname:St Maurice;given-names:M.</infon><infon key="pub-id_pmid">17717183</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">317</infon><infon key="year">2007</infon><offset>47579</offset><text>Domain architecture of pyruvate carboxylase, a biotin-dependent multifunctional enzyme</text></passage><passage><infon key="fpage">120</infon><infon key="lpage">124</infon><infon key="name_0">surname:Tran;given-names:T. H.</infon><infon key="pub-id_pmid">25383525</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">518</infon><infon key="year">2015</infon><offset>47666</offset><text>Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase</text></passage><passage><infon key="fpage">9626</infon><infon key="lpage">9631</infon><infon key="name_0">surname:Kim;given-names:C. W.</infon><infon key="pub-id_pmid">20457939</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">107</infon><infon key="year">2010</infon><offset>47753</offset><text>Induced polymerization of mammalian acetyl-CoA carboxylase by MIG12 provides a tertiary level of regulation of fatty acid synthesis</text></passage><passage><infon key="fpage">973</infon><infon key="lpage">982</infon><infon key="name_0">surname:Ray;given-names:H.</infon><infon key="name_1">surname:Moreau;given-names:K.</infon><infon key="name_2">surname:Dizin;given-names:E.</infon><infon key="name_3">surname:Callebaut;given-names:I.</infon><infon key="name_4">surname:Venezia;given-names:N. D.</infon><infon key="pub-id_pmid">16698035</infon><infon key="section_type">REF</infon><infon key="source">J. Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">359</infon><infon key="year">2006</infon><offset>47885</offset><text>ACCA phosphopeptide recognition by the BRCT repeats of BRCA1</text></passage><passage><infon key="fpage">183</infon><infon key="lpage">190</infon><infon key="name_0">surname:Davies;given-names:S. P.</infon><infon key="name_1">surname:Sim;given-names:A. T.</infon><infon key="name_2">surname:Hardie;given-names:D. G.</infon><infon key="pub-id_pmid">1967580</infon><infon key="section_type">REF</infon><infon key="source">Eur. J. Biochem.</infon><infon key="type">ref</infon><infon key="volume">187</infon><infon key="year">1990</infon><offset>47946</offset><text>Location and function of three sites phosphorylated on rat acetyl-CoA carboxylase by the AMP-activated protein kinase</text></passage><passage><infon key="fpage">22162</infon><infon key="lpage">22168</infon><infon key="name_0">surname:Ha;given-names:J.</infon><infon key="name_1">surname:Daniel;given-names:S.</infon><infon key="name_2">surname:Broyles;given-names:S. S.</infon><infon key="name_3">surname:Kim;given-names:K. H.</infon><infon key="pub-id_pmid">7915280</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">269</infon><infon key="year">1994</infon><offset>48064</offset><text>Critical phosphorylation sites for acetyl-CoA carboxylase activity</text></passage><passage><infon key="fpage">187</infon><infon key="lpage">192</infon><infon key="name_0">surname:Cho;given-names:Y. S.</infon><infon key="pub-id_pmid">19900410</infon><infon key="section_type">REF</infon><infon key="source">Biochem. Biophys. Res. Commun.</infon><infon key="type">ref</infon><infon key="volume">391</infon><infon key="year">2010</infon><offset>48131</offset><text>Molecular mechanism for the regulation of human ACC2 through phosphorylation by AMPK</text></passage><passage><infon key="fpage">301</infon><infon key="lpage">305</infon><infon key="name_0">surname:Ficarro;given-names:S. B.</infon><infon key="pub-id_pmid">11875433</infon><infon key="section_type">REF</infon><infon key="source">Nat. Biotechnol.</infon><infon key="type">ref</infon><infon key="volume">20</infon><infon key="year">2002</infon><offset>48216</offset><text>Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae</text></passage><passage><infon key="fpage">19509</infon><infon key="lpage">19515</infon><infon key="name_0">surname:Woods;given-names:A.</infon><infon key="pub-id_pmid">7913470</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">269</infon><infon key="year">1994</infon><offset>48310</offset><text>Yeast SNF1 is functionally related to mammalian AMP-activated protein kinase and regulates acetyl-CoA carboxylase in vivo</text></passage><passage><infon key="fpage">1682</infon><infon key="lpage">1686</infon><infon key="name_0">surname:Holt;given-names:L. J.</infon><infon key="pub-id_pmid">19779198</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">325</infon><infon key="year">2009</infon><offset>48432</offset><text>Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution</text></passage><passage><infon key="fpage">31228</infon><infon key="lpage">31236</infon><infon key="name_0">surname:Diacovich;given-names:L.</infon><infon key="pub-id_pmid">12048195</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">277</infon><infon key="year">2002</infon><offset>48521</offset><text>Kinetic and structural analysis of a new group of Acyl-CoA carboxylases found in Streptomyces coelicolor A3(2)</text></passage><passage><infon key="fpage">723</infon><infon key="lpage">727</infon><infon key="name_0">surname:Wei;given-names:J.</infon><infon key="name_1">surname:Tong;given-names:L.</infon><infon key="pub-id_pmid">26458104</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">526</infon><infon key="year">2015</infon><offset>48632</offset><text>Crystal structure of the 500-kDa yeast acetyl-CoA carboxylase holoenzyme dimer</text></passage><passage><infon key="fpage">769</infon><infon key="lpage">780</infon><infon key="name_0">surname:Bernado;given-names:P.</infon><infon key="pub-id_pmid">19844700</infon><infon key="section_type">REF</infon><infon key="source">Eur. Biophys. J.</infon><infon key="type">ref</infon><infon key="volume">39</infon><infon key="year">2010</infon><offset>48711</offset><text>Effect of interdomain dynamics on the structure determination of modular proteins by small-angle scattering</text></passage><passage><infon key="fpage">9</infon><infon key="lpage">19</infon><infon key="name_0">surname:Smith;given-names:J. L.</infon><infon key="name_1">surname:Skiniotis;given-names:G.</infon><infon key="name_2">surname:Sherman;given-names:D. H.</infon><infon key="pub-id_pmid">25791608</infon><infon key="section_type">REF</infon><infon key="source">Curr. Opin. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">31</infon><infon key="year">2015</infon><offset>48819</offset><text>Architecture of the polyketide synthase module: surprises from electron cryo-microscopy</text></passage><passage><infon key="fpage">1315</infon><infon key="lpage">1322</infon><infon key="name_0">surname:Maier;given-names:T.</infon><infon key="name_1">surname:Leibundgut;given-names:M.</infon><infon key="name_2">surname:Ban;given-names:N.</infon><infon key="pub-id_pmid">18772430</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">321</infon><infon key="year">2008</infon><offset>48907</offset><text>The crystal structure of a mammalian fatty acid synthase</text></passage><passage><infon key="fpage">190</infon><infon key="lpage">197</infon><infon key="name_0">surname:Brignole;given-names:E. J.</infon><infon key="name_1">surname:Smith;given-names:S.</infon><infon key="pub-id_pmid">19151726</infon><infon key="section_type">REF</infon><infon key="source">Nat. Struct. Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">16</infon><infon key="year">2009</infon><offset>48964</offset><text>Asturias FJ. Conformational flexibility of metazoan fatty acid synthase enables catalysis</text></passage><passage><infon key="fpage">234</infon><infon key="lpage">240</infon><infon key="name_0">surname:Strieker;given-names:M.</infon><infon key="name_1">surname:Tanovic;given-names:A.</infon><infon key="name_2">surname:Marahiel;given-names:M. A.</infon><infon key="pub-id_pmid">20153164</infon><infon key="section_type">REF</infon><infon key="source">Curr. Opin. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">20</infon><infon key="year">2010</infon><offset>49054</offset><text>Nonribosomal peptide synthetases: structures and dynamics</text></passage><passage><infon key="fpage">16615</infon><infon key="lpage">16623</infon><infon key="name_0">surname:Patel;given-names:M. S.</infon><infon key="name_1">surname:Nemeria;given-names:N. S.</infon><infon key="name_2">surname:Furey;given-names:W.</infon><infon key="name_3">surname:Jordan;given-names:F.</infon><infon key="pub-id_pmid">24798336</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">289</infon><infon key="year">2014</infon><offset>49112</offset><text>The pyruvate dehydrogenase complexes: structure-based function and regulation</text></passage><passage><infon key="fpage">1021</infon><infon key="lpage">1032</infon><infon key="name_0">surname:Fitzgerald;given-names:D. J.</infon><infon key="pub-id_pmid">17117155</infon><infon key="section_type">REF</infon><infon key="source">Nat. Methods</infon><infon key="type">ref</infon><infon key="volume">3</infon><infon key="year">2006</infon><offset>49190</offset><text>Protein complex expression by using multigene baculoviral vectors</text></passage><passage><infon key="fpage">125</infon><infon key="lpage">132</infon><infon key="name_0">surname:Kabsch;given-names:W.</infon><infon key="pub-id_pmid">20124692</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>49256</offset><text>XDS</text></passage><passage><infon key="fpage">53</infon><infon key="lpage">64</infon><infon key="name_0">surname:Zhang;given-names:K. Y.</infon><infon key="name_1">surname:Cowtan;given-names:K.</infon><infon key="name_2">surname:Main;given-names:P.</infon><infon key="pub-id_pmid">18488305</infon><infon key="section_type">REF</infon><infon key="source">Methods Enzymol.</infon><infon key="type">ref</infon><infon key="volume">277</infon><infon key="year">1997</infon><offset>49260</offset><text>Combining constraints for electron-density modification</text></passage><passage><infon key="fpage">235</infon><infon key="lpage">242</infon><infon key="name_0">surname:Winn;given-names:M. D.</infon><infon key="pub-id_pmid">21460441</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">67</infon><infon key="year">2011</infon><offset>49316</offset><text>Overview of the CCP4 suite and current developments</text></passage><passage><infon key="fpage">213</infon><infon key="lpage">221</infon><infon key="name_0">surname:Adams;given-names:P. D.</infon><infon key="pub-id_pmid">20124702</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>49368</offset><text>PHENIX: a comprehensive Python-based system for macromolecular structure solution</text></passage><passage><infon key="fpage">486</infon><infon key="lpage">501</infon><infon key="name_0">surname:Emsley;given-names:P.</infon><infon key="name_1">surname:Lohkamp;given-names:B.</infon><infon key="name_2">surname:Scott;given-names:W. G.</infon><infon key="name_3">surname:Cowtan;given-names:K.</infon><infon key="pub-id_pmid">20383002</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>49450</offset><text>Features and development of Coot</text></passage><passage><infon key="fpage">112</infon><infon key="lpage">122</infon><infon key="name_0">surname:Sheldrick;given-names:G. M.</infon><infon key="pub-id_pmid">18156677</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. A</infon><infon key="type">ref</infon><infon key="volume">64</infon><infon key="year">2008</infon><offset>49483</offset><text>A short history of SHELX</text></passage><passage><infon key="fpage">2256</infon><infon key="lpage">2268</infon><infon key="name_0">surname:Krissinel;given-names:E.</infon><infon key="name_1">surname:Henrick;given-names:K.</infon><infon key="pub-id_pmid">15572779</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D</infon><infon key="type">ref</infon><infon key="volume">60</infon><infon key="year">2004</infon><offset>49508</offset><text>Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions</text></passage><passage><infon key="fpage">843</infon><infon key="lpage">844</infon><infon key="name_0">surname:Pape;given-names:T.</infon><infon key="name_1">surname:Schneider;given-names:T. R.</infon><infon key="section_type">REF</infon><infon key="source">J. Appl. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">37</infon><infon key="year">2004</infon><offset>49612</offset><text>HKL2MAP: a graphical user interface for macromolecular phasing with SHELX programs</text></passage><passage><infon key="fpage">ra3</infon><infon key="name_0">surname:Olsen;given-names:J. V.</infon><infon key="pub-id_pmid">20068231</infon><infon key="section_type">REF</infon><infon key="source">Sci. Signal.</infon><infon key="type">ref</infon><infon key="volume">3</infon><infon key="year">2010</infon><offset>49695</offset><text>Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis</text></passage><passage><infon key="fpage">253</infon><infon key="lpage">262</infon><infon key="name_0">surname:Bian;given-names:Y.</infon><infon key="pub-id_pmid">24275569</infon><infon key="section_type">REF</infon><infon key="source">J. Proteomics</infon><infon key="type">ref</infon><infon key="volume">96</infon><infon key="year">2014</infon><offset>49796</offset><text>An enzyme assisted RP-RPLC approach for in-depth analysis of human liver phosphoproteome</text></passage><passage><infon key="fpage">1346</infon><infon key="lpage">1351</infon><infon key="name_0">surname:Cantin;given-names:G. T.</infon><infon key="pub-id_pmid">18220336</infon><infon key="section_type">REF</infon><infon key="source">J. Proteome Res.</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2008</infon><offset>49885</offset><text>Combining protein-based IMAC, peptide-based IMAC, and MudPIT for efficient phosphoproteomic analysis</text></passage><passage><infon key="fpage">658</infon><infon key="lpage">674</infon><infon key="name_0">surname:McCoy;given-names:A. J.</infon><infon key="pub-id_pmid">19461840</infon><infon key="section_type">REF</infon><infon key="source">J. Appl. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">40</infon><infon key="year">2007</infon><offset>49986</offset><text>Phaser crystallographic software</text></passage><passage><infon key="fpage">W252</infon><infon key="lpage">W258</infon><infon key="name_0">surname:Biasini;given-names:M.</infon><infon key="pub-id_pmid">24782522</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">42</infon><infon key="year">2014</infon><offset>50019</offset><text>SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information</text></passage><passage><infon key="fpage">700</infon><infon key="lpage">712</infon><infon key="name_0">surname:Pei;given-names:J.</infon><infon key="name_1">surname:Grishin;given-names:N. V.</infon><infon key="pub-id_pmid">11524371</infon><infon key="section_type">REF</infon><infon key="source">Bioinformatics</infon><infon key="type">ref</infon><infon key="volume">17</infon><infon key="year">2001</infon><offset>50115</offset><text>AL2CO: calculation of positional conservation in a protein sequence alignment</text></passage><passage><infon key="fpage">539</infon><infon key="name_0">surname:Sievers;given-names:F.</infon><infon key="pub-id_pmid">21988835</infon><infon key="section_type">REF</infon><infon key="source">Mol. Syst. Biol.</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2011</infon><offset>50193</offset><text>Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega</text></passage><passage><infon key="fpage">342</infon><infon key="lpage">350</infon><infon key="name_0">surname:Petoukhov;given-names:M. V.</infon><infon key="pub-id_pmid">25484842</infon><infon key="section_type">REF</infon><infon key="source">J. Appl. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">45</infon><infon key="year">2012</infon><offset>50292</offset><text>New developments in the program package for small-angle scattering data analysis</text></passage><passage><infon key="fpage">1727</infon><infon key="lpage">1739</infon><infon key="name_0">surname:Skou;given-names:S.</infon><infon key="name_1">surname:Gillilan;given-names:R. E.</infon><infon key="name_2">surname:Ando;given-names:N.</infon><infon key="pub-id_pmid">24967622</infon><infon key="section_type">REF</infon><infon key="source">Nat. Protoc.</infon><infon key="type">ref</infon><infon key="volume">9</infon><infon key="year">2014</infon><offset>50373</offset><text>Synchrotron-based small-angle X-ray scattering of proteins in solution</text></passage><passage><infon key="fpage">642</infon><infon key="lpage">657</infon><infon key="name_0">surname:Jacques;given-names:D. A.</infon><infon key="name_1">surname:Trewhella;given-names:J.</infon><infon key="pub-id_pmid">20120026</infon><infon key="section_type">REF</infon><infon key="source">Protein Sci.</infon><infon key="type">ref</infon><infon key="volume">19</infon><infon key="year">2010</infon><offset>50444</offset><text>Small-angle scattering for structural biology-Expanding the frontier while avoiding the pitfalls</text></passage><passage><infon key="fpage">1277</infon><infon key="lpage">1282</infon><infon key="name_0">surname:Konarev;given-names:P. V.</infon><infon key="name_1">surname:Volkov;given-names:V. V.</infon><infon key="name_2">surname:Sokolova;given-names:A. V.</infon><infon key="name_3">surname:Koch;given-names:M. H. J.</infon><infon key="name_4">surname:Svergun;given-names:D. I.</infon><infon key="section_type">REF</infon><infon key="source">J. Appl. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">36</infon><infon key="year">2003</infon><offset>50541</offset><text>PRIMUS: a Windows PC-based system for small-angle scattering data analysis</text></passage><passage><infon key="fpage">101</infon><infon key="lpage">109</infon><infon key="name_0">surname:Fischer;given-names:H.</infon><infon key="name_1">surname:Neto;given-names:M. D.</infon><infon key="name_2">surname:Napolitano;given-names:H. B.</infon><infon key="name_3">surname:Polikarpov;given-names:I.</infon><infon key="name_4">surname:Craievich;given-names:A. F.</infon><infon key="section_type">REF</infon><infon key="source">J. Appl. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">43</infon><infon key="year">2010</infon><offset>50616</offset><text>Determination of the molecular weight of proteins in solution from a single small-angle X-ray scattering measurement on a relative scale</text></passage><passage><infon key="fpage">406</infon><infon key="lpage">418</infon><infon key="name_0">surname:Scheres;given-names:S. H.</infon><infon key="pub-id_pmid">22100448</infon><infon key="section_type">REF</infon><infon key="source">J. Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">415</infon><infon key="year">2012</infon><offset>50753</offset><text>A Bayesian view on cryo-EM structure determination</text></passage><passage><infon key="fpage">237</infon><infon key="lpage">240</infon><infon key="name_0">surname:Marabini;given-names:R.</infon><infon key="pub-id_pmid">8812978</infon><infon key="section_type">REF</infon><infon key="source">J. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">116</infon><infon key="year">1996</infon><offset>50804</offset><text>Xmipp: an image processing package for electron microscopy</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>50863</offset><text>Author contributions M.H. cloned, expressed, purified and crystallized fungal ACC constructs, determined their structure and carried out SAXS analysis. E.S. cloned, expressed and crystallized human ACC CD and determined its structure. EM analysis was carried out by E.S., M.H. and A.H. S.I. contributed to structural analysis and figure preparation. T.M. designed and supervised work and analysed crystallographic data; all authors contributed to manuscript preparation.</text></passage><passage><infon key="file">ncomms11196-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>51334</offset><text>The phosphorylated central domain of yeast ACC.</text></passage><passage><infon key="file">ncomms11196-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>51382</offset><text>(a) Schematic overview of the domain organization of eukaryotic ACCs. Crystallized constructs are indicated. (b) Cartoon representation of the SceCD crystal structure. CDN is linked by a four-helix bundle (CDL) to two α–β-fold domains (CDC1 and CDC2). The regulatory loop is shown as bold cartoon, and the phosphorylated Ser1157 is marked by a red triangle. The N- and C termini are indicated by spheres. (c) Superposition of CDC1 and CDC2 reveals highly conserved folds. (d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group. (e) Structural overview of HsaBT-CD. The attachment points to the N-terminal BCCP domain and the C-terminal CT domain are indicated with spheres. All colourings are according to scheme a.</text></passage><passage><infon key="file">ncomms11196-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>52228</offset><text>Architecture of the CD–CT core of fungal ACC.</text></passage><passage><infon key="file">ncomms11196-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>52276</offset><text>Cartoon representation of crystal structures of multidomain constructs of CthACC. One protomer is shown in colour and one in grey. Individual domains are labelled; the active site of CT and the position of the conserved regulatory phosphoserine site based on SceCD are indicated by an asterisk and a triangle, respectively.</text></passage><passage><infon key="file">ncomms11196-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>52600</offset><text>Variability of the connections of CDC2 to CT and CDC1 in fungal ACC.</text></passage><passage><infon key="file">ncomms11196-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>52669</offset><text>(a) Hinge properties of the CDC2–CT connection analysed by a CT-based superposition of eight instances of the CDC2-CT segment. For clarity, only one protomer of CthCD-CTCter1 is shown in full colour as reference. For other instances, CDC2 domains are shown in transparent tube representation with only one helix each highlighted. The range of hinge bending is indicated and the connection points between CDC2 and CT (blue) as well as between CDC1 and CDC2 (green and grey) are marked as spheres. (b) The interdomain interface of CDC1 and CDC2 exhibits only limited plasticity. Representation as in a, but the CDC1 and CDC2 are superposed based on CDC2. One protomer of CthΔBCCP is shown in colour, the CDL domains are omitted for clarity and the position of the phosphorylated serine based on SceCD is indicated with a red triangle. The connection points from CDC1 to CDC2 and to CDL are represented by green spheres.</text></passage><passage><infon key="file">ncomms11196-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>53591</offset><text>The conformational dynamics of fungal ACC.</text></passage><passage><infon key="file">ncomms11196-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>53634</offset><text>(a–c) Large-scale conformational variability of the CDN domain relative to the CDL/CDC1 domain. CthCD-CT1 (in colour) serves as reference, the compared structures (as indicated, numbers after construct name differentiate between individual protomers) are shown in grey. Domains other than CDN and CDL/CDC1 are omitted for clarity. The domains are labelled and the distances between the N termini of CDN (spheres) in the compared structures are indicated. (d) Schematic model of fungal ACC showing the intrinsic, regulated flexibility of CD in the phosphorylated inhibited or the non-phosphorylated activated state. Flexibility of the CDC2/CT and CDN/CDL hinges is illustrated by arrows. The Ser1157 phosphorylation site and the regulatory loop are schematically indicated in magenta.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>54420</offset><text>Crystallographic data collection and refinement statistics.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot; border=&quot;1&quot;&gt;&lt;colgroup&gt;&lt;col align=&quot;left&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;/colgroup&gt;&lt;thead valign=&quot;bottom&quot;&gt;&lt;tr&gt;&lt;th align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;&lt;bold&gt;Sce&lt;/bold&gt;&lt;/italic&gt;&lt;bold&gt;CD&lt;/bold&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;&lt;bold&gt;Sce&lt;/bold&gt;&lt;/italic&gt;&lt;bold&gt;CD Thimerosal&lt;/bold&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;&lt;bold&gt;Sce&lt;/bold&gt;&lt;/italic&gt;&lt;bold&gt;CD Eu&lt;/bold&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;&lt;bold&gt;Hsa&lt;/bold&gt;&lt;/italic&gt;&lt;bold&gt;BT-CD&lt;/bold&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;&lt;bold&gt;Hsa&lt;/bold&gt;&lt;/italic&gt;&lt;bold&gt;BT-CD Cd&lt;/bold&gt;&lt;sup&gt;&lt;bold&gt;2+&lt;/bold&gt;&lt;/sup&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;&lt;bold&gt;Cth&lt;/bold&gt;&lt;/italic&gt;&lt;bold&gt;CD-CT&lt;/bold&gt;&lt;sub&gt;&lt;bold&gt;Cter1&lt;/bold&gt;&lt;/sub&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;&lt;bold&gt;Cth&lt;/bold&gt;&lt;/italic&gt;&lt;bold&gt;CD-CT&lt;/bold&gt;&lt;sub&gt;&lt;bold&gt;Cter2&lt;/bold&gt;&lt;/sub&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;&lt;bold&gt;Cth&lt;/bold&gt;&lt;/italic&gt;&lt;bold&gt;CD-CT&lt;/bold&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;&lt;bold&gt;Cth&lt;/bold&gt;&lt;/italic&gt;Δ&lt;bold&gt;BCCP&lt;/bold&gt;&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign=&quot;top&quot;&gt;&lt;tr&gt;&lt;td colspan=&quot;10&quot; align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;Data collection&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Space group&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;P4&lt;sub&gt;3&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;P4&lt;sub&gt;3&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;P4&lt;sub&gt;3&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;I4&lt;sub&gt;1&lt;/sub&gt;22&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;I4&lt;sub&gt;1&lt;/sub&gt;22&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;P2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;P2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;P3&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;P6&lt;sub&gt;4&lt;/sub&gt;22&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Cell dimensions&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;  &lt;italic&gt;a, b, c&lt;/italic&gt; (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;110.86, 110.86, 131.12&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;111.22, 111.22, 131.49&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;108.65, 108.65, 127.36&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;267.27, 267.27, 210.61&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;267.67, 267.67, 210.46&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;97.66, 165.34, 219.23&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;100.17, 153.45, 249,24&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;295.02, 295.02, 189.52&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;462.20, 462.20, 204.64&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;  α, β, γ (°)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90, 90, 120&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90, 90, 120&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Resolution&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn1&quot;&gt;*&lt;/xref&gt; (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;3.0&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;3.4&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;4.0&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;3.7&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;4.1&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;3.6&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;4.5&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;7.2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;8.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;Merge&lt;/sub&gt;&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn2&quot;&gt;†&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;18.2 (389.6)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;20.5 (306.1)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;40.6 (327.0)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;7.5 (400.9)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;15 (730.5)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;14.5 (384.5)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;27.4 (225.6)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;5.6 (302.6)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;29.4 (381.7)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; CC ½&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn1&quot;&gt;*&lt;/xref&gt;&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn2&quot;&gt;†&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;100 (58.3)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.9 (42.6)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.9 (48.5)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;100 (59.4)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.8 (73.2)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.9 (50.9)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.5 (46.7)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;100 (33.3)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.7 (35)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;italic&gt;I&lt;/italic&gt;/&lt;italic&gt;σI&lt;/italic&gt;&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn2&quot;&gt;†&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;24.68 (1.46)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;7.99 (0.89)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;17.92 (1.85)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;21.24 (1.07)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;16.53 (1.41)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;10.61 (0.97)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;6.35 (1.00)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;18.95 (0.92)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;9.05 (0.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Completeness&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn2&quot;&gt;†&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.9 (99.9)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.6 (100)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.7 (96.8)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.8 (99.1)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.8 (99.7)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.7 (99.9)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.4 (98.6)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.6 (100)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.1 (99.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Redundancy&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn2&quot;&gt;†&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;39.1 (39.8)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;12.1 (14.3)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;81.6 (65.2)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;13.7 (13.7)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;20.9 (19.1)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;12.7 (13.5)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;6.1 (6.5)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;9.9 (10.4)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;18.5 (18.2)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td&gt; &lt;/td&gt;&lt;td&gt; &lt;/td&gt;&lt;td&gt; &lt;/td&gt;&lt;td&gt; &lt;/td&gt;&lt;td&gt; &lt;/td&gt;&lt;td&gt; &lt;/td&gt;&lt;td&gt; &lt;/td&gt;&lt;td&gt; &lt;/td&gt;&lt;td&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;10&quot; align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;Refinement&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Resolution&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn1&quot;&gt;*&lt;/xref&gt; (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;46.4–3.0&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;84.5–3.7&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;49.2–3.6&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;49.1–4.5&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;49.9–7.2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;50.0–8.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Reflections&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;16,928&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;40,647&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;41,799&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;23,340&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;14,046&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;12,111&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;work&lt;/sub&gt;/&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.20/0.24&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.35/0.38&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn3&quot;&gt;‡&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.20/0.24&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.24/0.24&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.23/0.25&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.30/0.32&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Number of atoms&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;  Protein&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;5,465&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;6,925&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;16,592&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;16,405&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;22,543&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;22,445&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;  Waters&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;43&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;  Ligand/ion&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;7&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;5&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;italic&gt;B&lt;/italic&gt;-factors&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;  Protein&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;130&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;158&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;226&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;275&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;272&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;250&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;  Waters&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;84&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;  Ligand/ion&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;189&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; R.m.s.d.&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;  RMS (angles, °)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.97&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.83&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.07&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.11&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.15&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.01&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;  RMS (bonds, Å)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.01&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.01&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;—&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.01&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.01&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.01&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.01&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>54480</offset><text> 	SceCD	SceCD Thimerosal	SceCD Eu	HsaBT-CD	HsaBT-CD Cd2+	CthCD-CTCter1	CthCD-CTCter2	CthCD-CT	CthΔBCCP	 	Data collection	 	 Space group	P43212	P43212	P43212	I4122	I4122	P212121	P212121	P31212	P6422	 	 Cell dimensions	 	 	 	 	 	 	 	 	 	 	  a, b, c (Å)	110.86, 110.86, 131.12	111.22, 111.22, 131.49	108.65, 108.65, 127.36	267.27, 267.27, 210.61	267.67, 267.67, 210.46	97.66, 165.34, 219.23	100.17, 153.45, 249,24	295.02, 295.02, 189.52	462.20, 462.20, 204.64	 	  α, β, γ (°)	90, 90, 90	90, 90, 90	90, 90, 90	90, 90, 90	90, 90, 90	90, 90, 90	90, 90, 90	90, 90, 120	90, 90, 120	 	 Resolution* (Å)	3.0	3.4	4.0	3.7	4.1	3.6	4.5	7.2	8.4	 	 RMerge†	18.2 (389.6)	20.5 (306.1)	40.6 (327.0)	7.5 (400.9)	15 (730.5)	14.5 (384.5)	27.4 (225.6)	5.6 (302.6)	29.4 (381.7)	 	 CC ½*†	100 (58.3)	99.9 (42.6)	99.9 (48.5)	100 (59.4)	99.8 (73.2)	99.9 (50.9)	99.5 (46.7)	100 (33.3)	99.7 (35)	 	 I/σI†	24.68 (1.46)	7.99 (0.89)	17.92 (1.85)	21.24 (1.07)	16.53 (1.41)	10.61 (0.97)	6.35 (1.00)	18.95 (0.92)	9.05 (0.9)	 	 Completeness†	99.9 (99.9)	99.6 (100)	99.7 (96.8)	99.8 (99.1)	99.8 (99.7)	99.7 (99.9)	99.4 (98.6)	99.6 (100)	99.1 (99.9)	 	 Redundancy†	39.1 (39.8)	12.1 (14.3)	81.6 (65.2)	13.7 (13.7)	20.9 (19.1)	12.7 (13.5)	6.1 (6.5)	9.9 (10.4)	18.5 (18.2)	 	 	 	 	 	 	 	 	 	 	 	 	Refinement	 	 Resolution* (Å)	46.4–3.0	 	 	84.5–3.7	 	49.2–3.6	49.1–4.5	49.9–7.2	50.0–8.4	 	 Reflections	16,928	—	—	40,647	—	41,799	23,340	14,046	12,111	 	 Rwork/Rfree	0.20/0.24	—	—	0.35/0.38‡	—	0.20/0.24	0.24/0.24	0.23/0.25	0.30/0.32	 	 Number of atoms	 	 	 	 	 	 	 	 	 	 	  Protein	5,465	 	 	6,925	 	16,592	16,405	22,543	22,445	 	  Waters	43	—	—	—	—	—	—	—	—	 	  Ligand/ion	7	—	—	5	—	—	—	—	—	 	 B-factors	 	 	 	 	 	 	 	 	 	 	  Protein	130	 	 	158	 	226	275	272	250	 	  Waters	84	—	—	—	—	—	—	—	—	 	  Ligand/ion	90	—	—	189	—	—	—	—	—	 	 R.m.s.d.	 	 	 	 	 	 	 	 	 	 	  RMS (angles, °)	0.97	—	—	0.83	—	1.07	1.11	1.15	1.01	 	  RMS (bonds, Å)	0.01	—	—	0.01	—	0.01	0.01	0.01	0.01	 	</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>56655</offset><text>*Resolution cutoffs determined based on internal correlation significant at the 0.1% level as calculated by XDS.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>56768</offset><text>†Highest-resolution shell is shown in parentheses.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>56821</offset><text>‡Modelled only as poly-alanine.</text></passage></document></collection>
diff --git a/raw_BioC_XML/PMC4841544_raw.xml b/raw_BioC_XML/PMC4841544_raw.xml
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+<?xml version="1.0" encoding="UTF-8"?>
+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection><source>PMC</source><date>20201223</date><key>pmc.key</key><document><id>4841544</id><infon key="license">CC BY</infon><passage><infon key="alt-title">Molecular Basis of NadR Regulation</infon><infon key="article-id_doi">10.1371/journal.ppat.1005557</infon><infon key="article-id_pmc">4841544</infon><infon key="article-id_pmid">27105075</infon><infon key="article-id_publisher-id">PPATHOGENS-D-15-00389</infon><infon key="elocation-id">e1005557</infon><infon key="issue">4</infon><infon key="license">This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.</infon><infon key="name_0">surname:Liguori;given-names:Alessia</infon><infon key="name_1">surname:Malito;given-names:Enrico</infon><infon key="name_10">surname:Nassif;given-names:Xavier</infon><infon key="name_2">surname:Lo Surdo;given-names:Paola</infon><infon key="name_3">surname:Fagnocchi;given-names:Luca</infon><infon key="name_4">surname:Cantini;given-names:Francesca</infon><infon key="name_5">surname:Haag;given-names:Andreas F.</infon><infon key="name_6">surname:Brier;given-names:Sébastien</infon><infon key="name_7">surname:Pizza;given-names:Mariagrazia</infon><infon key="name_8">surname:Delany;given-names:Isabel</infon><infon key="name_9">surname:Bottomley;given-names:Matthew J.</infon><infon key="notes">The atomic coordinates of the NadR structures (with or without 4-HPA) have been deposited in the Protein Data Bank (PDB) and have been released with codes 5aip and 5aiq.</infon><infon key="section_type">TITLE</infon><infon key="title">Data Availability</infon><infon key="type">front</infon><infon key="volume">12</infon><infon key="year">2016</infon><offset>0</offset><text>Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>125</offset><text> Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. Overall, this study deepens our molecular understanding of the sophisticated regulatory mechanisms of the expression of nadA and other genes governed by NadR, dependent on interactions with niche-specific signal molecules that may play important roles during meningococcal pathogenesis.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>2618</offset><text>Author Summary</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>2633</offset><text>Serogroup B meningococcus (MenB) causes fatal sepsis and invasive meningococcal disease, particularly in young children and adolescents, as highlighted by recent MenB outbreaks in universities of the United States and Canada. The Bexsero vaccine protects against MenB and has recently been approved in &gt; 35 countries worldwide. Neisseria adhesin A (NadA) present on the meningococcal surface can mediate binding to human cells and is one of the three MenB vaccine protein antigens. The amount of NadA exposed on the meningococcal surface also influences the antibody-mediated serum bactericidal response measured in vitro. A deep understanding of nadA expression is therefore important, otherwise the contribution of NadA to vaccine-induced protection against meningococcal meningitis may be underestimated. The abundance of surface-exposed NadA is regulated by the ligand-responsive transcriptional repressor NadR. Here, we present functional, biochemical and high-resolution structural data on NadR. Our studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by ‘conformational selection’ of inactive forms. These findings shed light on the regulation of NadR, a key MarR-family virulence factor of this important human pathogen.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>3999</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4012</offset><text>The ‘Reverse Vaccinology’ approach was pioneered to identify antigens for a protein-based vaccine against serogroup B Neisseria meningitidis (MenB), a human pathogen causing potentially-fatal sepsis and invasive meningococcal disease. Indeed, Reverse Vaccinology identified Neisseria adhesin A (NadA), a surface-exposed protein involved in epithelial cell invasion and found in ~30% of clinical isolates. Recently, we reported the crystal structure of NadA, providing insights into its biological and immunological functions. Recombinant NadA elicits a strong bactericidal immune response and is therefore included in the Bexsero vaccine that protects against MenB and which was recently approved in over 35 countries worldwide.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4745</offset><text>Previous studies revealed that nadA expression levels are mainly regulated by the Neisseria adhesin A Regulator (NadR). Although additional factors influence nadA expression, we focused on its regulation by NadR, the major mediator of NadA phase variable expression. Studies of NadR also have broader implications, since a genome-wide analysis of MenB wild-type and nadR knock-out strains revealed that NadR influences the regulation of &gt; 30 genes, including maf genes, from the multiple adhesin family. These genes encode a wide variety of proteins connected to many biological processes contributing to bacterial survival, adaptation in the host niche, colonization and invasion.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5427</offset><text>NadR belongs to the MarR (Multiple Antibiotic Resistance Regulator) family, a group of ligand-responsive transcriptional regulators ubiquitous in bacteria and archaea. MarR family proteins can promote bacterial survival in the presence of antibiotics, toxic chemicals, organic solvents or reactive oxygen species and can regulate virulence factor expression. MarR homologues can act either as transcriptional repressors or as activators. Although &gt; 50 MarR family structures are known, a molecular understanding of their ligand-dependent regulatory mechanisms is still limited, often hampered by lack of identification of their ligands and/or DNA targets. A potentially interesting exception comes from the ligand-free and salicylate-bound forms of the Methanobacterium thermoautotrophicum protein MTH313 which revealed that two salicylate molecules bind to one MTH313 dimer and induce large conformational changes, apparently sufficient to prevent DNA binding. However, the homologous archeal Sulfolobus tokodaii protein ST1710 presented essentially the same structure in ligand-free and salicylate-bound forms, apparently contrasting the mechanism proposed for MTH313. Despite these apparent differences, MTH313 and ST1710 bind salicylate in approximately the same site, between their dimerization and DNA-binding domains. However, it is unknown whether salicylate is a relevant in vivo ligand of either of these two proteins, which share ~20% sequence identity with NadR, rendering unclear the interpretation of these findings in relation to the regulatory mechanisms of NadR or other MarR family proteins.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>7037</offset><text>NadR binds the nadA promoter and represses gene transcription. NadR binds nadA on three different operators (OpI, OpII and OpIII). The DNA-binding activity of NadR is attenuated in vitro upon addition of various hydroxyphenylacetate (HPA) derivatives, including 4-HPA. 4-HPA is a small molecule derived from mammalian aromatic amino acid catabolism and is released in human saliva, where it has been detected at micromolar concentration. In the presence of 4-HPA, NadR is unable to bind the nadA promoter and nadA gene expression is induced. In vivo, the presence of 4-HPA in the host niche of N. meningitidis serves as an inducer of NadA production, thereby promoting bacterial adhesion to host cells. Further, we recently reported that 3Cl,4-HPA, produced during inflammation, is another inducer of nadA expression.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>7855</offset><text>Extending our previous studies based on hydrogen-deuterium exchange mass spectrometry (HDX-MS), here we sought to reveal the molecular mechanisms and effects of NadR/HPA interactions via X-ray crystallography, NMR spectroscopy and complementary biochemical and in vivo mutagenesis studies. We obtained detailed new insights into ligand specificity, how the ligand allosterically influences the DNA-binding ability of NadR, and the regulation of nadA expression, thus also providing a deeper structural understanding of the ligand-responsive MarR super-family. Moreover, these findings are important because the activity of NadR impacts the potential coverage provided by anti-NadA antibodies elicited by the Bexsero vaccine and influences host-bacteria interactions that contribute to meningococcal pathogenesis.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>8668</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8676</offset><text>NadR is dimeric and is stabilized by specific hydroxyphenylacetate ligands</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8751</offset><text>Recombinant NadR was produced in E. coli using an expression construct prepared from N. meningitidis serogroup B strain MC58. Standard chromatographic techniques were used to obtain a highly purified sample of NadR (see Materials and Methods). In analytical size-exclusion high-performance liquid chromatography (SE-HPLC) experiments coupled with multi-angle laser light scattering (MALLS), NadR presented a single species with an absolute molecular mass of 35 kDa (S1 Fig). These data showed that NadR was dimeric in solution, since the theoretical molecular mass of the NadR dimer is 33.73 kDa; and, there was no change in oligomeric state on addition of 4-HPA.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9415</offset><text>The thermal stability of NadR was examined using differential scanning calorimetry (DSC). Since ligand-binding often increases protein stability, we also investigated the effect of various HPAs (Fig 1A) on the melting temperature (Tm) of NadR. As a control of specificity, we also tested salicylate, a known ligand of some MarR proteins previously reported to increase the Tm of ST1710 and MTH313. The Tm of NadR was 67.4 ± 0.1°C in the absence of ligand, and was unaffected by salicylate. However, an increased thermal stability was induced by 4-HPA and, to a lesser extent, by 3-HPA. Interestingly, NadR displayed the greatest Tm increase upon addition of 3Cl,4-HPA (Table 1 and Fig 1B).</text></passage><passage><infon key="file">ppat.1005557.g001.jpg</infon><infon key="id">ppat.1005557.g001</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>10107</offset><text>Stability of NadR is increased by small molecule ligands.</text></passage><passage><infon key="file">ppat.1005557.g001.jpg</infon><infon key="id">ppat.1005557.g001</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>10165</offset><text>
+(A) Molecular structures of 3-HPA (MW 152.2), 4-HPA (MW 152.2), 3Cl,4-HPA (MW 186.6) and salicylic acid (MW 160.1). (B) DSC profiles, colored as follows: apo-NadR (violet), NadR+salicylate (red), NadR+3-HPA (green), NadR+4-HPA (blue), NadR+3Cl,4-HPA (pink). All DSC profiles are representative of triplicate experiments.</text></passage><passage><infon key="file">ppat.1005557.t001.xml</infon><infon key="id">ppat.1005557.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>10487</offset><text>Melting-point (Tm) and its ligand-induced increase (ΔTm) derived from DSC thermostability experiments.</text></passage><passage><infon key="file">ppat.1005557.t001.xml</infon><infon key="id">ppat.1005557.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>10594</offset><text>Dissociation constants (KD) of the NadR/ligand interactions from SPR steady-state binding experiments.</text></passage><passage><infon key="file">ppat.1005557.t001.xml</infon><infon key="id">ppat.1005557.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;colgroup span=&quot;1&quot;&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;/colgroup&gt;&lt;thead&gt;&lt;tr&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Ligand&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;T&lt;sub&gt;m&lt;/sub&gt; (°C)&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ΔT&lt;sub&gt;m&lt;/sub&gt; (°C)&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;K&lt;sub&gt;D&lt;/sub&gt; (mM)&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;No ligand&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;67.4 ± 0.1&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.a.&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.a.&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Salicylate&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;67.5 ± 0.1&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3-HPA&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;70.0 ± 0.1&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.7&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.7 ± 0.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4-HPA&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;70.7 ± 0.1&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.3&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.5 ± 0.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3Cl,4-HPA&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;71.3 ± 0.2&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.9&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.1 ± 0.1&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>10697</offset><text>Ligand	Tm (°C)	ΔTm (°C)	KD (mM)	 	No ligand	67.4 ± 0.1	n.a.	n.a.	 	Salicylate	67.5 ± 0.1	0	n.d.	 	3-HPA	70.0 ± 0.1	2.7	2.7 ± 0.1	 	4-HPA	70.7 ± 0.1	3.3	1.5 ± 0.1	 	3Cl,4-HPA	71.3 ± 0.2	3.9	1.1 ± 0.1	 	</text></passage><passage><infon key="file">ppat.1005557.t001.xml</infon><infon key="id">ppat.1005557.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>10911</offset><text>n.a.: not applicable; n.d.: not determinable</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10956</offset><text>NadR displays distinct binding affinities for hydroxyphenylacetate ligands</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11031</offset><text>To further investigate the binding of HPAs to NadR, we used surface plasmon resonance (SPR). The SPR sensorgrams revealed very fast association and dissociation events, typical of small molecule ligands, thus prohibiting a detailed study of binding kinetics. However, steady-state SPR analyses of the NadR-HPA interactions allowed determination of the equilibrium dissociation constants (KD) (Table 1 and S2 Fig). The interactions of 4-HPA and 3Cl,4-HPA with NadR exhibited KD values of 1.5 mM and 1.1 mM, respectively. 3-HPA showed a weaker interaction, with a KD of 2.7 mM, while salicylate showed only a very weak response that did not reach saturation, indicating a non-specific interaction with NadR. A ranking of these KD values showed that 3Cl,4-HPA was the tightest binder, and thus matched the ranking of ligand-induced Tm increases observed in the DSC experiments. Although these KD values indicate rather weak interactions, they are similar to the values reported previously for the MarR/salicylate interaction (KD ~1 mM) and the MTH313/salicylate interaction (KD 2–3 mM), and approximately 20-fold tighter than the ST1710/salicylate interaction (KD ~20 mM).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>12203</offset><text>Crystal structures of holo-NadR and apo-NadR</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12248</offset><text>To fully characterize the NadR/HPA interactions, we sought to determine crystal structures of NadR in ligand-bound (holo) and ligand-free (apo) forms. First, we crystallized NadR (a selenomethionine-labelled derivative) in the presence of a 200-fold molar excess of 4-HPA. The structure of the NadR/4-HPA complex was determined at 2.3 Å resolution using a combination of the single-wavelength anomalous dispersion (SAD) and molecular replacement (MR) methods, and was refined to R work/R free values of 20.9/26.0% (Table 2). Despite numerous attempts, we were unable to obtain high-quality crystals of NadR complexed with 3Cl,4-HPA, 3,4-HPA, 3-HPA or DNA targets. However, it was eventually possible to crystallize apo-NadR, and the structure was determined at 2.7 Å resolution by MR methods using the NadR/4-HPA complex as the search model. The apo-NadR structure was refined to R work/R free values of 19.1/26.8% (Table 2).</text></passage><passage><infon key="file">ppat.1005557.t002.xml</infon><infon key="id">ppat.1005557.t002</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>13176</offset><text>Data collection and refinement statistics for NadR structures.</text></passage><passage><infon key="file">ppat.1005557.t002.xml</infon><infon key="id">ppat.1005557.t002</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;colgroup span=&quot;1&quot;&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;/colgroup&gt;&lt;thead&gt;&lt;tr&gt;&lt;th align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;th align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NadR SeMet + 4-HPA (SAD peak) (PDB code 5aip)&lt;/th&gt;&lt;th align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NadR apo-form (PDB code 5aiq)&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;bold&gt;&lt;italic&gt;Data collection&lt;/italic&gt;&lt;/bold&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Wavelength (Å)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.9792&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Beamline&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SLS (PXII-X10SA)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SLS (PXII-X10SA)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution range (Å)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;39.2–2.3&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;48.2–2.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Space group&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;P 43 21 2&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;P 43 21 2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Unit cell dimensions (Å)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;75.3, 75.3, 91.8&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;69.4, 69.4, 253.8&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Total reflections&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;291132 (41090)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;225521 (35809)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Unique reflections&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;12320 (1773)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;17700 (2780)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Multiplicity&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;23.6 (23.2)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;12.7 (12.8)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Completeness (%)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100.0 (100.00)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.9 (99.7)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Mean I/sigma(I)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;25.5 (9.0)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;22.6 (3.8)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Wilson B-factor&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;23.9&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;49.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;italic&gt;R&lt;/italic&gt;
+&lt;sub&gt;sym&lt;/sub&gt;
+&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t002fn002&quot;&gt;*&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;10.9 (39.4)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;11.4 (77.6)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;italic&gt;R&lt;/italic&gt;
+&lt;sub&gt;meas&lt;/sub&gt;
+&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t002fn003&quot;&gt;**&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;11.3&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;11.8&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;bold&gt;&lt;italic&gt;Refinement&lt;/italic&gt;&lt;/bold&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;italic&gt;R&lt;/italic&gt;
+&lt;sub&gt;work&lt;/sub&gt;
+&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t002fn004&quot;&gt;
+&lt;sup&gt;♯&lt;/sup&gt;
+&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;20.9&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;21.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;italic&gt;R&lt;/italic&gt;
+&lt;sub&gt;free&lt;/sub&gt;
+&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t002fn005&quot;&gt;
+&lt;sup&gt;♯♯&lt;/sup&gt;
+&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;26.0&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;27.2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;italic&gt;Number of atoms&lt;/italic&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  Non-hydrogen atoms&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2263&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4163&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  Macromolecules&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2207&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4144&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  Ligands&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;11&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  Water&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;45&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;19&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Protein residues&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;275&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;521&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;RMS(bonds)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.008&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.003&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;RMS(angles)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.09&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.823&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;italic&gt;Ramachandran (%)&lt;/italic&gt;
+&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t002fn006&quot;&gt;
+&lt;sup&gt;§&lt;/sup&gt;
+&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  Favored&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;98.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  Outliers&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Clash score&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5.0&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.9&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;italic&gt;Average B-factor&lt;/italic&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  Macromolecules&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;34.8&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;53.3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  Ligands&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;32.9&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;-&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  Solvent&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;37.3 (H&lt;sub&gt;2&lt;/sub&gt;O)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;29.0 (H&lt;sub&gt;2&lt;/sub&gt;O)&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>13239</offset><text>	NadR SeMet + 4-HPA (SAD peak) (PDB code 5aip)	NadR apo-form (PDB code 5aiq)	 	Data collection			 	Wavelength (Å)	0.9792	1.0	 	Beamline	SLS (PXII-X10SA)	SLS (PXII-X10SA)	 	Resolution range (Å)	39.2–2.3	48.2–2.7	 	Space group	P 43 21 2	P 43 21 2	 	Unit cell dimensions (Å)	75.3, 75.3, 91.8	69.4, 69.4, 253.8	 	Total reflections	291132 (41090)	225521 (35809)	 	Unique reflections	12320 (1773)	17700 (2780)	 	Multiplicity	23.6 (23.2)	12.7 (12.8)	 	Completeness (%)	100.0 (100.00)	99.9 (99.7)	 	Mean I/sigma(I)	25.5 (9.0)	22.6 (3.8)	 	Wilson B-factor	23.9	49.1	 	Rsym*	10.9 (39.4)	11.4 (77.6)	 	Rmeas**	11.3	11.8	 	Refinement			 	Rwork♯	20.9	21.7	 	Rfree♯♯	26.0	27.2	 	Number of atoms			 	  Non-hydrogen atoms	2263	4163	 	  Macromolecules	2207	4144	 	  Ligands	11	0	 	  Water	45	19	 	Protein residues	275	521	 	RMS(bonds)	0.008	0.003	 	RMS(angles)	1.09	0.823	 	Ramachandran (%)§			 	  Favored	100	98.4	 	  Outliers	0	0	 	Clash score	5.0	3.9	 	Average B-factor			 	  Macromolecules	34.8	53.3	 	  Ligands	32.9	-	 	  Solvent	37.3 (H2O)	29.0 (H2O)	 	</text></passage><passage><infon key="file">ppat.1005557.t002.xml</infon><infon key="id">ppat.1005557.t002</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>14314</offset><text>Statistics for the highest-resolution shell are shown in parentheses.</text></passage><passage><infon key="file">ppat.1005557.t002.xml</infon><infon key="id">ppat.1005557.t002</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>14384</offset><text>*R
+sym = Σhkl Σi |Ii(hkl)—&lt;I(hkl)&gt;| / Σhkl Σi Ii(hkl)</text></passage><passage><infon key="file">ppat.1005557.t002.xml</infon><infon key="id">ppat.1005557.t002</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>14454</offset><text>** R
+meas = redundancy-independent (multiplicity-weighted) R
+merge as reported from AIMLESS.</text></passage><passage><infon key="file">ppat.1005557.t002.xml</infon><infon key="id">ppat.1005557.t002</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>14547</offset><text>
+♯
+R
+work = Σ||F(obs)|- |F(calc)||/Σ|F(obs)|</text></passage><passage><infon key="file">ppat.1005557.t002.xml</infon><infon key="id">ppat.1005557.t002</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>14600</offset><text>
+♯♯
+R
+free = as for R
+work, calculated for 5.0% of the total reflections, chosen at random, and omitted from refinement.</text></passage><passage><infon key="file">ppat.1005557.t002.xml</infon><infon key="id">ppat.1005557.t002</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>14725</offset><text>
+§ Values obtained using Molprobity.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14763</offset><text>The asymmetric unit of the NadR/4-HPA crystals (holo-NadR) contained one NadR homodimer, while the apo-NadR crystals contained two homodimers. In the apo-NadR crystals, the two homodimers were related by a rotation of ~90°; the observed association of the two dimers was presumably merely an effect of crystal packing, since the interface between the two homodimers is small (&lt; 550 Å2 of buried surface area), and is not predicted to be physiologically relevant by the PISA software. Moreover, our SE-HPLC/MALLS analyses (see above) revealed that in solution NadR is dimeric, and previous studies using native mass spectrometry (MS) revealed dimers, not tetramers.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15430</offset><text>The NadR homodimer bound to 4-HPA has a dimerization interface mostly involving the top of its ‘triangular’ form, while the two DNA-binding domains are located at the base (Fig 2A). High-quality electron density maps allowed clear identification of the bound ligand, 4-HPA (Fig 2B). The overall structure of NadR shows dimensions of ~50 × 65 × 50 Å and a large homodimer interface that buries a total surface area of ~ 4800 Å2. Each NadR monomer consists of six α-helices and two short β-strands, with helices α1, α5, and α6 forming the dimer interface. Helices α3 and α4 form a helix-turn-helix motif, followed by the “wing motif” comprised of two short antiparallel β-strands (β1-β2) linked by a relatively long and flexible loop. Interestingly, in the α4-β2 region, the stretch of residues from R64-R91 presents seven positively-charged side chains, all available for potential interactions with DNA. Together, these structural elements constitute the winged helix-turn-helix (wHTH) DNA-binding domain and, together with the dimeric organization, are the hallmarks of MarR family structures.</text></passage><passage><infon key="file">ppat.1005557.g002.jpg</infon><infon key="id">ppat.1005557.g002</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>16568</offset><text>The crystal structure of NadR in complex with 4-HPA.</text></passage><passage><infon key="file">ppat.1005557.g002.jpg</infon><infon key="id">ppat.1005557.g002</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>16621</offset><text>
+(A) The holo-NadR homodimer is depicted in green and blue for chains A and B respectively, while yellow sticks depict the 4-HPA ligand (labelled). For simplicity, secondary structure elements are labelled for chain B only. Red dashes show hypothetical positions of chain B residues 88–90 that were not modeled due to lack of electron density. (B) A zoom into the pocket occupied by 4-HPA shows that the ligand contacts both chains A and B; blue mesh shows electron density around 4-HPA calculated from a composite omit map (omitting 4-HPA), using phenix. The map is contoured at 1σ and the figure was prepared with a density mesh carve factor of 1.7, using Pymol (www.pymol.org).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>17306</offset><text>A single conserved leucine residue (L130) is crucial for dimerization</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17376</offset><text>The NadR dimer interface is formed by at least 32 residues, which establish numerous inter-chain salt bridges or hydrogen bonds, and many hydrophobic packing interactions (Fig 3A and 3B). To determine which residues were most important for dimerization, we studied the interface in silico and identified several residues as potential mediators of key stabilizing interactions. Using site-directed mutagenesis, a panel of eight mutant NadR proteins was prepared (including mutations H7A, S9A, N11A, D112A, R114A, Y115A, K126A, L130K and L133K), sufficient to explore the entire dimer interface. Each mutant NadR protein was purified, and then its oligomeric state was examined by analytical SE-HPLC. Almost all the mutants showed the same elution profile as the wild-type (WT) NadR protein. Only the L130K mutation induced a notable change in the oligomeric state of NadR (Fig 3C). Further, in SE-MALLS analyses, the L130K mutant displayed two distinct species in solution, approximately 80% being monomeric (a 19 kDa species), and only 20% retaining the typical native dimeric state (a 35 kDa species) (Fig 3D), demonstrating that Leu130 is crucial for stable dimerization. It is notable that L130 is usually present as Leu, or an alternative bulky hydrophobic amino acid (e.g. Phe, Val), in many MarR family proteins, suggesting a conserved role in stabilizing the dimer interface. In contrast, most of the other residues identified in the NadR dimer interface were poorly conserved in the MarR family.</text></passage><passage><infon key="file">ppat.1005557.g003.jpg</infon><infon key="id">ppat.1005557.g003</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>18880</offset><text>Analysis of the NadR dimer interface.</text></passage><passage><infon key="file">ppat.1005557.g003.jpg</infon><infon key="id">ppat.1005557.g003</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>18918</offset><text>
+(A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). (C) SE-HPLC analyses of all mutant forms of NadR are compared with the wild-type (WT) protein. The WT and most of the mutants show a single elution peak with an absorbance maximum at 17.5 min. Only the mutation L130K has a noteworthy effect on the oligomeric state, inducing a second peak with a longer retention time and a second peak maximum at 18.6 min. To a much lesser extent, the L133K mutation also appears to induce a ‘shoulder’ to the main peak, suggesting very weak ability to disrupt the dimer. (D) SE-HPLC/MALLS analyses of the L130K mutant, shows 20% dimer and 80% monomer. The curves plotted correspond to Absorbance Units (mAU) at 280nm wavelength (green), light scattering (red), and refractive index (blue).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>20621</offset><text>The holo-NadR structure presents only one occupied ligand-binding pocket</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20694</offset><text>The NadR/4-HPA structure revealed the ligand-binding site nestled between the dimerization and DNA-binding domains (Fig 2). The ligand showed a different position and orientation compared to salicylate complexed with MTH313 and ST1710 (see Discussion). The binding pocket was almost entirely filled by 4-HPA and one water molecule, although there also remained a small tunnel 2-4Å in diameter and 5-6Å long leading from the pocket (proximal to the 4-hydroxyl position) to the protein surface. The tunnel was lined with rather hydrophobic amino acids, and did not contain water molecules. Unexpectedly, only one monomer of the holo-NadR homodimer contained 4-HPA in the binding pocket, whereas the corresponding pocket of the other monomer was unoccupied by ligand, despite the large excess of 4-HPA used in the crystallization conditions.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21535</offset><text>Inspection of the protein-ligand interaction network revealed no bonds from NadR backbone groups to the ligand, but several key side chain mediated hydrogen (H)-bonds and ionic interactions, most notably between the carboxylate group of 4-HPA and Ser9 of chain A (SerA9), and chain B residues TrpB39, ArgB43 and TyrB115 (Fig 4A). At the other ‘end’ of the ligand, the 4-hydroxyl group was proximal to AspB36, with which it may establish an H-bond (see bond distances in Table 3). The water molecule observed in the pocket was bound by the carboxylate group and the side chains of SerA9 and AsnA11.</text></passage><passage><infon key="file">ppat.1005557.g004.jpg</infon><infon key="id">ppat.1005557.g004</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>22137</offset><text>Atomic details of NadR/HPA interactions.</text></passage><passage><infon key="file">ppat.1005557.g004.jpg</infon><infon key="id">ppat.1005557.g004</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>22178</offset><text>
+A) A stereo-view zoom into the binding pocket showing side chain sticks for all interactions between NadR and 4-HPA. Green and blue ribbons depict NadR chains A and B, respectively. 4-HPA is shown in yellow sticks, with oxygen atoms in red. A water molecule is shown by the red sphere. H-bonds up to 3.6Å are shown as dashed lines. The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually). Residues AsnA11 and ArgB18 likely make indirect yet local contributions to ligand binding, mainly by stabilizing the position of AspB36. Bond distances for interacting polar atoms are provided in Table 3. Side chains mediating hydrophobic interactions are shown in orange. (B) A model was prepared to visualize putative interactions of 3Cl,4-HPA (pink) with NadR, revealing the potential for additional contacts (dashed lines) of the chloro moiety (green stick) with LeuB29 and AspB36.</text></passage><passage><infon key="file">ppat.1005557.t003.xml</infon><infon key="id">ppat.1005557.t003</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>23247</offset><text>List of 4-HPA atoms bound to NadR via ionic interactions and/or H-bonds.</text></passage><passage><infon key="file">ppat.1005557.t003.xml</infon><infon key="id">ppat.1005557.t003</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;colgroup span=&quot;1&quot;&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;/colgroup&gt;&lt;thead&gt;&lt;tr&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4-HPA atom&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NadR residue/atom&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Distance (Å)&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;O2&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;TrpB39/NE1&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.83&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;O2&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ArgB43/NH1&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.76&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;O1&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ArgB43/NH1&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.84&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;O1&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SerA9/OG&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.75&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;O1&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;TyrB115/OH&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.50&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;O2&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Water (&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t003fn001&quot;&gt;*&lt;/xref&gt;Ser9/Asn11)&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.88&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;OH&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;AspB36/OD1/OD2&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.6/3.7&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>23320</offset><text>4-HPA atom	NadR residue/atom	Distance (Å)	 	O2	TrpB39/NE1	2.83	 	O2	ArgB43/NH1	2.76	 	O1	ArgB43/NH1	3.84	 	O1	SerA9/OG	2.75	 	O1	TyrB115/OH	2.50	 	O2	Water (*Ser9/Asn11)	2.88	 	OH	AspB36/OD1/OD2	3.6/3.7	 	</text></passage><passage><infon key="file">ppat.1005557.t003.xml</infon><infon key="id">ppat.1005557.t003</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>23527</offset><text>* Bond distance between the ligand carboxylate group and the water molecule, which in turn makes H-bond to the SerA9 and AsnA11 side chains.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23668</offset><text>In addition to the H-bonds involving the carboxylate and hydroxyl groups of 4-HPA, binding of the phenyl moiety appeared to be stabilized by several van der Waals’ contacts, particularly those involving the hydrophobic side chain atoms of LeuB21, MetB22, PheB25, LeuB29 and ValB111 (Fig 4A). Notably, the phenyl ring of PheB25 was positioned parallel to the phenyl ring of 4-HPA, potentially forming π-π parallel-displaced stacking interactions. Consequently, residues in the 4-HPA binding pocket are mostly contributed by NadR chain B, and effectively created a polar ‘floor’ and a hydrophobic ‘ceiling’, which house the ligand. Collectively, this mixed network of polar and hydrophobic interactions endows NadR with a strong recognition pattern for HPAs, with additional medium-range interactions potentially established with the hydroxyl group at the 4-position.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>24546</offset><text>Structure-activity relationships: molecular basis of enhanced stabilization by 3Cl,4-HPA</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24635</offset><text>We modelled the binding of other HPAs by in silico superposition onto 4-HPA in the holo-NadR structure, and thereby obtained molecular explanations for the binding specificities of diverse ligands. For example, similar to 4-HPA, the binding of 3Cl,4-HPA could involve multiple bonds towards the carboxylate group of the ligand and some to the 4-hydroxyl group. Additionally, the side chains of LeuB29 and AspB36 would be only 2.6–3.5 Å from the chlorine atom, thus providing van der Waals’ interactions or H-bonds to generate the additional binding affinity observed for 3Cl,4-HPA (Fig 4B). The presence of a single hydroxyl group at position 2, as in 2-HPA, rather than at position 4, would eliminate the possibility of favorable interactions with AspB36, resulting in the lack of NadR regulation by 2-HPA described previously. Finally, salicylate is presumably unable to specifically bind NadR due to the 2-hydroxyl substitution and the shorter aliphatic chain connecting its carboxylate group (Fig 1A): the compound simply seems too small to simultaneously establish the network of beneficial bonds observed in the NadR/HPA interactions.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>25781</offset><text>Analysis of the pockets reveals the molecular basis for asymmetric binding and stoichiometry</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>25874</offset><text>We attempted to investigate further the binding stoichiometry using solution-based techniques. However, studies based on tryptophan fluorescence were confounded by the fluorescence of the HPA ligands, and isothermal titration calorimetry (ITC) was unfeasible due to the need for very high concentrations of NadR in the ITC chamber (due to the relatively low affinity), which exceeded the solubility limits of the protein. However, it was possible to calculate the binding stoichiometry of the NadR-HPA interactions using an SPR-based approach. In SPR, the signal measured is proportional to the total molecular mass proximal to the sensor surface; consequently, if the molecular weights of the interactors are known, then the stoichiometry of the resulting complex can be determined. This approach relies on the assumption that the captured protein (‘the ligand’, according to SPR conventions) is 100% active and freely-accessible to potential interactors (‘the analytes’). This assumption is likely valid for this pair of interactors, for two main reasons. Firstly, NadR is expected to be covalently immobilized on the sensor chip as a dimer in random orientations, since it is a stable dimer in solution and has sixteen lysines well-distributed around its surface, all able to act as potential sites for amine coupling to the chip, and none of which are close to the ligand-binding pocket. Secondly, the HPA analytes are all very small (MW 150–170, Fig 1A) and therefore are expected to be able to diffuse readily into all potential binding sites, irrespective of the random orientations of the immobilized NadR dimers on the chip.The stoichiometry of the NadR-HPA interactions was determined using Eq 1 (see Materials and Methods), and revealed stoichiometries of 1.13 for 4-HPA, 1.02 for 3-HPA, and 1.21 for 3Cl,4-HPA, strongly suggesting that one NadR dimer bound to 1 HPA analyte molecule.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>27780</offset><text>The crystallographic data, supported by the SPR studies of binding stoichiometry, revealed the lack of a second 4-HPA molecule in the homodimer, suggesting negative co-operativity, a phenomenon previously described for the MTH313/salicylate interaction and for other MarR family proteins. To explore the molecular basis of asymmetry in holo-NadR, we superposed its ligand-free monomer (chain A) onto the ligand-occupied monomer (chain B). Overall, the superposition revealed a high degree of structural similarity (Cα root mean square deviation (rmsd) of 1.5Å), though on closer inspection a rotational difference of ~9 degrees along the long axis of helix α6 was observed, suggesting that 4-HPA induced a slight conformational change (Fig 5A). However, since residues of helix α6 were not directly involved in ligand binding, an explanation for the lack of 4-HPA in monomer A did not emerge by analyzing only these backbone atom positions, suggesting that a more complex series of allosteric events may occur. Indeed, we noted interesting differences in the side chains of Met22, Phe25 and Arg43, which in monomer B are used to contact the ligand while in monomer A they partially occupied the pocket and collectively reduced its volume significantly. Specifically, upon analysis with the CASTp software, the pocket in chain B containing the 4-HPA exhibited a total volume of approximately 370 Å3, while the pocket in chain A was occupied by these three side chains that adopted ‘inward’ positions and thereby divided the space into a few much smaller pockets, each with volume &lt; 50 Å3, evidently rendering chain A unfavorable for ligand binding. Most notably, atomic clashes between the ligand and the side chains of MetA22, PheA25 and ArgA43 would occur if 4-HPA were present in the monomer A pocket (Fig 5B). Subsequently, analyses of the pockets in apo-NadR revealed that in the absence of ligand the long Arg43 side chain was always in the open ‘outward’ position compatible with binding to the 4-HPA carboxylate group. In contrast, the apo-form Met22 and Phe25 residues were still encroaching the spaces of the 4-hydroxyl group and the phenyl ring of the ligand, respectively (Fig 5C). The ‘outward’ position of Arg43 generated an open apo-form pocket with volume approximately 380Å3. Taken together, these observations suggest that Arg43 is a major determinant of ligand binding, and that its ‘inward’ position inhibits the binding of 4-HPA to the empty pocket of holo-NadR.</text></passage><passage><infon key="file">ppat.1005557.g005.jpg</infon><infon key="id">ppat.1005557.g005</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>30286</offset><text>Structural differences of NadR in ligand-bound or free forms.</text></passage><passage><infon key="file">ppat.1005557.g005.jpg</infon><infon key="id">ppat.1005557.g005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>30348</offset><text>
+(A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. In these crystals, the ArgA43 side chain showed two alternate conformations, modelled with 50% occupancy in each state, as indicated by the two ‘mirrored’ arrows. The inner conformer is the one that would display major clashes if 4-HPA were present. (C) Comparison of the empty pocket from holo-NadR (green residues) with the four empty pockets of apo-NadR (grey residues), shows that in the absence of 4-HPA the Arg43 side chain is always observed in the ‘outward’ conformation.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>31380</offset><text>Finally, we applied 15N heteronuclear solution NMR spectroscopy to examine the interaction of 4-HPA with apo NadR. We collected NMR spectra on NadR in the presence and absence of 4-HPA (see Materials and Methods). The 1H-15N TROSY-HSQC spectrum of apo-NadR, acquired at 25°C, displayed approximately 140 distinct peaks (Fig 6A), most of which correspond to backbone amide N-H groups. The broad spectral dispersion and the number of peaks observed, which is close to the number of expected backbone amide N-H groups for this polypeptide, confirmed that apo-NadR is well-folded under these conditions and exhibits one conformation appreciable on the NMR timescale, i.e. in the NMR experiments at 25°C, two or more distinct conformations of apo-NadR monomers were not readily apparent. Upon the addition of 4-HPA, over 45 peaks showed chemical shift perturbations, i.e. changed position in the spectrum or disappeared, while the remaining peaks remained unchanged. This observation showed that 4-HPA was able to bind NadR and induce notable changes in specific regions of the protein.</text></passage><passage><infon key="file">ppat.1005557.g006.jpg</infon><infon key="id">ppat.1005557.g006</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>32464</offset><text>NMR spectra of NadR in the presence and absence of 4-HPA.</text></passage><passage><infon key="file">ppat.1005557.g006.jpg</infon><infon key="id">ppat.1005557.g006</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>32522</offset><text>
+(A) Superposition of two 1H-15N TROSY-HSQC spectra recorded at 25°C on apo-NadR (cyan) and on NadR in the presence of 4-HPA (red). (B,C) Overlay of selected regions of the 1H-15N TROSY-HSQC spectra acquired at 25°C of apo-NadR (cyan) and NadR/4-HPA (red) superimposed with the spectra acquired at 10°C of apo-NadR (blue) and NadR/4-HPA (green). The spectra acquired at 10°C are excluded from panel A for simplicity.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>32943</offset><text>However, in the presence of 4-HPA, the 1H-15N TROSY-HSQC spectrum of NadR displayed approximately 140 peaks, as for apo-NadR, i.e. two distinct stable conformations (that might have potentially revealed the molecular asymmetry observed crystallographically) were not notable. Considering the small size, fast diffusion and relatively low binding affinity of 4-HPA, it would not be surprising if the ligand associates and dissociates rapidly on the NMR time scale, resulting in only one set of peaks whose chemical shifts represent the average environment of the bound and unbound states. Interestingly, by cooling the samples to 10°C, we observed that a number of those peaks strongly affected by 4-HPA (and therefore likely to be in the ligand-binding site) demonstrated evidence of peak splitting, i.e. a tendency to become two distinct peaks rather than one single peak (Fig 6B and 6C). These doubled peaks may therefore reveal that the cooler temperature partially trapped the existence in solution of two distinct states, in presence or absence of 4-HPA, with minor conformational differences occurring at least in proximity to the binding pocket. Although more comprehensive NMR experiments and full chemical shift assignment of the spectra would be required to precisely define this multi-state behavior, the NMR data clearly demonstrate that NadR exhibits conformational flexibility which is modulated by 4-HPA in solution.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>34376</offset><text>Apo-NadR structures reveal intrinsic conformational flexibility</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>34440</offset><text>The apo-NadR crystal structure contained two homodimers in the asymmetric unit (chains A+B and chains C+D). Upon overall structural superposition, these dimers revealed a few minor differences in the α6 helix (a major component of the dimer interface) and the helices α4-α5 (the DNA binding region), and an rmsd of 1.55Å (Fig 7A). Similarly, the entire holo-homodimer could be closely superposed onto each of the apo-homodimers, showing rmsd values of 1.29Å and 1.31Å, and with more notable differences in the α6 helix positions (Fig 7B). The slightly larger rmsd between the two apo-homodimers, rather than between apo- and holo-homodimers, further indicate that apo-NadR possesses a notable degree of intrinsic conformational flexibility.</text></passage><passage><infon key="file">ppat.1005557.g007.jpg</infon><infon key="id">ppat.1005557.g007</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>35197</offset><text>Overall apo- and holo-NadR structures are similar.</text></passage><passage><infon key="file">ppat.1005557.g007.jpg</infon><infon key="id">ppat.1005557.g007</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>35248</offset><text>
+(A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. Here, larger differences are observed in the α6 helices (top).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>35520</offset><text>4-HPA stabilizes concerted conformational changes in NadR that prevent DNA-binding</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>35603</offset><text>To further investigate the conformational rearrangements of NadR, we performed local structural alignments using only a subset of residues in the DNA-binding helix (α4). By selecting and aligning residues Arg64-Ala77 of one α4 helix per dimer, superposition of the holo-homodimer onto the two apo-homodimers revealed differences in the monomer conformations of each structure. While one monomer from each structure was closely superimposable (Fig 8A, left side), the second monomer displayed quite large differences (Fig 8A, right side). Most notably, the position of the DNA-binding helix α4 shifted by as much as 6 Å (Fig 8B). Accordingly, helix α4 was also found to be one of the most dynamic regions in previous HDX-MS analyses of apo-NadR in solution.</text></passage><passage><infon key="file">ppat.1005557.g008.jpg</infon><infon key="id">ppat.1005557.g008</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>36376</offset><text>Structural comparisons of NadR and modelling of interactions with DNA.</text></passage><passage><infon key="file">ppat.1005557.g008.jpg</infon><infon key="id">ppat.1005557.g008</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>36447</offset><text>
+(A) The holo-homodimer structure is shown as green and blue cartoons, for chain A and B, respectively, while the two homodimers of apo-NadR are both cyan and pale blue for chains A/C and B/D, respectively. The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). The α4 helices aligned closely, Cα rmsd 0.2Å for 14 residues. (B) The relative positions of the α4 helices of the 4-HPA-bound holo homodimer chain B (blue), and of apo homodimers AB and CD (showing chains B and D) in pale blue. Dashes indicate the Ala77 Cα atoms, in the most highly shifted region of the ‘non-fixed’ α4 helix. (C) The double-stranded DNA molecule (grey cartoon) from the OhrR-ohrA complex is shown after superposition with NadR, to highlight the expected positions of the NadR α4 helices in the DNA major grooves. The proteins share ~30% amino acid sequence identity. For clarity, only the α4 helices are shown in panels (B) and (C). (D) Upon comparison with the experimentally-determined OhrR:ohrA structure (grey), the α4 helix of holo-NadR (blue) is shifted ~8Å out of the major groove.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>37749</offset><text>However, structural comparisons revealed that the shift of holo-NadR helix α4 induced by the presence of 4-HPA was also accompanied by several changes at the holo dimer interface, while such extensive structural differences were not observed in the apo dimer interfaces, particularly notable when comparing the α6 helices (S3 Fig). In summary, compared to ligand-stabilized holo-NadR, apo-NadR displayed an intrinsic flexibility focused in the DNA-binding region. This was also evident in the greater disorder (i.e. less well-defined electron density) in the β1-β2 loops of the apo dimers (density for 16 residues per dimer was missing) compared to the holo dimer (density for only 3 residues was missing).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>38470</offset><text>In holo-NadR, the distance separating the two DNA-binding α4 helices was 32 Å, while in apo-NadR it was 29 Å for homodimer AB, and 34 Å for homodimer CD (Fig 8C). Thus, the apo-homodimer AB presented the DNA-binding helices in a conformation similar to that observed in the protein:DNA complex of OhrR:ohrA from Bacillus subtilis (Fig 8C). Interestingly, OhrR contacts ohrA across 22 base pairs (bp), and similarly the main NadR target sites identified in the nadA promoter (the operators Op I and Op II) both span 22 bp. Pairwise superpositions showed that the NadR apo-homodimer AB was the most similar to OhrR (rmsd 2.6 Å), while the holo-homodimer was the most divergent (rmsd 3.3 Å) (Fig 8C). Assuming the same DNA-binding mechanism is used by OhrR and NadR, the apo-homodimer AB seems ideally pre-configured for DNA binding, while 4-HPA appeared to stabilize holo-NadR in a conformation poorly suited for DNA binding. Specifically, in addition to the different inter-helical translational distances, the α4 helices in the holo-NadR homodimer were also reoriented, resulting in movement of α4 out of the major groove, by up to 8Å, and presumably preventing efficient DNA binding in the presence of 4-HPA (Fig 8D). When aligned with OhrR, the apo-homodimer CD presented yet another different intermediate conformation (rmsd 2.9Å), apparently not ideally pre-configured for DNA binding, but which in solution can presumably readily adopt the AB conformation due to the intrinsic flexibility described above.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>39993</offset><text>NadR residues His7, Ser9, Asn11 and Phe25 are essential for regulation of NadA expression in vivo </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>40092</offset><text>While previous studies had correctly suggested the involvement of several NadR residues in ligand binding, the crystal structures presented here revealed additional residues with previously unknown roles in dimerization and/or binding to 4-HPA. To explore the functional involvement of these residues, we characterized the behavior of four new NadR mutants (H7A, S9A, N11A and F25A) in an in vivo assay using the previously described MC58-Δ1843 nadR-null mutant strain, which was complemented either by wild-type nadR or by the nadR mutants. NadA protein abundance levels were assessed by Western blotting to evaluate the ability of the NadR mutants to repress the nadA promoter, in the presence or absence of 4-HPA. The nadR H7A, S9A and F25A complemented strains showed hyper-repression of nadA expression in vivo, i.e. these mutants repressed nadA more efficiently than the NadR WT protein, either in the presence or absence of 4-HPA, while complementation with wild-type nadR resulted in high production of NadA only in the presence of 4-HPA (Fig 9). Interestingly, and on the contrary, the nadR N11A complemented strain showed hypo-repression (i.e. exhibited high expression of nadA both in absence and presence of 4-HPA). This mutagenesis data revealed that NadR residues His7, Ser9, Asn11 and Phe25 play key roles in the ligand-mediated regulation of NadR; they are each involved in the controlled de-repression of the nadA promoter and synthesis of NadA in response to 4-HPA in vivo.</text></passage><passage><infon key="file">ppat.1005557.g009.jpg</infon><infon key="id">ppat.1005557.g009</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>41588</offset><text>Structure-based point mutations shed light on ligand-induced regulation of NadR.</text></passage><passage><infon key="file">ppat.1005557.g009.jpg</infon><infon key="id">ppat.1005557.g009</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>41669</offset><text>Western blot analyses of wild-type (WT) strain (lanes 1–2) or isogenic nadR knockout strains (ΔNadR) complemented to express the indicated NadR WT or mutant proteins (lanes 3–12) or not complemented (lanes 13–14), grown in the presence (even lanes) or absence (odd lanes) of 5mM 4-HPA, showing NadA and NadR expression. Complementation of ΔNadR with WT NadR enables induction of nadA expression by 4-HPA. The H7A, S9A and F25A mutants efficiently repress nadA expression but are less ligand-responsive than WT NadR. The N11A mutant does not efficiently repress nadA expression either in presence or absence of 4-HPA. (The protein abundance levels of the meningococcal factor H binding protein (fHbp) were used as a gel loading control).</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>42414</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>42425</offset><text>NadA is a surface-exposed meningococcal protein contributing to pathogenesis, and is one of three main antigens present in the vaccine Bexsero. A detailed understanding of the in vitro repression of nadA expression by the transcriptional regulator NadR is important, both because it is a relevant disease-related model of how small-molecule ligands can regulate MarR family proteins and thereby impact bacterial virulence, and because nadA expression levels are linked to the prediction of vaccine coverage. The repressive activity of NadR can be relieved by hydroxyphenylacetate (HPA) ligands, and HDX-MS studies previously indicated that 4-HPA stabilizes dimeric NadR in a configuration incompatible with DNA binding. Despite these and other studies, the molecular mechanisms by which ligands regulate MarR family proteins are relatively poorly understood and likely differ depending on the specific ligand. Given the importance of NadR-mediated regulation of NadA levels in the contexts of meningococcal pathogenesis, we sought to characterize NadR, and its interaction with ligands, at atomic resolution.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>43534</offset><text>Firstly, we confirmed that NadR is dimeric in solution and demonstrated that it retains its dimeric state in the presence of 4-HPA, indicating that induction of a monomeric status is not the manner by which 4-HPA regulates NadR. These observations were in agreement with (i) a previous study of NadR performed using SEC and mass spectrometry, and (ii) crystallographic studies showing that several MarR homologues are dimeric. We also used structure-guided site-directed mutagenesis to identify an important conserved residue, Leu130, which stabilizes the NadR dimer interface, knowledge of which may also inform future studies to explore the regulatory mechanisms of other MarR family proteins. Secondly, we assessed the thermal stability and unfolding of NadR in the presence or absence of ligands. All DSC profiles showed a single peak, suggesting that a single unfolding event simultaneously disrupted the dimer and the monomer. HPA ligands specifically increased the stability of NadR. The largest effects were induced by the naturally-occurring compounds 4-HPA and 3Cl,4-HPA, which, in SPR assays, were found to bind NadR with KD values of 1.5 mM and 1.1 mM, respectively. Although these NadR/HPA interactions appeared rather weak, their distinct affinities and specificities matched their in vitro effects and their biological relevance appears similar to previous proposals that certain small molecules, including some antibiotics, in the millimolar concentration range may be broad inhibitors of MarR family proteins. Indeed, 4-HPA is found in human saliva and 3Cl,4-HPA is produced during inflammatory processes, suggesting that these natural ligands are encountered by N. meningitidis in the mucosa of the oropharynx during infections. It is also possible that NadR responds to currently unidentified HPA analogues. Indeed, in the NadR/4-HPA complex there was a water molecule close to the carboxylate group and also a small unfilled tunnel ~5Å long, both factors suggesting that alternative larger ligands could occupy the pocket. It is conceivable that such putative ligands may establish different bonding networks, potentially binding in a 2:2 ratio, rather than the 1:2 ratio observed herein. The ability to respond to various ligands might enable NadR in vivo to orchestrate multiple response mechanisms and modulate expression of genes other than nadA. Ultimately, confirmation of the relevance of each ligand will require a deeper understanding of the available concentration in vivo in the host niche during bacterial colonization and inflammation.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>46104</offset><text>Here, we determined the first crystal structures of apo-NadR and holo-NadR. These experimentally-determined structures enabled a new detailed characterization of the ligand-binding pocket. In holo-NadR, 4-HPA interacted directly with at least 11 polar and hydrophobic residues. Several, but not all, of these interactions were predicted previously by homology modelling combined with ligand docking in silico. Subsequently, we established the functional importance of His7, Ser9, Asn11 and Phe25 in the in vitro response of meningococcus to 4-HPA, via site-directed mutagenesis. More unexpectedly, the crystal structure revealed that only one molecule of 4-HPA was bound per NadR dimer. We confirmed this stoichiometry in solution using SPR methods. We also used heteronuclear NMR spectroscopy to detect substantial conformational changes of NadR occurring in solution upon addition of 4-HPA. Moreover, NMR spectra at 10°C suggested the existence of two distinct conformations of NadR in the vicinity of the ligand-binding pocket. More powerfully, our unique crystallographic observation of this ‘occupied vs unoccupied site’ asymmetry in the NadR/4-HPA interaction is, to our knowledge, the first example reported for a MarR family protein. Structural analyses suggested that ‘inward’ side chain positions of Met22, Phe25 and especially Arg43 precluded binding of a second ligand molecule. Such a mechanism indicates negative cooperativity, which may enhance the ligand-responsiveness of NadR.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>47608</offset><text>Comparisons of the NadR/4-HPA complex with available MarR family/salicylate complexes revealed that 4-HPA has a previously unobserved binding mode. Briefly, in the M. thermoautotrophicum MTH313 dimer, one molecule of salicylate binds in the pocket of each monomer, though with two rather different positions and orientations, only one of which (site-1) is thought to be biologically relevant (Fig 10A). In the S. tokodaii protein ST1710, salicylate binds to the same position in each monomer of the dimer, in a site equivalent to the putative biologically relevant site of MTH313 (Fig 10B). Unlike other MarR family proteins which revealed multiple ligand binding interactions, we observed only 1 molecule of 4-HPA bound to NadR, suggesting a more specific and less promiscuous interaction. In NadR, the single molecule of 4-HPA binds in a position distinctly different from the salicylate binding site: translated by &gt; 10 Å and with a 180° inverted orientation (Fig 10C).</text></passage><passage><infon key="file">ppat.1005557.g010.jpg</infon><infon key="id">ppat.1005557.g010</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>48583</offset><text>NadR shows a ligand binding site distinct from other MarR homologues.</text></passage><passage><infon key="file">ppat.1005557.g010.jpg</infon><infon key="id">ppat.1005557.g010</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>48653</offset><text>
+(A) A structural alignment of MTH313 chains A and B shows that salicylate is bound in distinct locations in each monomer; site-1 (thought to be the biologically relevant site) and site-2 differ by ~7Å (indicated by black dotted line) and also by ligand orientation. (B) A structural alignment of MTH313 chain A and ST1710 (pink) (Cα rmsd 2.3Å), shows that they bind salicylate in equivalent sites (differing by only ~3Å) and with the same orientation. (C) Addition of holo-NadR (chain B, blue) to the alignment reveals that bound 4-HPA differs in position by &gt; 10 Å compared to salicylate, and adopts a novel orientation.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>49281</offset><text>Interestingly, a crystal structure was previously reported for a functionally-uncharacterized meningococcal homologue of NadR, termed NMB1585, which shares 16% sequence identity with NadR. The two structures can be closely aligned (rmsd 2.3 Å), but NMB1585 appears unsuited for binding HPAs, since its corresponding ‘pocket’ region is occupied by several bulky hydrophobic side chains. It can be speculated that MarR family members have evolved separately to engage distinct signaling molecules, thus enabling bacteria to use the overall conserved MarR scaffold to adapt and respond to diverse changing environmental stimuli experienced in their natural niches. Alternatively, it is possible that other MarR homologues (e.g. NMB1585) may have no extant functional binding pocket and thus may have lost the ability to respond to a ligand, acting instead as constitutive DNA-binding regulatory proteins.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>50188</offset><text>The apo-NadR crystal structures revealed two dimers with slightly different conformations, most divergent in the DNA-binding domain. It is not unusual for a crystal structure to reveal multiple copies of the same protein in very slightly different conformations, which are likely representative of the lowest-energy conformations sampled by the dynamic ensemble of molecular states occurring in solution, and which likely have only small energetic differences, as described previously for MexR (a MarR protein) or more recently for the solute-binding protein FhuD2. Further, the holo-NadR structure was overall more different from the two apo-NadR structures (rmsd values ~1.3Å), suggesting that the ligand selected and stabilized yet another conformation of NadR. These observations suggest that 4-HPA, and potentially other similar ligands, can shift the molecular equilibrium, changing the energy barriers that separate active and inactive states, and stabilizing the specific conformation of NadR poorly suited to bind DNA.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>51217</offset><text>Comparisons of the apo- and holo-NadR structures revealed that the largest differences occurred in the DNA-binding helix α4. The shift of helix α4 in holo-NadR was also accompanied by rearrangements at the dimer interface, involving helices α1, α5, and α6, and this holo-form appeared poorly suited for DNA-binding when compared with the known OhrR:ohrA complex. While some flexibility of helix α4 was also observed in the two apo-structures, concomitant changes in the dimer interfaces were not observed, possibly due to the absence of ligand. One of the two conformations of apo-NadR appeared ideally suited for DNA-binding. Overall, these analyses suggest that the apo-NadR dimer has a pre-existing equilibrium that samples a variety of conformations, some of which are compatible with DNA binding. This intrinsically dynamic nature underlies the possibility for different conformations to inter-convert or to be preferentially selected by a regulatory ligand, as generally described in the ‘conformational selection’ model for protein-ligand interactions (the Monod-Wyman-Changeux model), rather than an ‘induced fit’ model (Koshland-Nemethy-Filmer). The noted flexibility may also explain how NadR can adapt to bind various DNA target sequences with slightly different structural features. Subsequently, upon ligand binding, holo-NadR adopts a structure less suited for DNA-binding and this conformation is selected and stabilized by a network of protein-ligand interactions and concomitant rearrangements at the NadR holo dimer interface. In an alternative and less extensive manner, the binding of two salicylate molecules to the M. thermoautotrophicum protein MTH313 appeared to induce large changes in the wHTH domain, which was associated with reduced DNA-binding activity.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>53025</offset><text>Here we have presented two new crystal structures of the transcription factor, NadR, which regulates expression of the meningococcal surface protein, virulence factor and vaccine antigen NadA. Detailed structural analyses provided a molecular explanation for the ligand-responsive regulation by NadR on the majority of the promoters of meningococcal genes regulated by NadR, including nadA. Intriguingly, NadR exhibits a reversed regulatory mechanism on a second class of promoters, including mafA of the multiple adhesin family–i.e. NadR represses these genes in the presence but not absence of 4-HPA. The latter may influence the surface abundance or secretion of maf proteins, an emerging class of highly conserved meningococcal putative adhesins and toxins with many important roles. Further work is required to investigate how the two different promoter types influence the ligand-responsiveness of NadR during bacterial infection and may provide insights into the regulatory mechanisms occurring during these host-pathogen interactions. Ultimately, knowledge of the ligand-dependent activity of NadR will continue to deepen our understanding of nadA expression levels, which influence meningococcal pathogenesis.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>54246</offset><text>Materials and Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>54268</offset><text>Bacterial strains, culture conditions and mutant generation</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>54328</offset><text>In this study we used N. meningitidis MC58 wild type strain and related mutant derivatives. The MC58 isolate was kindly provided to us by Professor E. Richard Moxon, University of Oxford, UK, and was previously submitted to the Meningococcal Reference Laboratory, Manchester, UK. Strains were routinely cultured, stocked, and transformed as described previously. To generate N. meningitidis MC58 mutant strains expressing only the amino acid substituted forms of NadR, plasmids containing the sequence of nadR mutated to insert alanine codons to replace His7, Ser9, Asn11 or Phe25 were constructed using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene). The nadR gene (also termed NMB1843) was mutated in the pComEry-1843 plasmid using couples of mutagenic primers (forward and reverse). The resulting plasmids were named pComEry-1843H7A, -1843S9A, -1843N11A or -1843F25A, and contained a site-directed mutant allele of the nadR gene, in which the selected codons were respectively substituted by a GCG alanine codon, and were used for transformation of the MC-Δ1843 strain. Total lysates from single colonies of all transformants were used as a template for PCR analysis to confirm the correct insertion by double homologous recombinant event. When indicated, bacterial strains were grown in presence of 5 mM 4-HPA (MW 152, Sigma-Aldrich).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>55688</offset><text>Molecular cloning</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>55706</offset><text>The preparation of the expression construct enabling production of soluble NadR with an N-terminal His-tag followed by a thrombin cleavage site (MGSSHHHHHHSSGLVPR↓GSH-) (where the arrow indicates the cleavage site) and then NadR residues M1-S146 (Uniprot code Q7DD70), and methods to generate site-directed mutants, were described previously. Briefly, site-directed mutagenesis was performed using two overlapping primers containing the desired mutation to amplify pET15b containing several NadR variants. (Full oligonucleotide sequences of primers are available upon request). 1–10 ng of plasmid DNA template were amplified using Kapa HiFi DNA polymerase (Kapa Biosystems) and with the following cycling conditions: 98°C for 5 min, 15 cycles of (98°C for 30 s, 60°C for 30 s, 72°C for 6 min) followed by a final extension of 10 min at 72°C. Residual template DNA was digested by 30 min incubation with FastDigest DpnI (Thermo Scientific) at 37°C and 1 μl of this reaction was used to transform E. coli DH5α competent cells. The full recombinant tagged NadR protein generated contained 166 residues, with a theoretical MW of 18746, while after thrombin-cleavage the untagged protein contained 149 residues, with a theoretical MW of 16864.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>56957</offset><text>Protein production and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>56993</offset><text>The NadR expression constructs (wild-type or mutant clones) were transformed into E. coli BL21 (DE3) cells and were grown at 37°C in Luria-Bertani (LB) medium supplemented with 100 μg/mL ampicillin, until an OD600 of 0.5 was reached. Target protein production was induced by the addition of 1 mM IPTG followed by incubation with shaking overnight at 21°C. For production of the selenomethionine (SeMet) derivative form of NadR for crystallization studies, essentially the same procedure was followed, but using the E. coli B834 strain grown in a modified M9 minimal medium supplemented with 40 mg/L L-SeMet. For production of 15N-labeled NadR for NMR analyses, the EnPresso B Defined Nitrogen-free medium (Sigma-Aldrich) was used; in brief, BL21 (DE3) cells were grown in BioSilta medium at 30°C for 30 h, and production of the 15N-labeled NadR was enabled by the addition of 2.5 g/L 15NH4Cl and further incubation for 2 days.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>57924</offset><text>Cells were harvested by centrifugation (6400 g, 30 min, 4°C), resuspended in 20 mM HEPES pH 8.0, 300 mM NaCl, 20 mM imidazole, and were lysed by sonication (Qsonica Q700). Cell lysates were clarified by centrifugation at 2800 g for 30 min, and the supernatant was filtered using a 0.22 μm membrane (Corning filter system) prior to protein purification. NadR was purified by affinity chromatography using an AKTA purifier (GE Healthcare). All steps were performed at room temperature (18–26°C), unless stated otherwise. The filtered supernatant was loaded onto an Ni-NTA resin (5 mL column, GE Healthcare), and NadR was eluted using 4 steps of imidazole at 20, 30, 50 and 250 mM concentration, at a flow rate of 5 mL/min. Eluted fractions were examined by reducing and denaturing SDS-PAGE analysis. Fractions containing NadR were identified by a band migrating at ~17 kDa, and were pooled. The N-terminal 6-His tag was removed enzymatically using the Thrombin CleanCleave Kit (Sigma-Aldrich). Subsequently, the sample was reloaded on the Ni-NTA resin to capture the free His tag (or unprocessed tagged protein), thus allowing elution in the column flow-through of tagless NadR protein, which was used in all subsequent studies. The NadR sample was concentrated and loaded onto a HiLoad Superdex 75 (16/60) preparative size-exclusion chromatography (SEC) column equilibrated in buffer containing 20 mM HEPES pH 8.0, 150 mM NaCl, at a flow-rate of 1 mL/min. NadR protein was collected and the final yield of purified protein obtained from 0.5 L LB growth medium was approximately 8 mg (~2 mg protein per g wet biomass). Samples were used immediately for crystallization or analytical experiments, or were frozen for storage at -20°C.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>59661</offset><text>SE-HPLC/MALLS analyses</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>59684</offset><text>MALLS analyses were performed online with SE-HPLC using a Dawn TREOS MALLS detector (Wyatt Corp., Santa Barbara, CA, USA) and an incident laser wavelength of 658 nm. The intensity of the scattered light was measured at 3 angles simultaneously. Data analysis was performed using the Astra V software (Wyatt) to determine the weighted-average absolute molecular mass (MW), the polydispersity index (MW/Mn) and homogeneity (Mz/Mn) for each oligomer present in solution. Normalization of the MALLS detectors was performed in each analytical session by use of bovine serum albumin.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>60261</offset><text>Differential scanning calorimetry</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>60295</offset><text>The thermal stability of NadR proteins was assessed by differential scanning calorimetry (DSC) using a MicroCal VP-Capillary DSC instrument (GE Healthcare). NadR samples were prepared at a protein concentration of 0.5 mg/mL (~30 μM) in buffer containing 20 mM HEPES, 300 mM NaCl, pH 7.4, with or without 6 mM HPA or salicylate. The DSC temperature scan ranged from 10°C to 110°C, with a thermal ramping rate of 200°C per hour and a 4 second filter period. Data were analyzed by subtraction of the reference data for a sample containing buffer only, using the Origin 7 software. All experiments were performed in triplicate, and mean values of the melting temperature (Tm) were determined.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>60988</offset><text>Surface plasmon resonance (SPR)</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_3</infon><offset>61020</offset><text>Determination of equilibrium dissociation constant, KD </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>61076</offset><text>Surface plasmon resonance binding analyses were performed using a Biacore T200 instrument (GE Healthcare) equilibrated at 25°C. The ligand (NadR) was covalently immobilized by amine-coupling on a CM-5 sensor chip (GE Healthcare), using 20 μg/mL purified protein in 10 mM sodium acetate buffer pH 5, injected at 10 μl/min for 120 s until ~9000 response units (RU) were captured. A high level of ligand immobilization was required due to the small size of the analytes. An unmodified surface was used as the reference channel. Titrations with analytes (HPAs or salicylate) were performed with a flow-rate of 30 μl/min, injecting the compounds in a concentration range of 10 μM to 20 mM, using filtered running buffer containing Phosphate Buffered Saline (PBS) with 0.05% Tween-20, pH 7.4. Following each injection, sensor chip surfaces were regenerated with a 30-second injection of 10 mM Glycine pH 2.5. Each titration series contained 20 analyte injections and was performed in triplicate. Titration experiments with long injection phases (&gt; 15 mins) were used to enable steady-state analyses. Data were analyzed using the BIAcore T200 evaluation software and the steady-state affinity model. A buffer injection was subtracted from each curve, and reference sensorgrams were subtracted from experimental sensorgrams to yield curves representing specific binding. The equilibrium dissociation constant, KD, was determined from the plot of RUeq against analyte concentration (S2 Fig), as described previously. Determination of binding stoichiometry: From each plot of RUeq against analyte concentration, obtained from triplicate experiments, the Rmax value (maximum analyte binding capacity of the surface) was extrapolated from the experimental data (S2 Fig). Stoichiometry was calculated using the molecular weight of dimeric NadR as ligand molecule (MWligand) and the molecular weights of the HPA analyte molecules (MWanalyte), and the following equation:  where Rligand is recorded directly from the sensorgram during ligand immobilization prior to the titration series, as described previously. The stoichiometry derived therefore represented the number of HPA molecules bound to one dimeric NadR protein.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>63290</offset><text>Crystallization of NadR in the presence or absence of 4-HPA</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>63350</offset><text>Purified NadR was concentrated to 2.7 mg/mL (~160 μM) using a centrifugal concentration device (Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-10 membrane with cut-off size 10 kDa; Millipore) running at 600 g in a bench top centrifuge (Thermo Scientific IEC CL40R) refrigerated at 2–8°C. To prepare holo-NadR samples, HPA ligands were added at a 200-fold molar excess prior to the centrifugal concentration step. The concentrated holo- or apo-NadR was subjected to crystallization trials performed in 96-well low-profile Intelli-Plates (Art Robbins) or 96-well low-profile Greiner crystallization plates, using a nanodroplet sitting-drop vapour-diffusion format and mixing equal volumes (200 nL) of protein samples and crystallization buffers using a Gryphon robot (Art Robbins). Crystallization trays were incubated at 20°C. Crystals of apo-NadR were obtained in 50% PEG 3350 and 0.13 M di-Ammonium hydrogen citrate, whereas crystals of SeMet–NadR in complex with 4-HPA grew in condition H4 of the Morpheus screen (Molecular Dimensions), which contains 37.5% of the pre-mixed precipitant stock MPD_P1K_PEG 3350, buffer system 1 and 0.1 M amino acids, at pH 6.5. All crystals were mounted in cryo-loops using 10% ethylene glycol or 10% glycerol as cryo-protectant before cooling to 100 K for data collection.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>64673</offset><text>X-ray diffraction data collection and structure determination</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>64735</offset><text>X-ray diffraction data from crystals of apo-NadR and SeMet–NadR/4-HPA were collected on beamline PXII-X10SA of the Swiss Light Source (SLS) at the Paul Scherrer Institut (PSI), Villigen, Switzerland. All diffraction data were processed with XDS and programs from the CCP4 suite. Crystals of apo-NadR and 4-HPA-bound SeMet-NadR belonged to space group P43 21 2 (see Table 2). Apo-NadR crystals contained four molecules (two dimers) in the asymmetric unit (Matthews coefficient 2.25 Å3 Da−1, for a solvent content of 45%), while crystals of SeMet–NadR/4-HPA contained two molecules (one dimer) in the asymmetric unit (Matthews coefficient 1.98 Å3 Da−1, for a solvent content of 38%). In solving the holo-NadR structure, an initial and marginal molecular replacement (MR) solution was obtained using as template search model the crystal structure of the transcriptional regulator PA4135 (PBD entry 2FBI), with which NadR shares ~54% sequence identity. This solution was combined with SAD data to aid identification of two selenium sites in NadR, using autosol in phenix and this allowed generation of high-quality electron density maps that were used to build and refine the structure of the complex. Electron densities were clearly observed for almost the entire dimeric holo-NadR protein, except for a new N-terminal residues and residues 88–90 of chain B.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>66103</offset><text>The crystal structure of apo-NadR was subsequently solved by MR in Phaser at 2.7 Å, using the final refined model of SeMet-NadR/4-HPA as the search model. For apo-NadR, electron densities were clearly observed for almost the entire protein, although residues 84–91 of chains A, C, and D, and residues 84–90 of chain B lacked densities suggesting local disorder.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>66470</offset><text>Both structures were refined and rebuilt using phenix and Coot, and structural validation was performed using Molprobity. Data collection and refinement statistics are reported in Table 2. Atomic coordinates of the two NadR structures have been deposited in the Protein Data Bank, with entry codes 5aip (NadR bound to 4-HPA) and 5aiq (apo-NadR). All crystallographic software was compiled, installed and maintained by SBGrid.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>66896</offset><text>NMR spectroscopy</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>66913</offset><text>For heteronuclear NMR experiments, the NadR protein concentration used was 85 μM (~ 1.4 mg/mL) in a solution containing 100 mM sodium phosphate buffer (90% H2O and 10% D2O) and 200 mM NaCl, prepared in the apo-form or in the presence of a 200-fold molar excess of 4-HPA, at pH 6.5. The stability, integrity and dimeric state of the protein in the NMR buffer was confirmed by analytical SEC (Superdex 75, 10/300 column) prior to NMR studies. 1H-15N transverse relaxation-optimized spectroscopy (TROSY)-heteronuclear single quantum coherence (HSQC) experiments on apo-NadR and NadR in the presence of 4-HPA were acquired using an Avance 950 Bruker spectrometer, operating at a proton frequency of 949.2 MHz and equipped with triple resonance cryogenically-cooled probe at two different temperatures (298 K and 283 K). 1H-15N TROSY-HSQC experiments were recorded for 12 h, with a data size of 1024 x 232 points. Spectra were processed using the Bruker TopSpin 2.1 and 3.1 software packages.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>67902</offset><text>Western blot</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>67915</offset><text>Western blot analysis was performed as described previously.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>67976</offset><text>Supporting Information</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>67999</offset><text>References</text></passage><passage><infon key="fpage">1816</infon><infon key="issue">5459</infon><infon key="lpage">20</infon><infon key="name_0">surname:Pizza;given-names:M</infon><infon key="name_1">surname:Scarlato;given-names:V</infon><infon key="name_2">surname:Masignani;given-names:V</infon><infon key="name_3">surname:Giuliani;given-names:MM</infon><infon key="name_4">surname:Arico;given-names:B</infon><infon key="name_5">surname:Comanducci;given-names:M</infon><infon key="pub-id_pmid">10710308</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">287</infon><infon key="year">2000</infon><offset>68010</offset><text>Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing</text></passage><passage><infon key="fpage">966</infon><infon key="issue">7</infon><infon key="lpage">71</infon><infon key="name_0">surname:Bambini;given-names:S</infon><infon key="name_1">surname:De Chiara;given-names:M</infon><infon key="name_2">surname:Muzzi;given-names:A</infon><infon key="name_3">surname:Mora;given-names:M</infon><infon key="name_4">surname:Lucidarme;given-names:J</infon><infon key="name_5">surname:Brehony;given-names:C</infon><infon key="pub-id_doi">10.1128/CVI.00825-13</infon><infon key="pub-id_pmid">24807056</infon><infon key="section_type">REF</infon><infon key="source">Clin Vaccine Immunol</infon><infon key="type">ref</infon><infon key="volume">21</infon><infon key="year">2014</infon><offset>68108</offset><text> Neisseria adhesin A variation and revised nomenclature scheme</text></passage><passage><infon key="fpage">687</infon><infon key="issue">3</infon><infon key="lpage">98</infon><infon key="name_0">surname:Capecchi;given-names:B</infon><infon key="name_1">surname:Adu-Bobie;given-names:J</infon><infon key="name_2">surname:Di Marcello;given-names:F</infon><infon key="name_3">surname:Ciucchi;given-names:L</infon><infon key="name_4">surname:Masignani;given-names:V</infon><infon key="name_5">surname:Taddei;given-names:A</infon><infon key="pub-id_doi">10.1111/j.1365-2958.2004.04423.x</infon><infon key="pub-id_pmid">15660996</infon><infon key="section_type">REF</infon><infon key="source">Molecular microbiology</infon><infon key="type">ref</infon><infon key="volume">55</infon><infon key="year">2005</infon><offset>68171</offset><text> Neisseria meningitidis NadA is a new invasin which promotes bacterial adhesion to and penetration into human epithelial cells</text></passage><passage><infon key="fpage">1445</infon><infon key="issue">11</infon><infon key="lpage">54</infon><infon key="name_0">surname:Comanducci;given-names:M</infon><infon key="name_1">surname:Bambini;given-names:S</infon><infon key="name_2">surname:Brunelli;given-names:B</infon><infon key="name_3">surname:Adu-Bobie;given-names:J</infon><infon key="name_4">surname:Arico;given-names:B</infon><infon key="name_5">surname:Capecchi;given-names:B</infon><infon key="pub-id_pmid">12045242</infon><infon key="section_type">REF</infon><infon key="source">J Exp Med</infon><infon key="type">ref</infon><infon key="volume">195</infon><infon key="year">2002</infon><offset>68298</offset><text>NadA, a novel vaccine candidate of Neisseria meningitidis </text></passage><passage><infon key="fpage">17128</infon><infon key="issue">48</infon><infon key="lpage">33</infon><infon key="name_0">surname:Malito;given-names:E</infon><infon key="name_1">surname:Biancucci;given-names:M</infon><infon key="name_2">surname:Faleri;given-names:A</infon><infon key="name_3">surname:Ferlenghi;given-names:I</infon><infon key="name_4">surname:Scarselli;given-names:M</infon><infon key="name_5">surname:Maruggi;given-names:G</infon><infon key="pub-id_doi">10.1073/pnas.1419686111</infon><infon key="pub-id_pmid">25404323</infon><infon key="section_type">REF</infon><infon key="source">Proceedings of the National Academy of Sciences of the United States of America</infon><infon key="type">ref</infon><infon key="volume">111</infon><infon key="year">2014</infon><offset>68357</offset><text>Structure of the meningococcal vaccine antigen NadA and epitope mapping of a bactericidal antibody</text></passage><passage><infon key="fpage">15</infon><infon key="issue">1</infon><infon key="lpage">30</infon><infon key="name_0">surname:O'Ryan;given-names:M</infon><infon key="name_1">surname:Stoddard;given-names:J</infon><infon key="name_2">surname:Toneatto;given-names:D</infon><infon key="name_3">surname:Wassil;given-names:J</infon><infon key="name_4">surname:Dull;given-names:PM</infon><infon key="pub-id_doi">10.1007/s40265-013-0155-7</infon><infon key="pub-id_pmid">24338083</infon><infon key="section_type">REF</infon><infon key="source">Drugs</infon><infon key="type">ref</infon><infon key="volume">74</infon><infon key="year">2014</infon><offset>68456</offset><text>A multi-component meningococcal serogroup B vaccine (4CMenB): the clinical development program</text></passage><passage><infon key="fpage">1054</infon><infon key="issue">4</infon><infon key="lpage">67</infon><infon key="name_0">surname:Schielke;given-names:S</infon><infon key="name_1">surname:Huebner;given-names:C</infon><infon key="name_2">surname:Spatz;given-names:C</infon><infon key="name_3">surname:Nagele;given-names:V</infon><infon key="name_4">surname:Ackermann;given-names:N</infon><infon key="name_5">surname:Frosch;given-names:M</infon><infon key="pub-id_doi">10.1111/j.1365-2958.2009.06710.x</infon><infon key="pub-id_pmid">19400792</infon><infon key="section_type">REF</infon><infon key="source">Molecular microbiology</infon><infon key="type">ref</infon><infon key="volume">72</infon><infon key="year">2009</infon><offset>68551</offset><text>Expression of the meningococcal adhesin NadA is controlled by a transcriptional regulator of the MarR family</text></passage><passage><infon key="fpage">e56097</infon><infon key="issue">2</infon><infon key="name_0">surname:Cloward;given-names:JM</infon><infon key="name_1">surname:Shafer;given-names:WM</infon><infon key="pub-id_doi">10.1371/journal.pone.0056097</infon><infon key="pub-id_pmid">23409129</infon><infon key="section_type">REF</infon><infon key="source">PloS one</infon><infon key="type">ref</infon><infon key="volume">8</infon><infon key="year">2013</infon><offset>68660</offset><text>. MtrR control of a transcriptional regulatory pathway in Neisseria meningitidis that influences expression of a gene (nadA) encoding a vaccine candidate</text></passage><passage><infon key="fpage">e1000710</infon><infon key="issue">12</infon><infon key="name_0">surname:Metruccio;given-names:MM</infon><infon key="name_1">surname:Pigozzi;given-names:E</infon><infon key="name_2">surname:Roncarati;given-names:D</infon><infon key="name_3">surname:Berlanda Scorza;given-names:F</infon><infon key="name_4">surname:Norais;given-names:N</infon><infon key="name_5">surname:Hill;given-names:SA</infon><infon key="pub-id_doi">10.1371/journal.ppat.1000710</infon><infon key="pub-id_pmid">20041170</infon><infon key="section_type">REF</infon><infon key="source">PLoS Pathog</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2009</infon><offset>68814</offset><text>A novel phase variation mechanism in the meningococcus driven by a ligand-responsive repressor and differential spacing of distal promoter elements</text></passage><passage><infon key="fpage">460</infon><infon key="issue">2</infon><infon key="lpage">74</infon><infon key="name_0">surname:Fagnocchi;given-names:L</infon><infon key="name_1">surname:Pigozzi;given-names:E</infon><infon key="name_2">surname:Scarlato;given-names:V</infon><infon key="name_3">surname:Delany;given-names:I</infon><infon key="pub-id_doi">10.1128/JB.06161-11</infon><infon key="pub-id_pmid">22081399</infon><infon key="section_type">REF</infon><infon key="source">J Bacteriol</infon><infon key="type">ref</infon><infon key="volume">194</infon><infon key="year">2012</infon><offset>68962</offset><text>In the NadR Regulon, Adhesins and Diverse Meningococcal Functions Are Regulated in Response to Signals in Human Saliva</text></passage><passage><infon key="fpage">e1004592</infon><infon key="issue">1</infon><infon key="name_0">surname:Jamet;given-names:A</infon><infon key="name_1">surname:Jousset;given-names:AB</infon><infon key="name_2">surname:Euphrasie;given-names:D</infon><infon key="name_3">surname:Mukorako;given-names:P</infon><infon key="name_4">surname:Boucharlat;given-names:A</infon><infon key="name_5">surname:Ducousso;given-names:A</infon><infon key="pub-id_doi">10.1371/journal.ppat.1004592</infon><infon key="pub-id_pmid">25569427</infon><infon key="section_type">REF</infon><infon key="source">PLoS Pathog</infon><infon key="type">ref</infon><infon key="volume">11</infon><infon key="year">2015</infon><offset>69081</offset><text>A new family of secreted toxins in pathogenic Neisseria species</text></passage><passage><infon key="fpage">e01974</infon><infon key="issue">5</infon><infon key="lpage">14</infon><infon key="name_0">surname:Lamelas;given-names:A</infon><infon key="name_1">surname:Harris;given-names:SR</infon><infon key="name_2">surname:Roltgen;given-names:K</infon><infon key="name_3">surname:Dangy;given-names:JP</infon><infon key="name_4">surname:Hauser;given-names:J</infon><infon key="name_5">surname:Kingsley;given-names:RA</infon><infon key="pub-id_doi">10.1128/mBio.01974-14</infon><infon key="pub-id_pmid">25336458</infon><infon key="section_type">REF</infon><infon key="source">mBio</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2014</infon><offset>69145</offset><text>Emergence of a new epidemic Neisseria meningitidis serogroup A Clone in the African meningitis belt: high-resolution picture of genomic changes that mediate immune evasion</text></passage><passage><infon key="fpage">410</infon><infon key="issue">10</infon><infon key="lpage">3</infon><infon key="name_0">surname:Alekshun;given-names:MN</infon><infon key="name_1">surname:Levy;given-names:SB</infon><infon key="pub-id_pmid">10498949</infon><infon key="section_type">REF</infon><infon key="source">Trends Microbiol</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">1999</infon><offset>69317</offset><text>The mar regulon: multiple resistance to antibiotics and other toxic chemicals</text></passage><passage><infon key="fpage">R142</infon><infon key="issue">4</infon><infon key="lpage">3</infon><infon key="name_0">surname:Grove;given-names:A</infon><infon key="pub-id_doi">10.1016/j.cub.2013.01.013</infon><infon key="pub-id_pmid">23428319</infon><infon key="section_type">REF</infon><infon key="source">Curr Biol</infon><infon key="type">ref</infon><infon key="volume">23</infon><infon key="year">2013</infon><offset>69395</offset><text>MarR family transcription factors</text></passage><passage><infon key="fpage">153</infon><infon key="issue">2</infon><infon key="lpage">9</infon><infon key="name_0">surname:Ellison;given-names:DW</infon><infon key="name_1">surname:Miller;given-names:VL</infon><infon key="pub-id_doi">10.1016/j.mib.2006.02.003</infon><infon key="pub-id_pmid">16529980</infon><infon key="section_type">REF</infon><infon key="source">Curr Opin Microbiol</infon><infon key="type">ref</infon><infon key="volume">9</infon><infon key="year">2006</infon><offset>69429</offset><text>Regulation of virulence by members of the MarR/SlyA family</text></passage><passage><infon key="fpage">243</infon><infon key="issue">5</infon><infon key="lpage">54</infon><infon key="name_0">surname:Perera;given-names:IC</infon><infon key="name_1">surname:Grove;given-names:A</infon><infon key="pub-id_doi">10.1093/jmcb/mjq021</infon><infon key="pub-id_pmid">20716550</infon><infon key="section_type">REF</infon><infon key="source">J Mol Cell Biol</infon><infon key="type">ref</infon><infon key="volume">2</infon><infon key="year">2010</infon><offset>69488</offset><text>Molecular mechanisms of ligand-mediated attenuation of DNA binding by MarR family transcriptional regulators</text></passage><passage><infon key="fpage">655</infon><infon key="issue">3</infon><infon key="lpage">67</infon><infon key="name_0">surname:Saridakis;given-names:V</infon><infon key="name_1">surname:Shahinas;given-names:D</infon><infon key="name_2">surname:Xu;given-names:X</infon><infon key="name_3">surname:Christendat;given-names:D</infon><infon key="pub-id_doi">10.1016/j.jmb.2008.01.001</infon><infon key="pub-id_pmid">18272181</infon><infon key="section_type">REF</infon><infon key="source">Journal of molecular biology</infon><infon key="type">ref</infon><infon key="volume">377</infon><infon key="year">2008</infon><offset>69597</offset><text>Structural insight on the mechanism of regulation of the MarR family of proteins: high-resolution crystal structure of a transcriptional repressor from Methanobacterium thermoautotrophicum </text></passage><passage><infon key="fpage">4723</infon><infon key="issue">14</infon><infon key="lpage">35</infon><infon key="name_0">surname:Kumarevel;given-names:T</infon><infon key="name_1">surname:Tanaka;given-names:T</infon><infon key="name_2">surname:Umehara;given-names:T</infon><infon key="name_3">surname:Yokoyama;given-names:S</infon><infon key="pub-id_doi">10.1093/nar/gkp496</infon><infon key="pub-id_pmid">19509310</infon><infon key="section_type">REF</infon><infon key="source">Nucleic acids research</infon><infon key="type">ref</infon><infon key="volume">37</infon><infon key="year">2009</infon><offset>69787</offset><text>ST1710-DNA complex crystal structure reveals the DNA binding mechanism of the MarR family of regulators</text></passage><passage><infon key="fpage">116</infon><infon key="issue">1–3</infon><infon key="lpage">8</infon><infon key="name_0">surname:Takahama;given-names:U</infon><infon key="name_1">surname:Oniki;given-names:T</infon><infon key="name_2">surname:Murata;given-names:H</infon><infon key="pub-id_pmid">11997029</infon><infon key="section_type">REF</infon><infon key="source">FEBS Lett</infon><infon key="type">ref</infon><infon key="volume">518</infon><infon key="year">2002</infon><offset>69891</offset><text>The presence of 4-hydroxyphenylacetic acid in human saliva and the possibility of its nitration by salivary nitrite in the stomach</text></passage><passage><infon key="fpage">560</infon><infon key="issue">2</infon><infon key="lpage">9</infon><infon key="name_0">surname:Fagnocchi;given-names:L</infon><infon key="name_1">surname:Biolchi;given-names:A</infon><infon key="name_2">surname:Ferlicca;given-names:F</infon><infon key="name_3">surname:Boccadifuoco;given-names:G</infon><infon key="name_4">surname:Brunelli;given-names:B</infon><infon key="name_5">surname:Brier;given-names:S</infon><infon key="pub-id_doi">10.1128/IAI.01085-12</infon><infon key="pub-id_pmid">23230289</infon><infon key="section_type">REF</infon><infon key="source">Infect Immun</infon><infon key="type">ref</infon><infon key="volume">81</infon><infon key="year">2013</infon><offset>70022</offset><text>Transcriptional regulation of the nadA gene in Neisseria meningitidis impacts the prediction of coverage of a multicomponent meningococcal serogroup B vaccine</text></passage><passage><infon key="fpage">6738</infon><infon key="issue">34</infon><infon key="lpage">52</infon><infon key="name_0">surname:Brier;given-names:S</infon><infon key="name_1">surname:Fagnocchi;given-names:L</infon><infon key="name_2">surname:Donnarumma;given-names:D</infon><infon key="name_3">surname:Scarselli;given-names:M</infon><infon key="name_4">surname:Rappuoli;given-names:R</infon><infon key="name_5">surname:Nissum;given-names:M</infon><infon key="pub-id_doi">10.1021/bi300656w</infon><infon key="pub-id_pmid">22834735</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry</infon><infon key="type">ref</infon><infon key="volume">51</infon><infon key="year">2012</infon><offset>70181</offset><text>Structural Insight into the Mechanism of DNA-Binding Attenuation of the Neisserial Adhesin Repressor NadR by the Small Natural Ligand 4-Hydroxyphenylacetic Acid</text></passage><passage><infon key="fpage">5456</infon><infon key="issue">12</infon><infon key="lpage">60</infon><infon key="name_0">surname:Martin;given-names:RG</infon><infon key="name_1">surname:Rosner;given-names:JL</infon><infon key="pub-id_pmid">7777530</infon><infon key="section_type">REF</infon><infon key="source">Proceedings of the National Academy of Sciences of the United States of America</infon><infon key="type">ref</infon><infon key="volume">92</infon><infon key="year">1995</infon><offset>70342</offset><text>Binding of purified multiple antibiotic-resistance repressor protein (MarR) to mar operator sequences</text></passage><passage><infon key="fpage">774</infon><infon key="issue">3</infon><infon key="lpage">97</infon><infon key="name_0">surname:Krissinel;given-names:E</infon><infon key="name_1">surname:Henrick;given-names:K</infon><infon key="pub-id_doi">10.1016/j.jmb.2007.05.022</infon><infon key="pub-id_pmid">17681537</infon><infon key="section_type">REF</infon><infon key="source">Journal of molecular biology</infon><infon key="type">ref</infon><infon key="volume">372</infon><infon key="year">2007</infon><offset>70444</offset><text>Inference of macromolecular assemblies from crystalline state</text></passage><passage><infon key="fpage">271</infon><infon key="issue">2</infon><infon key="lpage">7</infon><infon key="name_0">surname:Mistrik;given-names:P</infon><infon key="name_1">surname:Moreau;given-names:F</infon><infon key="name_2">surname:Allen;given-names:JM</infon><infon key="pub-id_doi">10.1016/j.ab.2004.01.022</infon><infon key="pub-id_pmid">15051545</infon><infon key="section_type">REF</infon><infon key="source">Anal Biochem</infon><infon key="type">ref</infon><infon key="volume">327</infon><infon key="year">2004</infon><offset>70506</offset><text>BiaCore analysis of leptin-leptin receptor interaction: evidence for 1:1 stoichiometry</text></passage><passage><infon key="fpage">1</infon><infon key="issue">1</infon><infon key="lpage">17</infon><infon key="name_0">surname:Liang;given-names:J</infon><infon key="name_1">surname:Edelsbrunner;given-names:H</infon><infon key="name_2">surname:Fu;given-names:P</infon><infon key="name_3">surname:Sudhakar;given-names:PV</infon><infon key="name_4">surname:Subramaniam;given-names:S</infon><infon key="pub-id_pmid">9741840</infon><infon key="section_type">REF</infon><infon key="source">Proteins</infon><infon key="type">ref</infon><infon key="volume">33</infon><infon key="year">1998</infon><offset>70593</offset><text>Analytical shape computation of macromolecules: I. Molecular area and volume through alpha shape</text></passage><passage><infon key="fpage">131</infon><infon key="issue">1</infon><infon key="lpage">41</infon><infon key="name_0">surname:Hong;given-names:M</infon><infon key="name_1">surname:Fuangthong;given-names:M</infon><infon key="name_2">surname:Helmann;given-names:JD</infon><infon key="name_3">surname:Brennan;given-names:RG</infon><infon key="pub-id_doi">10.1016/j.molcel.2005.09.013</infon><infon key="pub-id_pmid">16209951</infon><infon key="section_type">REF</infon><infon key="source">Mol Cell</infon><infon key="type">ref</infon><infon key="volume">20</infon><infon key="year">2005</infon><offset>70690</offset><text>Structure of an OhrR-ohrA operator complex reveals the DNA binding mechanism of the MarR family</text></passage><passage><infon key="fpage">19490</infon><infon key="issue">45</infon><infon key="lpage">5</infon><infon key="name_0">surname:Donnelly;given-names:J</infon><infon key="name_1">surname:Medini;given-names:D</infon><infon key="name_2">surname:Boccadifuoco;given-names:G</infon><infon key="name_3">surname:Biolchi;given-names:A</infon><infon key="name_4">surname:Ward;given-names:J</infon><infon key="name_5">surname:Frasch;given-names:C</infon><infon key="pub-id_doi">10.1073/pnas.1013758107</infon><infon key="pub-id_pmid">20962280</infon><infon key="section_type">REF</infon><infon key="source">Proceedings of the National Academy of Sciences of the United States of America</infon><infon key="type">ref</infon><infon key="volume">107</infon><infon key="year">2010</infon><offset>70786</offset><text>Qualitative and quantitative assessment of meningococcal antigens to evaluate the potential strain coverage of protein-based vaccines</text></passage><passage><infon key="fpage">29114</infon><infon key="issue">40</infon><infon key="lpage">21</infon><infon key="name_0">surname:Mani;given-names:AR</infon><infon key="name_1">surname:Ippolito;given-names:S</infon><infon key="name_2">surname:Moreno;given-names:JC</infon><infon key="name_3">surname:Visser;given-names:TJ</infon><infon key="name_4">surname:Moore;given-names:KP</infon><infon key="pub-id_doi">10.1074/jbc.M704270200</infon><infon key="pub-id_pmid">17686770</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">282</infon><infon key="year">2007</infon><offset>70920</offset><text>The metabolism and dechlorination of chlorotyrosine in vivo</text></passage><passage><infon key="fpage">204</infon><infon key="issue">Pt 3</infon><infon key="lpage">9</infon><infon key="name_0">surname:Nichols;given-names:CE</infon><infon key="name_1">surname:Sainsbury;given-names:S</infon><infon key="name_2">surname:Ren;given-names:J</infon><infon key="name_3">surname:Walter;given-names:TS</infon><infon key="name_4">surname:Verma;given-names:A</infon><infon key="name_5">surname:Stammers;given-names:DK</infon><infon key="pub-id_doi">10.1107/S174430910900414X</infon><infon key="pub-id_pmid">19255465</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr Sect F Struct Biol Cryst Commun</infon><infon key="type">ref</infon><infon key="volume">65</infon><infon key="year">2009</infon><offset>70980</offset><text>The structure of NMB1585, a MarR-family regulator from Neisseria meningitidis </text></passage><passage><infon key="fpage">29253</infon><infon key="issue">32</infon><infon key="lpage">9</infon><infon key="name_0">surname:Lim;given-names:D</infon><infon key="name_1">surname:Poole;given-names:K</infon><infon key="name_2">surname:Strynadka;given-names:NC</infon><infon key="pub-id_doi">10.1074/jbc.M111381200</infon><infon key="pub-id_pmid">12034710</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">277</infon><infon key="year">2002</infon><offset>71059</offset><text>Crystal structure of the MexR repressor of the mexRAB-oprM multidrug efflux operon of Pseudomonas aeruginosa </text></passage><passage><infon key="fpage">683</infon><infon key="issue">3</infon><infon key="lpage">93</infon><infon key="name_0">surname:Mariotti;given-names:P</infon><infon key="name_1">surname:Malito;given-names:E</infon><infon key="name_2">surname:Biancucci;given-names:M</infon><infon key="name_3">surname:Lo Surdo;given-names:P</infon><infon key="name_4">surname:Mishra;given-names:RP</infon><infon key="name_5">surname:Nardi-Dei;given-names:V</infon><infon key="pub-id_doi">10.1042/BJ20121426</infon><infon key="pub-id_pmid">23113737</infon><infon key="section_type">REF</infon><infon key="source">The Biochemical journal</infon><infon key="type">ref</infon><infon key="volume">449</infon><infon key="year">2013</infon><offset>71169</offset><text>Structural and functional characterization of the Staphylococcus aureus virulence factor and vaccine candidate FhuD2</text></passage><passage><infon key="fpage">2017</infon><infon key="issue">12</infon><infon key="lpage">31</infon><infon key="name_0">surname:Podkowa;given-names:KJ</infon><infon key="name_1">surname:Briere;given-names:LA</infon><infon key="name_2">surname:Heinrichs;given-names:DE</infon><infon key="name_3">surname:Shilton;given-names:BH</infon><infon key="pub-id_doi">10.1021/bi401349d</infon><infon key="pub-id_pmid">24606332</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry</infon><infon key="type">ref</infon><infon key="volume">53</infon><infon key="year">2014</infon><offset>71286</offset><text>Crystal and solution structure analysis of FhuD2 from Staphylococcus aureus in multiple unliganded conformations and bound to ferrioxamine-B</text></passage><passage><infon key="fpage">19</infon><infon key="name_0">surname:Changeux;given-names:JP</infon><infon key="name_1">surname:Edelstein;given-names:S</infon><infon key="pub-id_doi">10.3410/B3-19</infon><infon key="pub-id_pmid">21941598</infon><infon key="section_type">REF</infon><infon key="source">F1000 biology reports</infon><infon key="type">ref</infon><infon key="volume">3</infon><infon key="year">2011</infon><offset>71427</offset><text>Conformational selection or induced fit? 50 years of debate resolved</text></passage><passage><infon key="fpage">514</infon><infon key="issue">8740</infon><infon key="lpage">7</infon><infon key="name_0">surname:McGuinness;given-names:BT</infon><infon key="name_1">surname:Clarke;given-names:IN</infon><infon key="name_2">surname:Lambden;given-names:PR</infon><infon key="name_3">surname:Barlow;given-names:AK</infon><infon key="name_4">surname:Poolman;given-names:JT</infon><infon key="name_5">surname:Jones;given-names:DM</infon><infon key="pub-id_pmid">1705642</infon><infon key="section_type">REF</infon><infon key="source">Lancet</infon><infon key="type">ref</infon><infon key="volume">337</infon><infon key="year">1991</infon><offset>71496</offset><text>Point mutation in meningococcal por A gene associated with increased endemic disease</text></passage><passage><infon key="fpage">1300</infon><infon key="issue">12</infon><infon key="lpage">5</infon><infon key="name_0">surname:Lo Surdo;given-names:P</infon><infon key="name_1">surname:Bottomley;given-names:MJ</infon><infon key="name_2">surname:Calzetta;given-names:A</infon><infon key="name_3">surname:Settembre;given-names:EC</infon><infon key="name_4">surname:Cirillo;given-names:A</infon><infon key="name_5">surname:Pandit;given-names:S</infon><infon key="pub-id_doi">10.1038/embor.2011.205</infon><infon key="pub-id_pmid">22081141</infon><infon key="section_type">REF</infon><infon key="source">EMBO Rep</infon><infon key="type">ref</infon><infon key="volume">12</infon><infon key="year">2011</infon><offset>71581</offset><text>Mechanistic implications for LDL receptor degradation from the PCSK9/LDLR structure at neutral pH</text></passage><passage><infon key="fpage">125</infon><infon key="issue">Pt 2</infon><infon key="lpage">32</infon><infon key="name_0">surname:Kabsch;given-names:W</infon><infon key="pub-id_doi">10.1107/S0907444909047337</infon><infon key="pub-id_pmid">20124692</infon><infon key="section_type">REF</infon><infon key="source">Acta crystallographica Section D, Biological crystallography</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>71679</offset><text>Xds</text></passage><passage><infon key="fpage">760</infon><infon key="issue">Pt 5</infon><infon key="lpage">3</infon><infon key="pub-id_pmid">15299374</infon><infon key="section_type">REF</infon><infon key="source">Acta crystallographica Section D, Biological crystallography</infon><infon key="type">ref</infon><infon key="volume">50</infon><infon key="year">1994</infon><offset>71683</offset><text>The CCP4 suite: programs for protein crystallography</text></passage><passage><infon key="fpage">213</infon><infon key="issue">Pt 2</infon><infon key="lpage">21</infon><infon key="name_0">surname:Adams;given-names:PD</infon><infon key="name_1">surname:Afonine;given-names:PV</infon><infon key="name_2">surname:Bunkoczi;given-names:G</infon><infon key="name_3">surname:Chen;given-names:VB</infon><infon key="name_4">surname:Davis;given-names:IW</infon><infon key="name_5">surname:Echols;given-names:N</infon><infon key="pub-id_doi">10.1107/S0907444909052925</infon><infon key="pub-id_pmid">20124702</infon><infon key="section_type">REF</infon><infon key="source">Acta crystallographica Section D, Biological crystallography</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>71736</offset><text>PHENIX: a comprehensive Python-based system for macromolecular structure solution</text></passage><passage><infon key="fpage">658</infon><infon key="issue">Pt 4</infon><infon key="lpage">74</infon><infon key="name_0">surname:McCoy;given-names:AJ</infon><infon key="name_1">surname:Grosse-Kunstleve;given-names:RW</infon><infon key="name_2">surname:Adams;given-names:PD</infon><infon key="name_3">surname:Winn;given-names:MD</infon><infon key="name_4">surname:Storoni;given-names:LC</infon><infon key="name_5">surname:Read;given-names:RJ</infon><infon key="pub-id_doi">10.1107/S0021889807021206</infon><infon key="pub-id_pmid">19461840</infon><infon key="section_type">REF</infon><infon key="source">J Appl Crystallogr</infon><infon key="type">ref</infon><infon key="volume">40</infon><infon key="year">2007</infon><offset>71818</offset><text>Phaser crystallographic software</text></passage><passage><infon key="fpage">486</infon><infon key="issue">Pt 4</infon><infon key="lpage">501</infon><infon key="name_0">surname:Emsley;given-names:P</infon><infon key="name_1">surname:Lohkamp;given-names:B</infon><infon key="name_2">surname:Scott;given-names:WG</infon><infon key="name_3">surname:Cowtan;given-names:K</infon><infon key="pub-id_doi">10.1107/S0907444910007493</infon><infon key="pub-id_pmid">20383002</infon><infon key="section_type">REF</infon><infon key="source">Acta crystallographica Section D, Biological crystallography</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>71851</offset><text>Features and development of Coot</text></passage><passage><infon key="fpage">12</infon><infon key="issue">Pt 1</infon><infon key="lpage">21</infon><infon key="name_0">surname:Chen;given-names:VB</infon><infon key="name_1">surname:Arendall;given-names:WB;suffix:3rd</infon><infon key="name_2">surname:Headd;given-names:JJ</infon><infon key="name_3">surname:Keedy;given-names:DA</infon><infon key="name_4">surname:Immormino;given-names:RM</infon><infon key="name_5">surname:Kapral;given-names:GJ</infon><infon key="pub-id_doi">10.1107/S0907444909042073</infon><infon key="pub-id_pmid">20057044</infon><infon key="section_type">REF</infon><infon key="source">Acta crystallographica Section D, Biological crystallography</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>71884</offset><text>MolProbity: all-atom structure validation for macromolecular crystallography</text></passage><passage><infon key="fpage">e01456</infon><infon key="name_0">surname:Morin;given-names:A</infon><infon key="name_1">surname:Eisenbraun;given-names:B</infon><infon key="name_2">surname:Key;given-names:J</infon><infon key="name_3">surname:Sanschagrin;given-names:PC</infon><infon key="name_4">surname:Timony;given-names:MA</infon><infon key="name_5">surname:Ottaviano;given-names:M</infon><infon key="pub-id_doi">10.7554/eLife.01456</infon><infon key="pub-id_pmid">24040512</infon><infon key="section_type">REF</infon><infon key="source">Elife</infon><infon key="type">ref</infon><infon key="volume">2</infon><infon key="year">2013</infon><offset>71961</offset><text>Collaboration gets the most out of software</text></passage><passage><infon key="fpage">72</infon><infon key="issue">Pt 1</infon><infon key="lpage">82</infon><infon key="name_0">surname:Evans;given-names:P</infon><infon key="pub-id_doi">10.1107/S0907444905036693</infon><infon key="pub-id_pmid">16369096</infon><infon key="section_type">REF</infon><infon key="source">Acta crystallographica Section D, Biological crystallography</infon><infon key="type">ref</infon><infon key="volume">62</infon><infon key="year">2006</infon><offset>72005</offset><text>Scaling and assessment of data quality</text></passage><passage><infon key="fpage">D292</infon><infon key="lpage">6</infon><infon key="name_0">surname:de Beer;given-names:TA</infon><infon key="name_1">surname:Berka;given-names:K</infon><infon key="name_2">surname:Thornton;given-names:JM</infon><infon key="name_3">surname:Laskowski;given-names:RA</infon><infon key="pub-id_doi">10.1093/nar/gkt940</infon><infon key="pub-id_pmid">24153109</infon><infon key="section_type">REF</infon><infon key="source">Nucleic acids research</infon><infon key="type">ref</infon><infon key="volume">42</infon><infon key="year">2014</infon><offset>72044</offset><text>PDBsum additions</text></passage></document></collection>
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+<collection><source>PMC</source><date>20201220</date><key>pmc.key</key><document><id>4848090</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.7554/eLife.15075</infon><infon key="article-id_pmc">4848090</infon><infon key="article-id_pmid">27058169</infon><infon key="article-id_publisher-id">15075</infon><infon key="elocation-id">e15075</infon><infon key="kwd">membrane signaling receptor kinase peptide hormone floral abscission plant development protein complexes &lt;i&gt;A. thaliana&lt;/i&gt;</infon><infon key="license">This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.</infon><infon key="name_0">surname:Santiago;given-names:Julia</infon><infon key="name_1">surname:Brandt;given-names:Benjamin</infon><infon key="name_10">surname:Hothorn;given-names:Michael</infon><infon key="name_11">surname:Butenko;given-names:Melinka A</infon><infon key="name_12">surname:Brandt;given-names:Benjamin</infon><infon key="name_13">surname:Santiago;given-names:Julia</infon><infon key="name_2">surname:Wildhagen;given-names:Mari</infon><infon key="name_3">surname:Hohmann;given-names:Ulrich</infon><infon key="name_4">surname:Hothorn;given-names:Ludwig A</infon><infon key="name_5">surname:Butenko;given-names:Melinka A</infon><infon key="name_6">surname:Hothorn;given-names:Michael</infon><infon key="name_7">surname:Zhang;given-names:Mingjie</infon><infon key="name_8">surname:Hothorn;given-names:Michael</infon><infon key="name_9">surname:Hothorn;given-names:Michael</infon><infon key="section_type">TITLE</infon><infon key="title">Author Keywords Research Organism</infon><infon key="type">front</infon><infon key="volume">5</infon><infon key="year">2016</infon><offset>0</offset><text>Mechanistic insight into a peptide hormone signaling complex mediating floral organ abscission</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>95</offset><text>Plants constantly renew during their life cycle and thus require to shed senescent and damaged organs. Floral abscission is controlled by the leucine-rich repeat receptor kinase (LRR-RK) HAESA and the peptide hormone IDA. It is unknown how expression of IDA in the abscission zone leads to HAESA activation. Here we show that IDA is sensed directly by the HAESA ectodomain. Crystal structures of HAESA in complex with IDA reveal a hormone binding pocket that accommodates an active dodecamer peptide. A central hydroxyproline residue anchors IDA to the receptor. The HAESA co-receptor SERK1, a positive regulator of the floral abscission pathway, allows for high-affinity sensing of the peptide hormone by binding to an Arg-His-Asn motif in IDA. This sequence pattern is conserved among diverse plant peptides, suggesting that plant peptide hormone receptors may share a common ligand binding mode and activation mechanism.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1019</offset><text>DOI: http://dx.doi.org/10.7554/eLife.15075.001</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>1066</offset><text>eLife digest</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1079</offset><text>Plants can shed their leaves, flowers or other organs when they no longer need them. But how does a leaf or a flower know when to let go? A receptor protein called HAESA is found on the surface of the cells that surround a future break point on the plant. When its time to shed an organ, a hormone called IDA instructs HAESA to trigger the shedding process. However, the molecular details of how IDA triggers organ shedding are not clear.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1518</offset><text>The shedding of floral organs (or leaves) can be easily studied in a model plant called Arabidopsis. Santiago et al. used protein biochemistry, structural biology and genetics to uncover how the IDA hormone activates HAESA. The experiments show that IDA binds directly to a canyon shaped pocket in HAESA that extends out from the surface of the cell. IDA binding to HAESA allows another receptor protein called SERK1 to bind to HAESA, which results in the release of signals inside the cell that trigger the shedding of organs.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>2046</offset><text>The next step following on from this work is to understand what signals are produced when IDA activates HAESA. Another challenge will be to find out where IDA is produced in the plant and what causes it to accumulate in specific places in preparation for organ shedding.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>2317</offset><text>DOI: http://dx.doi.org/10.7554/eLife.15075.002</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>2364</offset><text>Introduction</text></passage><passage><infon key="file">elife-15075-fig1-figsupp1.jpg</infon><infon key="id">fig1s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>2377</offset><text>The HAESA ectodomain folds into a superhelical assembly of 21 leucine-rich repeats.</text></passage><passage><infon key="file">elife-15075-fig1-figsupp1.jpg</infon><infon key="id">fig1s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>2461</offset><text>(A) SDS PAGE analysis of the purified Arabidopsis thaliana HAESA ectodomain (residues 20–620) obtained by secreted expression in insect cells. The calculated molecular mass is 65.7 kDa, the actual molecular mass obtained by mass spectrometry is 74,896 Da, accounting for the N-glycans. (B) Ribbon diagrams showing front (left panel) and side views (right panel) of the isolated HAESA LRR domain. The N- (residues 20–88) and C-terminal (residues 593–615) capping domains are shown in yellow, the central 21 LRR motifs are in blue and disulphide bonds are highlighted in green (in bonds representation). (C) Structure based sequence alignment of the 21 leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. Conserved hydrophobic residues are shaded in gray, N-glycosylation sites visible in our structures are highlighted in blue, cysteine residues involved in disulphide bridge formation in green. (D) Asn-linked glycans mask the N-terminal portion of the HAESA ectodomain. Oligomannose core structures (containing two N-actylglucosamines and three terminal mannose units) as found in Trichoplusia ni cells and in plants were modeled onto the seven glycosylation sites observed in our HAESA structures, to visualize the surface areas potentially not masked by carbohydrate. The HAESA ectodomain is shown in blue (in surface representation), the glycan structures are shown in yellow. Molecular surfaces were calculated with the program MSMS, with a probe radius of 1.5 Å.</text></passage><passage><infon key="file">elife-15075-fig1-figsupp1.jpg</infon><infon key="id">fig1s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>3975</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.15075.004</text></passage><passage><infon key="file">elife-15075-fig1-figsupp2.jpg</infon><infon key="id">fig1s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>4022</offset><text>Hydrophobic contacts and a hydrogen-bond network mediate the interaction between HAESA and the peptide hormone IDA.</text></passage><passage><infon key="file">elife-15075-fig1-figsupp2.jpg</infon><infon key="id">fig1s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>4138</offset><text>(A) Details of the IDA binding pocket. HAESA is shown in blue (ribbon diagram), the C-terminal Arg-His-Asn motif (left panel), the central Hyp anchor (center) and the N-terminal Pro-rich motif in IDA (right panel) are shown in yellow (in bonds representation). HAESA interface residues are shown as sticks, selected hydrogen bond interactions are denoted as dotted lines (in magenta). (B) View of the complete IDA (in bonds representation, in yellow) binding pocket in HAESA (surface view, in blue). Orientation as in (A). (C) Structure based sequence alignment of leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. Residues mediating hydrophobic interactions with the IDA peptide are highlighted in blue, residues contributing to hydrogen bond interactions and/or salt bridges are shown in red. The IDA binding pocket covers LRRs 2–14 and all residues originate from the inner surface of the HAESA superhelix.</text></passage><passage><infon key="file">elife-15075-fig1-figsupp2.jpg</infon><infon key="id">fig1s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>5090</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.15075.005</text></passage><passage><infon key="file">elife-15075-fig1-figsupp3.jpg</infon><infon key="id">fig1s3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>5137</offset><text>The IDA-HAESA and SERK1-HAESA complex interfaces are conserved among HAESA and HAESA-like proteins from different plant species.</text></passage><passage><infon key="file">elife-15075-fig1-figsupp3.jpg</infon><infon key="id">fig1s3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>5267</offset><text>Structure-based sequence alignment of the HAESA family members: Arabidopsis thaliana HAESA (Uniprot (http://www.uniprot.org) ID P47735), Arabidopsis thaliana HSL2 (Uniprot ID C0LGX3), Capsella rubella HAESA (Uniprot ID R0F2U6), Citrus clementina HSL2 (Uniprot ID V4U227), Vitis vinifera HAESA (Uniprot ID F6HM39). The alignment includes a secondary structure assignment calculated with the program DSSP and colored according to Figure 1, with the N- and C-terminal caps and the 21 LRR motifs indicated in orange and blue, respectively. Cysteine residues engaged in disulphide bonds are depicted in green. HAESA residues interacting with the IDA peptide and/or the SERK1 co-receptor kinase ectodomain are highlighted in blue and orange, respectively.</text></passage><passage><infon key="file">elife-15075-fig1-figsupp3.jpg</infon><infon key="id">fig1s3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>6017</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.15075.006</text></passage><passage><infon key="file">elife-15075-fig1.jpg</infon><infon key="id">fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>6064</offset><text>The peptide hormone IDA binds to the HAESA LRR ectodomain.</text></passage><passage><infon key="file">elife-15075-fig1.jpg</infon><infon key="id">fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>6123</offset><text>(A) Multiple sequence alignment of selected IDA family members. The conserved PIP motif is highlighted in yellow, the central Hyp in blue. The PKGV motif present in our N-terminally extended IDA peptide is highlighted in red. (B) Isothermal titration calorimetry of the HAESA ectodomain vs. IDA and including the synthetic peptide sequence. (C) Structure of the HAESA – IDA complex with HAESA shown in blue (ribbon diagram). IDA (in bonds representation, surface view included) is depicted in yellow. The peptide binding pocket covers HAESA LRRs 2–14. (D) Close-up view of the entire IDA (in yellow) peptide binding site in HAESA (in blue). Details of the interactions between the central Hyp anchor in IDA and the C-terminal Arg-His-Asn motif with HAESA are highlighted in (E) and (F), respectively. Hydrogren bonds are depicted as dotted lines (in magenta), a water molecule is shown as a red sphere.</text></passage><passage><infon key="file">elife-15075-fig1.jpg</infon><infon key="id">fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>7031</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.15075.003</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>7078</offset><text>During their growth, development and reproduction plants use cell separation processes to detach no-longer required, damaged or senescent organs. Abscission of floral organs in Arabidopsis is a model system to study these cell separation processes in molecular detail. The LRR-RKs HAESA (greek: to adhere to) and HAESA-LIKE 2 (HSL2) redundantly control floral abscission. Loss-of-function of the secreted small protein INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) causes floral organs to remain attached while its over-expression leads to premature shedding. Full-length IDA is proteolytically processed and a conserved stretch of 20 amino-acids (termed EPIP) can rescue the IDA loss-of-function phenotype (Figure 1A). It has been demonstrated that a dodecamer peptide within EPIP is able to activate HAESA and HSL2 in transient assays in tobacco cells. This sequence motif is highly conserved among IDA family members (IDA-LIKE PROTEINS, IDLs) and contains a central Pro residue, presumed to be post-translationally modified to hydroxyproline (Hyp; Figure 1A). The available genetic and biochemical evidence suggests that IDA and HAESA together control floral abscission, but it is poorly understood if IDA is directly sensed by the receptor kinase HAESA and how IDA binding at the cell surface would activate the receptor.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>8403</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8411</offset><text>IDA directly binds to the LRR domain of HAESA</text></passage><passage><infon key="file">elife-15075-fig2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>8457</offset><text>Active IDA-family peptide hormones are hydroxyprolinated dodecamers.</text></passage><passage><infon key="file">elife-15075-fig2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>8526</offset><text>Close-up views of (A) IDA, (B) the N-terminally extended PKGV-IDA and (C) IDL1 bound to the HAESA hormone binding pocket (in bonds representation, in yellow) and including simulated annealing 2Fo–Fc omit electron density maps contoured at 1.0 σ. Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. no detectable binding). (E) Structural superposition of the active IDA (in bonds representation, in gray) and IDL1 peptide (in yellow) hormones bound to the HAESA ectodomain. Root mean square deviation (r.m.s.d.) is 1.0 Å comparing 100 corresponding atoms.</text></passage><passage><infon key="file">elife-15075-fig2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>9382</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.15075.007</text></passage><passage><infon key="file">elife-15075-fig3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>9429</offset><text>The receptor kinase SERK1 acts as a HAESA co-receptor and promotes high-affinity IDA sensing.</text></passage><passage><infon key="file">elife-15075-fig3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>9523</offset><text>(A) Petal break-strength assays measure the force (expressed in gram equivalents) required to remove the petals from the flower of serk mutant plants compared to haesa/hsl2 mutant and Col-0 wild-type flowers. Petal break-strength is measured from positions 1 to 8 along the primary inflorescence where positions 1 is defined as the flower at anthesis (n=15, bars=SD). This treatment-by-position balanced two-way layout was analyzed separately per position, because of the serious interaction, by means of a Dunnett-type comparison against the Col-0 control, allowing for heterogeneous variances. Petal break-strength was found significantly increased in almost all positions (indicated with a *) for haesa/hsl2 and serk1-1 mutant plants with respect to the Col-0 control. Calculations were performed in R (version 3.2.3). (B) Analytical size-exclusion chromatography. The HAESA LRR domain elutes as a monomer (black dotted line), as does the isolated SERK1 ectodomain (blue dotted line). A HAESA – IDA – SERK1 complex elutes as an apparent heterodimer (red line), while a mixture of HAESA and SERK1 yields two isolated peaks that correspond to monomeric HAESA and SERK1, respectively (black line). Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). A SDS PAGE of the peak fractions is shown alongside. Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. (C) Isothermal titration calorimetry of wild-type and Hyp64→Pro IDA versus the HAESA and SERK1 ectodomains. The titration of IDA wild-type versus the isolated HAESA ectodomain from Figure 1B is shown for comparison (red line; n.d. no detectable binding) (D) Analytical size-exclusion chromatography in the presence of the IDA Hyp64→Pro mutant peptide reveals no complex formation between HAESA and SERK1 ectodomains. A SDS PAGE of the peak fractions is shown alongside. (E) In vitro kinase assays of the HAESA and SERK1 kinase domains. Wild-type HAESA and SERK1 kinase domains (KDs) exhibit auto-phosphorylation activities (lanes 1 + 3). Mutant (m) versions, which carry point mutations in their active sites (Asp837HAESA→Asn, Asp447SERK1→Asn) possess no autophosphorylation activity (lanes 2+4). Transphosphorylation activity from the active kinase to the mutated form can be observed in both directions (lanes 5+6). A coomassie-stained gel loading control is shown below.</text></passage><passage><infon key="file">elife-15075-fig3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>12096</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.15075.008</text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>12143</offset><text>Crystallographic data collection, phasing and refinement statistics for the isolated A. thaliana HAESA ectodomain.</text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>12258</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.15075.009</text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;thead&gt;&lt;tr&gt;&lt;th valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;th valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;HAESA NaI shortsoak&lt;/th&gt;&lt;th valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;HAESA apo&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;PDB-ID&lt;/bold&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5IXO&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;Data collection&lt;/bold&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Space group&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;P&lt;/italic&gt;3&lt;sub&gt;1&lt;/sub&gt; 21&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;P&lt;/italic&gt;3&lt;sub&gt;1&lt;/sub&gt; 21&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Cell dimensions&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;a&lt;/italic&gt;, &lt;italic&gt;b, c&lt;/italic&gt; (Å)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;148.55, 148.55, 58.30&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;149.87, 149.87, 58.48&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;α&lt;/italic&gt;, β, γ (°)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90, 90, 120&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90, 90, 120&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution (Å)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;48.63–2.39 (2.45–2.39)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;45.75–1.74 (1.85–1.74)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;R&lt;sub&gt;meas&lt;/sub&gt;&lt;sup&gt;#&lt;/sup&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.096 (0.866)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.038 (1.02)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;CC(1/2)&lt;italic&gt;&lt;sup&gt;#&lt;/sup&gt;&lt;/italic&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100/86.6&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100/75.6&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;I/σ I&lt;sup&gt;#&lt;/sup&gt;&lt;/italic&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;27.9 (4.9)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;18.7 (1.8)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Completeness (%)&lt;italic&gt;&lt;sup&gt;#&lt;/sup&gt;&lt;/italic&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.9 (98.6)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.6 (97.4)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Redundancy&lt;italic&gt;&lt;sup&gt;#&lt;/sup&gt;&lt;/italic&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;53.1 (29.9)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;14.4 (14.0)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Wilson B-factor (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;italic&gt;&lt;sup&gt;#&lt;/sup&gt;&lt;/italic&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;84.45&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;81.10&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;Refinement&lt;/bold&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution (Å)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;45.75 – 1.74&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;No. reflections&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;71,213&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;work/&lt;/sub&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt;&lt;sup&gt;$&lt;/sup&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.188/0.218&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;No. atoms&lt;/bold&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Protein/glycan&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4,533/126&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Water&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;71&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;Res. B-factors (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/bold&gt;&lt;sup&gt;$&lt;/sup&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Protein&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;77.54&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Glycan&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;95.98&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Water&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;73.20&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;R.m.s deviations&lt;/bold&gt;&lt;sup&gt;$&lt;/sup&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Bond lengths (Å)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.0095&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Bond angles (°)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.51&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>12305</offset><text>	HAESA NaI shortsoak	HAESA apo	 	PDB-ID		5IXO	 	Data collection			 	Space group	P31 21	P31 21	 	Cell dimensions			 	a, b, c (Å)	148.55, 148.55, 58.30	149.87, 149.87, 58.48	 	α, β, γ (°)	90, 90, 120	90, 90, 120	 	Resolution (Å)	48.63–2.39 (2.45–2.39)	45.75–1.74 (1.85–1.74)	 	Rmeas#	0.096 (0.866)	0.038 (1.02)	 	CC(1/2)#	100/86.6	100/75.6	 	I/σ I#	27.9 (4.9)	18.7 (1.8)	 	Completeness (%)#	99.9 (98.6)	99.6 (97.4)	 	Redundancy#	53.1 (29.9)	14.4 (14.0)	 	Wilson B-factor (Å2)#	84.45	81.10	 	Refinement			 	Resolution (Å)		45.75 – 1.74	 	No. reflections		71,213	 	Rwork/Rfree$		0.188/0.218	 	No. atoms			 	Protein/glycan		4,533/126	 	Water		71	 	Res. B-factors (Å2)$			 	Protein		77.54	 	Glycan		95.98	 	Water		73.20	 	R.m.s deviations$			 	Bond lengths (Å)		0.0095	 	Bond angles (°)		1.51	 	</text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>13117</offset><text>Highest resolution shell is shown in parenthesis.</text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>13167</offset><text>#As defined in XDS.</text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>13187</offset><text>$As defined in Refmac5.</text></passage><passage><infon key="file">tbl2.xml</infon><infon key="id">tbl2</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>13211</offset><text>Crystallographic data collection and refinement statistics for the HAESA – IDA, – PKGV-IDA, – IDL1 and – IDA – SERK1 complexes.</text></passage><passage><infon key="file">tbl2.xml</infon><infon key="id">tbl2</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>13349</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.15075.010</text></passage><passage><infon key="file">tbl2.xml</infon><infon key="id">tbl2</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;thead&gt;&lt;tr&gt;&lt;th valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;th valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;HAESA – IDA&lt;/th&gt;&lt;th valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;HAESA – PKGV-IDA&lt;/th&gt;&lt;th valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;HAESA – IDL1&lt;/th&gt;&lt;th valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;HAESA – IDA – SERK1&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;PDB-ID&lt;/bold&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5IXQ&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5IXT&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5IYN&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5IYX&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;Data collection&lt;/bold&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Space group&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;P&lt;/italic&gt;3&lt;sub&gt;1&lt;/sub&gt; 21&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;P&lt;/italic&gt;3&lt;sub&gt;1&lt;/sub&gt; 21&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;P&lt;/italic&gt;3&lt;sub&gt;1&lt;/sub&gt; 21&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;P&lt;/italic&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Cell dimensions&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;italic&gt;a&lt;/italic&gt;, &lt;italic&gt;b, c&lt;/italic&gt; (Å)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;148.55, 148.55, 58.30&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;148.92, 148.92, 58.02&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;150.18, 150.18, 60.07&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;74.51, 100.46, 142.76&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;α&lt;/italic&gt;, β, γ (°)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90, 90, 120&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90, 90, 120&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90, 90, 120&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90, 90, 90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution (Å)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;48.54–1.86 &lt;break/&gt;(1.97–1.86)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;48.75–1.94 (2,06–1.94)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;49.16–2.56 (2.72–2.56)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;47.59–2.43 (2.57–2.43)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;R&lt;sub&gt;meas&lt;/sub&gt;&lt;sup&gt;#&lt;/sup&gt;&lt;/italic&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.057 (1.35)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.037 (0.97)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.056 (1.27)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.113 (1.37)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;CC(1/2)&lt;italic&gt;&lt;sup&gt;#&lt;/sup&gt;&lt;/italic&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100/77.9&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100/80.3&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100/89.5&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100/77.6&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;I&lt;/italic&gt;/σ&lt;italic&gt;I&lt;sup&gt;#&lt;/sup&gt;&lt;/italic&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;16.7 (2.0)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;20.9 (2.4)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;26.0 (1.9)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;16.12 (2.0)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Completeness&lt;italic&gt;&lt;sup&gt;#&lt;/sup&gt;&lt;/italic&gt; (%)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.8 (98.6)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.4 (97.9)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.5 (98.8)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.4 (96.4s&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Redundancy&lt;italic&gt;&lt;sup&gt;#&lt;/sup&gt;&lt;/italic&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;20.3 (19.1)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;11.2 (11.1)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;14.7 (14.7)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;9.7 (9.3)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Wilson B-factor (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;italic&gt;&lt;sup&gt;#&lt;/sup&gt;&lt;/italic&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;80.0&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;81.7&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;89.5&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;59.3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;Refinement&lt;/bold&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution (Å)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;48.54–1.86&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;48.75–1.94&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;49.16–2.56&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;47.59–2.43&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;No. reflections&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;58,551&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;51,557&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;23,835&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;38,969&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;work/&lt;/sub&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt;&lt;sup&gt;$&lt;/sup&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.190/0.209&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.183/0.208&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.199/0.236&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.199/0.235&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;No. atoms&lt;/bold&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Protein/Glycan&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4,541/176&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4,545/176&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4,499/176&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5,965/168&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Peptide&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;93&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;93&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;112&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Water&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;39&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;40&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;9&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;136&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;Res. B-factors (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/bold&gt;&lt;sup&gt;$&lt;/sup&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Protein/Glycan&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;79.48/109.02&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;79.63/113.24&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;102.12/132.49&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;60.05/73.48&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Peptide&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;87.19&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;89.50&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;125.74&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;51.06&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Water&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;75.32&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;71.92&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;74.65&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;51.47&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;R.m.s deviations&lt;/bold&gt;&lt;sup&gt;$&lt;/sup&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Bond lengths (Å)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.0087&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.0091&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.0081&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.0074&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Bond angles (°)&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.48&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.47&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.36&lt;/td&gt;&lt;td valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.34&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>13396</offset><text>	HAESA – IDA	HAESA – PKGV-IDA	HAESA – IDL1	HAESA – IDA – SERK1	 	PDB-ID	5IXQ	5IXT	5IYN	5IYX	 	Data collection					 	Space group	P31 21	P31 21	P31 21	P212121	 	Cell dimensions					 	a, b, c (Å)	148.55, 148.55, 58.30	148.92, 148.92, 58.02	150.18, 150.18, 60.07	74.51, 100.46, 142.76	 	α, β, γ (°)	90, 90, 120	90, 90, 120	90, 90, 120	90, 90, 90	 	Resolution (Å)	48.54–1.86 (1.97–1.86)	48.75–1.94 (2,06–1.94)	49.16–2.56 (2.72–2.56)	47.59–2.43 (2.57–2.43)	 	Rmeas#	0.057 (1.35)	0.037 (0.97)	0.056 (1.27)	0.113 (1.37)	 	CC(1/2)#	100/77.9	100/80.3	100/89.5	100/77.6	 	I/σI#	16.7 (2.0)	20.9 (2.4)	26.0 (1.9)	16.12 (2.0)	 	Completeness# (%)	99.8 (98.6)	99.4 (97.9)	99.5 (98.8)	99.4 (96.4s	 	Redundancy#	20.3 (19.1)	11.2 (11.1)	14.7 (14.7)	9.7 (9.3)	 	Wilson B-factor (Å2)#	80.0	81.7	89.5	59.3	 	Refinement					 	Resolution (Å)	48.54–1.86	48.75–1.94	49.16–2.56	47.59–2.43	 	No. reflections	58,551	51,557	23,835	38,969	 	Rwork/Rfree$	0.190/0.209	0.183/0.208	0.199/0.236	0.199/0.235	 	No. atoms					 	Protein/Glycan	4,541/176	4,545/176	4,499/176	5,965/168	 	Peptide	93	93	90	112	 	Water	39	40	9	136	 	Res. B-factors (Å2)$					 	Protein/Glycan	79.48/109.02	79.63/113.24	102.12/132.49	60.05/73.48	 	Peptide	87.19	89.50	125.74	51.06	 	Water	75.32	71.92	74.65	51.47	 	R.m.s deviations$					 	Bond lengths (Å)	0.0087	0.0091	0.0081	0.0074	 	Bond angles (°)	1.48	1.47	1.36	1.34	 	</text></passage><passage><infon key="file">tbl2.xml</infon><infon key="id">tbl2</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>14804</offset><text>Highest resolution shell is shown in parenthesis.</text></passage><passage><infon key="file">tbl2.xml</infon><infon key="id">tbl2</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>14854</offset><text>#As defined in XDS.</text></passage><passage><infon key="file">tbl2.xml</infon><infon key="id">tbl2</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>14874</offset><text>$As defined in Refmac5.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14898</offset><text>We purified the HAESA ectodomain (residues 20–620) from baculovirus-infected insect cells (Figure 1—figure supplement 1A, see Materials and methods) and quantified the interaction of the ~75 kDa glycoprotein with synthetic IDA peptides using isothermal titration calorimetry (ITC). A Hyp-modified dodecamer comprising the highly conserved PIP motif in IDA (Figure 1A) interacts with HAESA with 1:1 stoichiometry (N) and with a dissociation constant (Kd) of ~20 μM (Figure 1B). We next determined crystal structures of the apo HAESA ectodomain and of a HAESA-IDA complex, at 1.74 and 1.86 Å resolution, respectively (Figure 1C; Figure 1—figure supplement 1B–D; Tables 1,2). IDA binds in a completely extended conformation along the inner surface of the HAESA ectodomain, covering LRRs 2–14 (Figure 1C,D, Figure 1—figure supplement 2). The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8–10, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1—figure supplement 3). The restricted size of the Hyp pocket suggests that IDA does not require arabinosylation of Hyp64IDA for activity in vivo, a modification that has been reported for Hyp residues in plant CLE peptide hormones. The C-terminal Arg-His-Asn motif in IDA maps to a cavity formed by HAESA LRRs 11–14 (Figure 1D,F). The COO- group of Asn69IDA is in direct contact with Arg407HAESA and Arg409HAESA and HAESA cannot bind a C-terminally extended IDA-SFVN peptide (Figures 1D,F, 2D). This suggests that the conserved Asn69IDA may constitute the very C-terminus of the mature IDA peptide in planta and that active IDA is generated by proteolytic processing from a longer pre-protein. Mutation of Arg417HSL2 (which corresponds to Arg409HAESA) causes a loss-of-function phenotype in HSL2, which indicates that the peptide binding pockets in different HAESA receptors have common structural and sequence features. Indeed, we find many of the residues contributing to the formation of the IDA binding surface in HAESA to be conserved in HSL2 and in other HAESA-type receptors in different plant species (Figure 1—figure supplement 3). A N-terminal Pro-rich motif in IDA makes contacts with LRRs 2–6 of the receptor (Figure 1D, Figure 1—figure supplement 2A–C). Other hydrophobic and polar interactions are mediated by Ser62IDA, Ser65IDA and by backbone atoms along the IDA peptide (Figure 1D, Figure 1—figure supplement 2A–C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>17511</offset><text>HAESA specifically senses IDA-family dodecamer peptides</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17567</offset><text>We next investigated whether HAESA binds N-terminally extended versions of IDA. We obtained a structure of HAESA in complex with a PKGV-IDA peptide at 1.94 Å resolution (Table 2). In this structure, no additional electron density accounts for the PKGV motif at the IDA N-terminus (Figure 2A,B). Consistently, PKGV-IDA and IDA have similar binding affinities in our ITC assays, further indicating that HAESA senses a dodecamer peptide comprising residues 58-69IDA (Figure 2D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18046</offset><text>We next tested if HAESA binds other IDA peptide family members. IDL1, which can rescue IDA loss-of-function mutants when introduced in abscission zone cells, can also be sensed by HAESA, albeit with lower affinity (Figure 2D). A 2.56 Å co-crystal structure with IDL1 reveals that different IDA family members use a common binding mode to interact with HAESA-type receptors (Figure 2A–C,E, Table 2). We do not detect interaction between HAESA and a synthetic peptide missing the C-terminal Asn69IDA (ΔN69), highlighting the importance of the polar interactions between the IDA carboxy-terminus and Arg407HAESA/Arg409HAESA (Figures 1F, 2D). Replacing Hyp64IDA, which is common to all IDLs, with proline impairs the interaction with the receptor, as does the Lys66IDA/Arg67IDA → Ala double-mutant discussed below (Figure 1A, 2D). Notably, HAESA can discriminate between IDLs and functionally unrelated dodecamer peptides with Hyp modifications, such as CLV3 (Figures 2D, 7).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>19026</offset><text>The co-receptor kinase SERK1 allows for high-affinity IDA sensing</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19092</offset><text>Our binding assays reveal that IDA family peptides are sensed by the isolated HAESA ectodomain with relatively weak binding affinities (Figures 1B, 2A–D). It has been recently reported that SOMATIC EMBRYOGENESIS RECEPTOR KINASES (SERKs) are positive regulators of floral abscission and can interact with HAESA and HSL2 in an IDA-dependent manner. As all five SERK family members appear to be expressed in the Arabidopsis abscission zone, we quantified their relative contribution to floral abscission in Arabidopsis using a petal break-strength assay. Our experiments suggest that among the SERK family members, SERK1 is a positive regulator of floral abscission. We found that the force required to remove the petals of serk1-1 mutants is significantly higher than that needed for wild-type plants, as previously observed for haesa/hsl2 mutants, and that floral abscission is delayed in serk1-1 (Figure 3A). The serk2-2, serk3-1, serk4-1 and serk5-1 mutant lines showed a petal break-strength profile not significantly different from wild-type plants. Possibly because SERKs have additional roles in plant development such as in pollen formation and brassinosteroid signaling, we found that higher-order SERK mutants exhibit pleiotropic phenotypes in the flower, rendering their analysis and comparison by quantitative petal break-strength assays difficult. We thus focused on analyzing the contribution of SERK1 to HAESA ligand sensing and receptor activation.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20558</offset><text>In vitro, the LRR ectodomain of SERK1 (residues 24–213) forms stable, IDA-dependent heterodimeric complexes with HAESA in size exclusion chromatography experiments (Figure 3B). We next quantified the contribution of SERK1 to IDA recognition by HAESA. We found that HAESA senses IDA with a ~60 fold higher binding affinity in the presence of SERK1, suggesting that SERK1 is involved in the specific recognition of the peptide hormone (Figure 3C). We next titrated SERK1 into a solution containing only the HAESA ectodomain. In this case, there was no detectable interaction between receptor and co-receptor, while in the presence of IDA, SERK1 strongly binds HAESA with a dissociation constant in the mid-nanomolar range (Figure 3C). This suggests that IDA itself promotes receptor – co-receptor association, as previously described for the steroid hormone brassinolide and for other LRR-RK complexes. Importantly, hydroxyprolination of IDA is critical for HAESA-IDA-SERK1 complex formation (Figure 3C,D). Our calorimetry experiments now reveal that SERKs may render HAESA, and potentially other receptor kinases, competent for high-affinity sensing of their cognate ligands.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21738</offset><text>Upon IDA binding at the cell surface, the kinase domains of HAESA and SERK1, which have been shown to be active protein kinases, may interact in the cytoplasm to activate each other. Consistently, the HAESA kinase domain can transphosphorylate SERK1 and vice versa in in vitro transphosphorylation assays (Figure 3E). Together, our genetic and biochemical experiments implicate SERK1 as a HAESA co-receptor in the Arabidopsis abscission zone.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>22181</offset><text>SERK1 senses a conserved motif in IDA family peptides</text></passage><passage><infon key="file">elife-15075-fig4.jpg</infon><infon key="id">fig4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>22235</offset><text>Crystal structure of a HAESA – IDA – SERK1 signaling complex.</text></passage><passage><infon key="file">elife-15075-fig4.jpg</infon><infon key="id">fig4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>22301</offset><text>(A) Overview of the ternary complex with HAESA in blue (surface representation), IDA in yellow (bonds representation) and SERK1 in orange (surface view). (B) The HAESA ectodomain undergoes a conformational change upon SERK1 co-receptor binding. Shown are Cα traces of a structural superposition of the unbound (yellow) and SERK1-bound (blue) HAESA ectodomains (r.m.s.d. is 1.5 Å between 572 corresponding Cα atoms). SERK1 (in orange) and IDA (in red) are shown alongside. The conformational change in the C-terminal LRRs and capping domain is indicated by an arrow. (C) SERK1 forms an integral part of the receptor's peptide binding pocket. The N-terminal capping domain of SERK1 (in orange) directly contacts the C-terminal part of IDA (in yellow, in bonds representation) and the receptor HAESA (in blue). Polar contacts of SERK1 with IDA are shown in magenta, with the HAESA LRR domain in gray. (D) Details of the zipper-like SERK1-HAESA interface. Ribbon diagrams of HAESA (in blue) and SERK1 (in orange) are shown with selected interface residues (in bonds representation). Polar interactions are highlighted as dotted lines (in magenta).</text></passage><passage><infon key="file">elife-15075-fig4.jpg</infon><infon key="id">fig4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>23453</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.15075.011</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23500</offset><text>To understand in molecular terms how SERK1 contributes to high-affinity IDA recognition, we solved a 2.43 Å crystal structure of the ternary HAESA – IDA – SERK1 complex (Figure 4A, Table 2). HAESA LRRs 16–21 and its C-terminal capping domain undergo a conformational change upon SERK1 binding (Figure 4B). The SERK1 ectodomain interacts with the IDA peptide binding site using a loop region (residues 51-59SERK1) from its N-terminal cap (Figure 4A,C). SERK1 loop residues establish multiple hydrophobic and polar contacts with Lys66IDA and the C-terminal Arg-His-Asn motif in IDA (Figure 4C). SERK1 LRRs 1–5 and its C-terminal capping domain form an additional zipper-like interface with residues originating from HAESA LRRs 15–21 and from the HAESA C-terminal cap (Figure 4D). SERK1 binds HAESA using these two distinct interaction surfaces (Figure 1—figure supplement 3), with the N-cap of the SERK1 LRR domain partially covering the IDA peptide binding cleft.</text></passage><passage><infon key="file">elife-15075-fig5.jpg</infon><infon key="id">fig5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>24478</offset><text>The IDA C-terminal motif is required for HAESA-SERK1 complex formation and for IDA bioactivity.</text></passage><passage><infon key="file">elife-15075-fig5.jpg</infon><infon key="id">fig5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>24574</offset><text>(A) Size exclusion chromatography experiments similar to Figure 3B,D reveal that IDA mutant peptides targeting the C-terminal motif do not form biochemically stable HAESA-IDA-SERK1 complexes. Deletion of the C-terminal Asn69IDA completely inhibits complex formation. Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. Left panel: IDA K66A/R67A; center: IDA ΔN69, right panel: SDS-PAGE of peak fractions. Note that the HAESA and SERK1 input lanes have already been shown in Figure 3D. (B) Isothermal titration thermographs of wild-type and mutant IDA peptides titrated into a HAESA - SERK1 mixture in the cell. Table summaries for calorimetric binding constants and stoichoimetries for different IDA peptides binding to the HAESA – SERK1 ectodomain mixture ( ± fitting errors; n.d. no detectable binding) are shown alongside. (C) Quantitative petal break-strength assay for Col-0 wild-type flowers and 35S::IDA wild-type and 35S::IDA K66A/R67A mutant flowers. Petal break is measured from positions 1 to 8 along the primary inflorescence where positions 1 is defined as the flower at anthesis (n=15, bars=SD). The three treatment groups in this unbalanced one-way layout were compared by Tukey’s all-pairs comparison procedure using the package multcomp in R (version 3.2.3). 35S::IDA plants showed significantly increased abscission compared to Col-0 controls in inflorescence positions 2 and 3 (a). Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. 15 out of 15 35S::IDA plants, 0 out of 15 Col-0 plants and 0 out of 15 35S::IDA K66A/R67A double-mutant plants, showed an enlarged abscission zone, respectively (3 independent lines were analyzed).</text></passage><passage><infon key="file">elife-15075-fig5.jpg</infon><infon key="id">fig5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27046</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.15075.012</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>27093</offset><text>The four C-terminal residues in IDA (Lys66IDA-Asn69IDA) are conserved among IDA family members and are in direct contact with SERK1 (Figures 1A, 4C). We thus assessed their contribution to HAESA – SERK1 complex formation. Deletion of the buried Asn69IDA completely inhibits receptor – co-receptor complex formation and HSL2 activation (Figure 5A,B). A synthetic Lys66IDA/Arg67IDA → Ala mutant peptide (IDA K66A/R66A) showed a 10 fold reduced binding affinity when titrated in a HAESA/SERK1 protein solution (Figures 5A,B, 2D). We over-expressed full-length wild-type IDA or this Lys66IDA/Arg67IDA → Ala double-mutant to similar levels in Col-0 Arabidopsis plants (Figure 5D). We found that over-expression of wild-type IDA leads to early floral abscission and an enlargement of the abscission zone (Figure 5C–E). In contrast, over-expression of the IDA Lys66IDA/Arg67IDA → Ala double mutant significantly delays floral abscission when compared to wild-type control plants, suggesting that the mutant IDA peptide has reduced activity in planta (Figure 5C–E). Comparison of 35S::IDA wild-type and mutant plants further indicates that mutation of Lys66IDA/Arg67IDA → Ala may cause a weak dominant negative effect (Figure 5C–E). In agreement with our structures and biochemical assays, this experiment suggests a role of the conserved IDA C-terminus in the control of floral abscission.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>28494</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>28505</offset><text>In contrast to animal LRR receptors, plant LRR-RKs harbor spiral-shaped ectodomains and thus they require shape-complementary co-receptor proteins for receptor activation. For a rapidly growing number of plant signaling pathways, SERK proteins act as these essential co-receptors (; ). SERK1 has been previously reported as a positive regulator in plant embryogenesis, male sporogenesis, brassinosteroid signaling and in phytosulfokine perception. Recent findings by and our mechanistic studies now also support a positive role for SERK1 in floral abscission. As serk1-1 mutant plants show intermediate abscission phenotypes when compared to haesa/hsl2 mutants, SERK1 likely acts redundantly with other SERKs in the abscission zone (Figure 3A). It has been previously suggested that SERK1 can inhibit cell separation. However our results show that SERK1 also can activate this process upon IDA sensing, indicating that SERKs may fulfill several different functions in the course of the abscission process.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>29513</offset><text>While the sequence of the mature IDA peptide has not been experimentally determined in planta, our HAESA-IDA complex structures and calorimetry assays suggest that active IDLs are hydroxyprolinated dodecamers. It will be thus interesting to see if proteolytic processing of full-length IDA in vivo is regulated in a cell-type or tissue-specific manner. The central Hyp residue in IDA is found buried in the HAESA peptide binding surface and thus this post-translational modification may regulate IDA bioactivity. Our comparative structural and biochemical analysis further suggests that IDLs share a common receptor binding mode, but may preferably bind to HAESA, HSL1 or HSL2 in different plant tissues and organs.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>30229</offset><text>In our quantitative biochemical assays, the presence of SERK1 dramatically increases the HAESA binding specificity and affinity for IDA. This observation is consistent with our complex structure in which receptor and co-receptor together form the IDA binding pocket. The fact that SERK1 specifically interacts with the very C-terminus of IDLs may allow for the rational design of peptide hormone antagonists, as previously demonstrated for the brassinosteroid pathway. Importantly, our calorimetry assays reveal that the SERK1 ectodomain binds HAESA with nanomolar affinity, but only in the presence of IDA (Figure 3C). This ligand-induced formation of a receptor – co-receptor complex may allow the HAESA and SERK1 kinase domains to efficiently trans-phosphorylate and activate each other in the cytoplasm. It is of note that our reported binding affinities for IDA and SERK1 have been measured using synthetic peptides and the isolated HAESA and SERK1 ectodomains, and thus might differ in the context of the full-length, membrane-embedded signaling complex.</text></passage><passage><infon key="file">elife-15075-fig6.jpg</infon><infon key="id">fig6</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>31293</offset><text>SERK1 uses partially overlapping surface areas to activate different plant signaling receptors.</text></passage><passage><infon key="file">elife-15075-fig6.jpg</infon><infon key="id">fig6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>31389</offset><text>(A) Structural comparison of plant steroid and peptide hormone membrane signaling complexes. Left panel: Ribbon diagram of HAESA (in blue), SERK1 (in orange) and IDA (in bonds and surface represention). Right panel: Ribbon diagram of the plant steroid receptor BRI1 (in blue) bound to brassinolide (in gray, in bonds representation) and to SERK1, shown in the same orientation (PDB-ID. 4lsx). (B) View of the inner surface of the SERK1 LRR domain (PDB-ID 4lsc, surface representation, in gray). A ribbon diagram of SERK1 in the same orientation is shown alongside. Residues interacting with the HAESA or BRI1 LRR domains are shown in orange or magenta, respectively.</text></passage><passage><infon key="file">elife-15075-fig6.jpg</infon><infon key="id">fig6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>32056</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.15075.013</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>32103</offset><text>Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A). Several residues in the SERK1 N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B). In addition, residues 53-55SERK1 from the SERK1 N-terminal cap mediate specific interactions with the IDA peptide (Figures 4C, 6B). These residues are not involved in the sensing of the steroid hormone brassinolide. In both cases however, the co-receptor completes the hormone binding pocket. This fact together with the largely overlapping SERK1 binding surfaces in HAESA and BRI1 allows us to speculate that SERK1 may promote high-affinity peptide hormone and brassinosteroid sensing by simply slowing down dissociation of the ligand from its cognate receptor.</text></passage><passage><infon key="file">elife-15075-fig7.jpg</infon><infon key="id">fig7</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>33296</offset><text>Different plant peptide hormone families contain a C-terminal (Arg)-His-Asn motif, which in IDA represents the co-receptor recognition site.</text></passage><passage><infon key="file">elife-15075-fig7.jpg</infon><infon key="id">fig7</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>33437</offset><text>Structure-guided multiple sequence alignment of IDA and IDA-like peptides with other plant peptide hormone families, including CLAVATA3 – EMBRYO SURROUNDING REGION-RELATED (CLV3/CLE), ROOT GROWTH FACTOR – GOLVEN (RGF/GLV), PRECURSOR GENE PROPEP1 (PEP1) from Arabidopsis thaliana. The conserved (Arg)-His-Asn motif is highlighted in red, the central Hyp residue in IDLs and CLEs is marked in blue.</text></passage><passage><infon key="file">elife-15075-fig7.jpg</infon><infon key="id">fig7</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>33838</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.15075.014</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>33885</offset><text>Our experiments reveal that SERK1 recognizes a C-terminal Arg-His-Asn motif in IDA. Importantly, this motif can also be found in other peptide hormone families (Figure 7). Among these are the CLE peptides regulating stem cell maintenance in the shoot and the root. It is interesting to note, that CLEs in their mature form are also hydroxyprolinated dodecamers, which bind to a surface area in the BARELY ANY MERISTEM 1 receptor that would correspond to part of the IDA binding cleft in HAESA. Diverse plant peptide hormones may thus also bind their LRR-RK receptors in an extended conformation along the inner surface of the LRR domain and may also use small, shape-complementary co-receptors for high-affinity ligand binding and receptor activation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>34637</offset><text>Materials and methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>34659</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34695</offset><text>Synthetic genes coding for the Arabidopsis thaliana HAESA (residues 20–620) and SERK1 ectodomains (residues 24–213, carrying Asn115→Asp and Asn163→Gln mutations), codon optimized for expression in Trichoplusia ni (Geneart, Germany), were cloned into a modified pBAC-6 transfer vector (Novagen, Billerica, MA), providing an azurocidin signal peptide and a C-terminal TEV (tobacco etch virus protease) cleavable Strep-9xHis tandem affinity tag. Recombinant baculoviruses were generated by co-transfecting transfer vectors with linearised baculovirus DNA (ProFold-ER1, AB vector, San Diego, CA) followed by viral amplification in Spodoptera frugiperda Sf9 cells. The HAESA and SERK1 ectodomains were individually expressed in Trichoplusia ni Tnao38 cells using a multiplicity of infection of 3, and harvested from the medium 2 days post infection by tangential flow filtration using 30 kDa MWCO and 10 kDa MWCO (molecular weight cut-off) filter membranes (GE Healthcare Life Sciences, Pittsburgh, PA), respectively. Proteins were purified separately by sequential Ni2+ (HisTrap HP, GE Healthcare) and Strep (Strep-Tactin Superflow high-capacity, IBA, Germany) affinity chromatography. Next, affinity tags were removed by incubating the purified proteins with recombinant Strep-tagged TEV protease in 1:100 molar ratio. The cleaved tag and the protease were separated from HAESA and SERK1 by a second Strep affinity step. The purified HAESA ectodomain was incubated with a synthetic IDA peptide (YVPIPPSA-Hyp-SKRHN, the N-terminal Tyr residue was added to allow for peptide quantification by UV absorbance) and the SERK1 ectodomain in 1:1:1.5 molar ratio. The HAESA-IDA-SERK1 complex was purified by size exclusion chromatography on a Superdex 200 HR10/30 column (GE Healthcare) equilibrated in 20 mM citric acid pH 5.0, 100 mM NaCl). Peak fractions containing the complex were concentrated to ~10 mg/mL and immediately used for crystallization. About 0.2 mg of purified HAESA and 0.1 mg of purified SERK1 protein were obtained from 1 L of insect cell culture, respectively.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>36785</offset><text>Crystallization and data collection</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>36821</offset><text>Hexagonal crystals of the isolated HAESA ectodomain developed at room-temperature in hanging drops composed of 1.0 μL of protein solution (5.5 mg/mL) and 1.0 μL of crystallization buffer (21% PEG 3,350, 0.2 M MgCl2 · 6 H2O, 0.1 M citric acid pH 4.0), suspended above 1.0 mL of crystallization buffer. For structure solution crystals were derivatized and cryo-protected by serial transfer into crystallization buffer supplemented with 0.5 M NaI and 15% ethylene glycol and cryo-cooled in liquid nitrogen. Redundant single-wavelength anomalous diffraction (SAD) data to 2.39 Å resolution were collected at beam-line PXII at the Swiss Light Source (SLS), Villigen, CH with λ=1.7 Å. A native data set to 1.74 Å resolution was collected on a crystal from the same drop cryo-protected by serial transfer into crystallization buffer supplemented with 15% (v/v) ethylene glycol only (λ=1.0 Å; Table 1).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>37745</offset><text>HAESA complexes with IDA (PIPPSA-Hyp-SKRHN), PKGV-IDA (YPKGVPIPPSA-Hyp-SKRHN) and IDL1 (LVPPSG-Hyp-SMRHN) peptide hormones were obtained by soaking apo crystals in crystallization buffer containing the respective synthetic peptide at a final concentration of 15 mM. Soaked crystals diffracted to 1.86 Å (HAESA – IDA), 1.94 Å (HAESA-PKGV-IDA) and 2.56 Å resolution (HAESA – IDL1), respectively (Table 2). Orthorhombic crystals of the HAESA-IDA-SERK1 complex developed in 18% PEG 8000, MgCl2 · 6 H2O, 0.1 M citric acid and diffracted to 2.43 Å resolution (Table 2). Data processing and scaling was done in XDS (version: Nov 2014).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>38395</offset><text>Structure solution and refinement</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38429</offset><text>The SAD method was used to determine the structure of the isolated HAESA ectodomain. SHELXD located 32 iodine sites (CC All/Weak 37.7/14.9). 20 consistent sites were input into the program SHARP for phasing and identification of 8 additional sites at 2.39 Å resolution. Refined heavy atom sites and phases were provided to PHENIX.AUTOBUILD for density modification and automated model building. The structure was completed in alternating cycles of model building in COOT and restrained TLS refinement in REFMAC5 (version 5.8.0107) against an isomorphous high resolution native data set. Crystals contain one HAESA monomer per asymmetric unit with a solvent content of ~55%, the final model comprises residues 20 – 615. The refined structure has excellent stereochemistry, with 93.8% of all residues in the favored region of the Ramachandran plot, no outliers and a PHENIX.MOLPROBITY score of 1.34 (Table 1).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>39342</offset><text>The HAESA – IDA – SERK1 complex structure was determined by molecular replacement with the program PHASER, using the isolated HAESA and SERK1 (PDB-ID: 4LSC) LRR domain structures as search models. The solution comprises one HASEA-IDA-SERK1 complex in the asymmetric unit. The structure was completed in iterative cycles of manual model-building in COOT and restrained TLS refinement in REFMAC5. Amino acids whose side-chain position could not be modeled with confidence were truncated to alanine (0.6 – 1% of total residues), the stereochemistry of N-linked glycan structures was assessed with the CCP4 program PRIVATEER-VALIDATE. The refined model has 94.44% of all residues in the favored region of the Ramachandran plot, no outliers and a PHENIX.MOLPROBITY score of 1.17 (Table 2). Structural visualization was done with POVScript+ and POV-Ray (http://www.povray.org).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>40220</offset><text>Size-exclusion chromatography</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>40250</offset><text>Gel filtration experiments were performed using a Superdex 200 HR 10/30 column (GE Healthcare) pre-equilibrated in 20 mM citric acid (pH 5) and 100 mM NaCl. 100 μL of the isolated HAESA ectodomain (5.5 mg/mL), of the purified SERK1 LRR domain (3 mg/mL) or of mixtures of HAESA and SERK1 (either in the presence or absence of synthetic wild-type IDA, wild-type IDL1 or mutant IDA peptides at a concentration of 25 μM; 10 mg/mL; samples contained HAESA and SERK1 in 1:1 molar ratio) were loaded sequentially onto the column and elution at 0.5 mL/min was monitored by ultraviolet absorbance at 280 nm.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>40861</offset><text>Isothermal titration calorimetry</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>40894</offset><text>ITC experiments were performed using a Nano ITC (TA Instruments, New Castle, DE) with a 1.0 mL standard cell and a 250 μL titration syringe. Proteins were dialyzed extensively against ITC buffer (20 mM citric acid pH 5.0, 100 mM NaCl) and synthetic wild-type or point-mutant peptides (with wild-type IDA sequence YVPIPPSA-Hyp-SKRHN, PKGV-IDA YPKGVPIPPSA-Hyp-SKRHN, IDA-SFVN YPIPPSA-Hyp-SKRHNSFVN, IDL1 YLVPPSG-Hyp-SMRHN and CLV3 sequence YRTV-Hyp-SG-Hyp-DPLHH) were dissolved in ITC buffer prior to all titrations. Molar protein concentrations for SERK1 and HAESA were calculated using their molar extinction coefficient and a molecular weight of 27,551 and 74,896 Da, respectively (determined by MALDI-TOF mass spectrometry). Experiments were performed at 25°C. A typical experiment consisted of injecting 10 μL aliquots of peptide solution (250 μM) into 20 μM HAESA. The concentrations for the complex titrations were 150 μM of ligand (either wild-type or point-mutant IDA peptides) in the syringe and 10 μM of a 1:1 HAESA – SERK1 protein mixture in the cell at time intervals of 150 s to ensure that the titration peak returned to the baseline. Binding of SERK1 to HAESA was assessed by titrating SERK1 (100 μM) into a solution containing HAESA (10 μM) in the pre- or absence of 150 μM wild-type IDA peptide. ITC data were corrected for the heat of dilution by subtracting the mixing enthalpies for titrant solution injections into protein free ITC buffer. Data were analyzed using the NanoAnalyze program (version 2.3.6) as provided by the manufacturer.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>42490</offset><text>In vitro kinase trans-phosphorylation assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>42534</offset><text>Coding sequences of SERK1 kinase domain (SERK1-KD) (residues 264–625) and HAESA-KD (residues 671–969) were cloned into a modified pET (Novagen) vector providing an TEV-cleavable N-terminal 8xHis-StrepII-Thioredoxin tag. Point mutations were introduced into the SERK1 (Asp447→Asn; mSERK1) and HAESA (Asp837→Asn; mHAESA) coding sequences by site directed mutagenesis, thereby rendering the kinases inactive. The plasmids were transformed into E.coli Rosetta 2 (DE3) (Novagen). Protein expression was induced by adding IPTG to final concentration of 0.5 mM to cell cultures grown to an OD600 = 0.6. Cells were then incubated at 16°C for 18 hr, pelleted by centrifugation at 5000 x g and 4°C for 15 min, and resuspended in buffer A (20 mM Tris-HCl pH 8, 500 mM NaCl, 4 mM MgCl2 and 2 mM β-Mercaptoethanol) supplemented with 15 mM Imidazole and 0.1% (v/v) Igepal. After cell lysis by sonication, cell debris was removed by centrifugation at 35,000 x g and 4°C for 30 min. The recombinant proteins were isolated by Co2+ metal affinity purification using a combination of batch and gravity flow approaches (HIS-Select Cobalt Affinity Gel, Sigma, St. Louis, MO). After washing the resin with the wash buffer (buffer A + 15 mM Imidazole) proteins were eluted in buffer A supplemented with 250 mM Imidazole. All elutions were then dialyzed against 20 mM Tris-HCl pH 8, 250 mM NaCl, 4 mM MgCl2 and 0.5 mM TCEP. For SERK1-KD and mSERK1-KD the 8xHis-StrepII-Thioredoxin tag was removed with 6xHis tagged TEV protease. TEV and the cleaved tag were removed by a second metal affinity purification step. Subsequently, all proteins were purified by gel filtration on a Superdex 200 10/300 GL column equilibrated in 20 mM Tris pH 8, 250 mM NaCl, 4 mM MgCl2 and 0.5 mM TCEP. Peak fractions were collected and concentrated using Amicon Ultra centrifugation devices (10,000 MWCO). For in vitro kinase assays, 1 μg of HAESA-KD, 0.25 μg of SERK1-KD and 2 μg of mSERK1 and mHAESA were used in a final reaction volume of 20 μl. The reaction buffer consisted of 20 mM Tris pH 8, 250 mM NaCl, 4 mM MgCl2 and 0.5 mM TCEP. The reactions were started by the addition of 4 μCi [γ-32P]-ATP (Perkin-Elmer, Waltham, MA), incubated at room temperature for 45 min and stopped by the addition of 6x SDS-loading dye immediately followed by incubating the samples at 95°C. Proteins of the whole reaction were subsequently separated via SDS-PAGE in 4–15% gradient gels (TGX, Biorad, Hercules, CA) and stained with Instant Blue (Expedeon, San Diego, CA). After pictures were taken of the stained gel, 32P-derived signals were visualized by exposing the gel to an X-ray film (Fuji, SuperRX, Valhalla, NY).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>45274</offset><text>Plant material and generation of transgenic lines</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>45324</offset><text>35S::IDA wild-type and 35S::IDA (R66 → Ala/K67 → Ala) over-expressing transgenic lines in Col-0 background were generated as follows: The constructs were introduced in the destination vector pB7m34GW2 and transferred to A. tumefaciens strain pGV2260. Plants were transformed using the floral dip method. Transformants were selected in medium supplemented with BASTA up to the T3 generation. For phenotyping, plants were grown at 21°C with 50% humidity and a 16h light: 8 hr dark cycle.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>45816</offset><text>RNA analyses</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>45829</offset><text>Plants were grown on ½ Murashige and Skoog (MS) plates supplemented with 1% sucrose. After 7 d, ∼30 to 40 seedlings were collected and frozen in liquid nitrogen. Total RNA was extracted using a RNeasy plant mini kit (Qiagen, Valencia, CA), and 1 μg of the RNA solution obtained was reverse-transcribed using the SuperScritpVILO cDNA synthesis kit (Invitrogen, Grand Island, NY). RT-qPCR amplifications and measurements were performed using a 7900HT Fast Real Time PCR-System by Applied Biosystems (Carlsbad, CA). RT-qPCR amplifications were monitored using SYBR-Green fluorescent stain (Applied Biosystems). Relative quantification of gene expression data was performed using the 2−ΔΔCT (or comparative CT) method. Expression levels were normalized using the CT values obtained for the actin2 gene (forward: TGCCAATCTACGAGGGTTTC; reverse: TTCTCGATGGAAGAGCTGGT). For detection and amplification of IDA sequence we used specific primers (forward: TCGTACGATGATGGTTCTGC; reverse: GAATGGGAACGCCTTTAGGT). The presence of a single PCR product was further verified by dissociation analysis in all amplifications. All quantifications were made in quadruplicates on RNA samples obtained from three independent experiments.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>47053</offset><text>Petal break measurements</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>47078</offset><text>serk1-1, serk2-2, serk3-1, serk4-1 and serk5-1 and Col-0 wild-type plants were grown in growth chambers at 22°C under long days (16 hr day/8 hr dark) at a light intensity of 100 µE·m-2·sec-1. Petal break-strength was quantified as the force in gram equivalents required for removal of a petal from a flower when the plants had a minimum of twenty flowers and siliques. Measurements were performed using a load transducer as described in. Break-strength was measured for 15 plants and a minimum of 15 measurements at each position.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">title_1</infon><offset>47617</offset><text>Funding Information</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>47637</offset><text>This paper was supported by the following grants:</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>47687</offset><text>  to Michael Hothorn.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>47709</offset><text>  to Michael Hothorn.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>47731</offset><text>  to Michael Hothorn.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>47753</offset><text>  to Melinka A Butenko.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>47777</offset><text>  to Benjamin Brandt.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>47799</offset><text>  to Julia Santiago.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">title_1</infon><offset>47820</offset><text>Additional information</text></passage><passage><infon key="section_type">COMP_INT</infon><infon key="type">title_1</infon><offset>47843</offset><text>Competing interests</text></passage><passage><infon key="section_type">COMP_INT</infon><infon key="type">footnote</infon><offset>47863</offset><text>The authors declare that no competing interests exist.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">title_1</infon><offset>47918</offset><text>Author contributions</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>47939</offset><text>JS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>48058</offset><text>BB, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>48154</offset><text>MW, Acquisition of data, Analysis and interpretation of data.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>48216</offset><text>UH, Acquisition of data, Analysis and interpretation of data.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>48278</offset><text>LAH, Analysis and interpretation of data, Drafting or revising the article.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>48354</offset><text>MAB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>48474</offset><text>MH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>48593</offset><text>References</text></passage><passage><infon key="fpage">5253</infon><infon key="lpage">5261</infon><infon key="name_0">surname:Aalen;given-names:RB</infon><infon key="name_1">surname:Wildhagen;given-names:M</infon><infon key="name_2">surname:Stø;given-names:IM</infon><infon key="name_3">surname:Butenko;given-names:MA</infon><infon key="pub-id_doi">10.1093/jxb/ert338</infon><infon key="pub-id_pmid">24151306</infon><infon key="section_type">REF</infon><infon key="source">Journal of Experimental Botany</infon><infon key="type">ref</infon><infon key="volume">64</infon><infon key="year">2013</infon><offset>48604</offset><text>Ida: A peptide ligand regulating cell separation processes in Arabidopsis</text></passage><passage><infon key="fpage">3337</infon><infon key="lpage">3349</infon><infon key="name_0">surname:Albrecht;given-names:C</infon><infon key="name_1">surname:Russinova;given-names:E</infon><infon key="name_2">surname:Hecht;given-names:V</infon><infon key="name_3">surname:Baaijens;given-names:E</infon><infon key="name_4">surname:de Vries;given-names:S</infon><infon key="pub-id_doi">10.1105/tpc.105.036814</infon><infon key="pub-id_pmid">16284305</infon><infon key="section_type">REF</infon><infon key="source">The Plant Cell</infon><infon key="type">ref</infon><infon key="volume">17</infon><infon key="year">2005</infon><offset>48678</offset><text>The Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASES1 and 2 control male sporogenesis</text></passage><passage><infon key="fpage">611</infon><infon key="lpage">619</infon><infon key="name_0">surname:Albrecht;given-names:C</infon><infon key="name_1">surname:Russinova;given-names:E</infon><infon key="name_2">surname:Kemmerling;given-names:B</infon><infon key="name_3">surname:Kwaaitaal;given-names:M</infon><infon key="name_4">surname:de Vries;given-names:SC</infon><infon key="pub-id_doi">10.1104/pp.108.123216</infon><infon key="pub-id_pmid">18667726</infon><infon key="section_type">REF</infon><infon key="source">Plant Physiology</infon><infon key="type">ref</infon><infon key="volume">148</infon><infon key="year">2008</infon><offset>48780</offset><text>Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE proteins serve brassinosteroid-dependent and -independent signaling pathways</text></passage><passage><infon key="fpage">31</infon><infon key="lpage">43</infon><infon key="name_0">surname:Bojar;given-names:D</infon><infon key="name_1">surname:Martinez;given-names:J</infon><infon key="name_2">surname:Santiago;given-names:J</infon><infon key="name_3">surname:Rybin;given-names:V</infon><infon key="name_4">surname:Bayliss;given-names:R</infon><infon key="name_5">surname:Hothorn;given-names:M</infon><infon key="pub-id_doi">10.1111/tpj.12445</infon><infon key="pub-id_pmid">24461462</infon><infon key="section_type">REF</infon><infon key="source">The Plant Journal</infon><infon key="type">ref</infon><infon key="volume">78</infon><infon key="year">2014</infon><offset>48907</offset><text>Crystal structures of the phosphorylated BRI1 kinase domain and implications for brassinosteroid signal initiation</text></passage><passage><infon key="fpage">R225</infon><infon key="lpage">R226</infon><infon key="name_0">surname:Brandt;given-names:B</infon><infon key="name_1">surname:Hothorn;given-names:M</infon><infon key="pub-id_doi">10.1016/j.cub.2015.12.014</infon><infon key="pub-id_pmid">27003880</infon><infon key="section_type">REF</infon><infon key="source">Current Biology</infon><infon key="type">ref</infon><infon key="volume">26</infon><infon key="year">2016</infon><offset>49022</offset><text>SERK co-receptor kinases</text></passage><passage><infon key="fpage">2023</infon><infon key="lpage">2030</infon><infon key="name_0">surname:Bricogne;given-names:G</infon><infon key="name_1">surname:Vonrhein;given-names:C</infon><infon key="name_2">surname:Flensburg;given-names:C</infon><infon key="name_3">surname:Schiltz;given-names:M</infon><infon key="name_4">surname:Paciorek;given-names:W</infon><infon key="pub-id_doi">10.1107/S0907444903017694</infon><infon key="pub-id_pmid">14573958</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallographica. Section D, Biological Crystallography</infon><infon key="type">ref</infon><infon key="volume">59</infon><infon key="year">2003</infon><offset>49047</offset><text>Generation, representation and flow of phase information in structure determination: Recent developments in and around SHARP 2.0</text></passage><passage><infon key="fpage">2296</infon><infon key="lpage">2307</infon><infon key="name_0">surname:Butenko;given-names:MA</infon><infon key="name_1">surname:Patterson;given-names:SE</infon><infon key="name_2">surname:Grini;given-names:PE</infon><infon key="name_3">surname:Stenvik;given-names:GE</infon><infon key="name_4">surname:Amundsen;given-names:SS</infon><infon key="name_5">surname:Mandal;given-names:A</infon><infon key="name_6">surname:Aalen;given-names:RB</infon><infon key="pub-id_doi">10.1105/tpc.014365</infon><infon key="pub-id_pmid">12972671</infon><infon key="section_type">REF</infon><infon key="source">The Plant Cell</infon><infon key="type">ref</infon><infon key="volume">15</infon><infon key="year">2003</infon><offset>49176</offset><text>Inflorescence deficient in abscission controls floral organ abscission in Arabidopsis and identifies a novel family of putative ligands in plants</text></passage><passage><infon key="fpage">255</infon><infon key="lpage">263</infon><infon key="name_0">surname:Butenko;given-names:MA</infon><infon key="name_1">surname:Vie;given-names:AK</infon><infon key="name_2">surname:Brembu;given-names:T</infon><infon key="name_3">surname:Aalen;given-names:RB</infon><infon key="name_4">surname:Bones;given-names:AM</infon><infon key="pub-id_doi">10.1016/j.tplants.2009.02.002</infon><infon key="pub-id_pmid">19362511</infon><infon key="section_type">REF</infon><infon key="source">Trends in Plant Science</infon><infon key="type">ref</infon><infon key="volume">14</infon><infon key="year">2009</infon><offset>49322</offset><text>Plant peptides in signalling: Looking for new partners</text></passage><passage><infon key="fpage">1838</infon><infon key="lpage">1847</infon><infon key="name_0">surname:Butenko;given-names:MA</infon><infon key="name_1">surname:Wildhagen;given-names:M</infon><infon key="name_2">surname:Albert;given-names:M</infon><infon key="name_3">surname:Jehle;given-names:A</infon><infon key="name_4">surname:Kalbacher;given-names:H</infon><infon key="name_5">surname:Aalen;given-names:RB</infon><infon key="name_6">surname:Felix;given-names:G</infon><infon key="pub-id_doi">10.1105/tpc.113.120071</infon><infon key="pub-id_pmid">24808051</infon><infon key="section_type">REF</infon><infon key="source">The Plant Cell</infon><infon key="type">ref</infon><infon key="volume">26</infon><infon key="year">2014</infon><offset>49377</offset><text>Tools and strategies to match peptide-ligand receptor pairs</text></passage><passage><infon key="fpage">15629</infon><infon key="lpage">15634</infon><infon key="name_0">surname:Cho;given-names:SK</infon><infon key="name_1">surname:Larue;given-names:CT</infon><infon key="name_2">surname:Chevalier;given-names:D</infon><infon key="name_3">surname:Wang;given-names:H</infon><infon key="name_4">surname:Jinn;given-names:TL</infon><infon key="name_5">surname:Zhang;given-names:S</infon><infon key="name_6">surname:Walker;given-names:JC</infon><infon key="pub-id_doi">10.1073/pnas.0805539105</infon><infon key="pub-id_pmid">18809915</infon><infon key="section_type">REF</infon><infon key="source">Proceedings of the National Academy of Sciences of the United States of America</infon><infon key="type">ref</infon><infon key="volume">105</infon><infon key="year">2008</infon><offset>49437</offset><text>Regulation of floral organ abscission in Arabidopsis thaliana</text></passage><passage><infon key="fpage">2057</infon><infon key="lpage">2067</infon><infon key="name_0">surname:Clark;given-names:SE</infon><infon key="name_1">surname:Running;given-names:MP</infon><infon key="name_2">surname:Meyerowitz;given-names:EM</infon><infon key="section_type">REF</infon><infon key="source">Development</infon><infon key="type">ref</infon><infon key="volume">121</infon><infon key="year">1995</infon><offset>49499</offset><text>CLAVATA3 is a specific regulator of shoot and floral meristem development affecting the same processes as CLAVATA1</text></passage><passage><infon key="fpage">735</infon><infon key="lpage">743</infon><infon key="name_0">surname:Clough;given-names:SJ</infon><infon key="name_1">surname:Bent;given-names:AF</infon><infon key="pub-id_doi">10.1046/j.1365-313x.1998.00343.x</infon><infon key="pub-id_pmid">10069079</infon><infon key="section_type">REF</infon><infon key="source">The Plant Journal</infon><infon key="type">ref</infon><infon key="volume">16</infon><infon key="year">1998</infon><offset>49614</offset><text>Floral dip: A simplified method for agrobacterium-mediated transformation of Arabidopsis thaliana</text></passage><passage><infon key="fpage">3350</infon><infon key="lpage">3361</infon><infon key="name_0">surname:Colcombet;given-names:J</infon><infon key="name_1">surname:Boisson-Dernier;given-names:A</infon><infon key="name_2">surname:Ros-Palau;given-names:R</infon><infon key="name_3">surname:Vera;given-names:CE</infon><infon key="name_4">surname:Schroeder;given-names:JI</infon><infon key="pub-id_doi">10.1105/tpc.105.036731</infon><infon key="pub-id_pmid">16284306</infon><infon key="section_type">REF</infon><infon key="source">The Plant Cell</infon><infon key="type">ref</infon><infon key="volume">17</infon><infon key="year">2005</infon><offset>49712</offset><text>Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASES1 and 2 are essential for tapetum development and microspore maturation</text></passage><passage><infon key="fpage">W375</infon><infon key="lpage">W383</infon><infon key="name_0">surname:Davis;given-names:IW</infon><infon key="name_1">surname:Leaver-Fay;given-names:A</infon><infon key="name_10">surname:Richardson;given-names:DC</infon><infon key="name_2">surname:Chen;given-names:VB</infon><infon key="name_3">surname:Block;given-names:JN</infon><infon key="name_4">surname:Kapral;given-names:GJ</infon><infon key="name_5">surname:Wang;given-names:X</infon><infon key="name_6">surname:Murray;given-names:LW</infon><infon key="name_7">surname:Arendall;given-names:WB</infon><infon key="name_8">surname:Snoeyink;given-names:J</infon><infon key="name_9">surname:Richardson;given-names:JS</infon><infon key="pub-id_doi">10.1093/nar/gkm216</infon><infon key="pub-id_pmid">17452350</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Research</infon><infon key="type">ref</infon><infon key="volume">35</infon><infon key="year">2007</infon><offset>49834</offset><text>Molprobity: All-atom contacts and structure validation for proteins and nucleic acids</text></passage><passage><infon key="fpage">2126</infon><infon key="lpage">2132</infon><infon key="name_0">surname:Emsley;given-names:P</infon><infon key="name_1">surname:Cowtan;given-names:K</infon><infon key="pub-id_doi">10.1107/S0907444904019158</infon><infon key="pub-id_pmid">15572765</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallographica Section D Biological Crystallography</infon><infon key="type">ref</infon><infon key="volume">60</infon><infon key="year">2004</infon><offset>49920</offset><text>Coot : Model-building tools for molecular graphics</text></passage><passage><infon key="fpage">944</infon><infon key="lpage">947</infon><infon key="name_0">surname:Fenn;given-names:TD</infon><infon key="name_1">surname:Ringe;given-names:D</infon><infon key="name_2">surname:Petsko;given-names:GA</infon><infon key="pub-id_doi">10.1107/S0021889803006721</infon><infon key="section_type">REF</infon><infon key="source">Journal of Applied Crystallography</infon><infon key="type">ref</infon><infon key="volume">36</infon><infon key="year">2003</infon><offset>49971</offset><text>Povscript+ : A program for model and data visualization using persistence of vision ray-tracing</text></passage><passage><infon key="elocation-id">e15075</infon><infon key="name_0">surname:Gou;given-names:X</infon><infon key="name_1">surname:Yin;given-names:H</infon><infon key="name_2">surname:He;given-names:K</infon><infon key="name_3">surname:Du;given-names:J</infon><infon key="name_4">surname:Yi;given-names:J</infon><infon key="name_5">surname:Xu;given-names:S</infon><infon key="name_6">surname:Lin;given-names:H</infon><infon key="name_7">surname:Clouse;given-names:SD</infon><infon key="name_8">surname:Li;given-names:J</infon><infon key="pub-id_doi">10.1371/journal.pgen.1002452</infon><infon key="section_type">REF</infon><infon key="source">PLoS Genetics</infon><infon key="type">ref</infon><infon key="volume">8</infon><infon key="year">2012</infon><offset>50067</offset><text>Genetic evidence for an indispensable role of somatic embryogenesis receptor kinases in brassinosteroid signaling</text></passage><passage><infon key="elocation-id">e15075</infon><infon key="name_0">surname:Hashimoto;given-names:Y</infon><infon key="name_1">surname:Zhang;given-names:S</infon><infon key="name_2">surname:Blissard;given-names:GW</infon><infon key="pub-id_doi">10.1186/1472-6750-10-50</infon><infon key="section_type">REF</infon><infon key="source">BMC Biotechnology</infon><infon key="type">ref</infon><infon key="volume">10</infon><infon key="year">2010</infon><offset>50181</offset><text>Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae), is permissive for AcMNPV infection and produces high levels of recombinant proteins</text></passage><passage><infon key="fpage">793</infon><infon key="lpage">800</infon><infon key="name_0">surname:Hasler;given-names:M</infon><infon key="name_1">surname:Hothorn;given-names:LA</infon><infon key="pub-id_doi">10.1002/bimj.200710466</infon><infon key="pub-id_pmid">18932141</infon><infon key="section_type">REF</infon><infon key="source">Biometrical Journal</infon><infon key="type">ref</infon><infon key="volume">50</infon><infon key="year">2008</infon><offset>50366</offset><text>Multiple contrast tests in the presence of heteroscedasticity</text></passage><passage><infon key="fpage">803</infon><infon key="lpage">816</infon><infon key="name_0">surname:Hecht;given-names:V</infon><infon key="name_1">surname:Vielle-Calzada;given-names:JP</infon><infon key="name_2">surname:Hartog;given-names:MV</infon><infon key="name_3">surname:Schmidt;given-names:ED</infon><infon key="name_4">surname:Boutilier;given-names:K</infon><infon key="name_5">surname:Grossniklaus;given-names:U</infon><infon key="name_6">surname:de Vries;given-names:SC</infon><infon key="pub-id_doi">10.1104/pp.010324</infon><infon key="pub-id_pmid">11706164</infon><infon key="section_type">REF</infon><infon key="source">Plant Physiology</infon><infon key="type">ref</infon><infon key="volume">127</infon><infon key="year">2001</infon><offset>50428</offset><text>The Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 gene is expressed in developing ovules and embryos and enhances embryogenic competence in culture</text></passage><passage><infon key="fpage">346</infon><infon key="lpage">363</infon><infon key="name_0">surname:Hothorn;given-names:T</infon><infon key="name_1">surname:Bretz;given-names:F</infon><infon key="name_2">surname:Westfall;given-names:P</infon><infon key="pub-id_doi">10.1002/bimj.200810425</infon><infon key="pub-id_pmid">18481363</infon><infon key="section_type">REF</infon><infon key="source">Biometrical Journal</infon><infon key="type">ref</infon><infon key="volume">50</infon><infon key="year">2008</infon><offset>50582</offset><text>Simultaneous inference in general parametric models</text></passage><passage><infon key="fpage">467</infon><infon key="lpage">471</infon><infon key="name_0">surname:Hothorn;given-names:M</infon><infon key="name_1">surname:Belkhadir;given-names:Y</infon><infon key="name_2">surname:Dreux;given-names:M</infon><infon key="name_3">surname:Dabi;given-names:T</infon><infon key="name_4">surname:Noel;given-names:JP</infon><infon key="name_5">surname:Wilson;given-names:IA</infon><infon key="name_6">surname:Chory;given-names:J</infon><infon key="pub-id_doi">10.1038/nature10153</infon><infon key="pub-id_pmid">21666665</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">474</infon><infon key="year">2011</infon><offset>50634</offset><text>Structural basis of steroid hormone perception by the receptor kinase BRI1</text></passage><passage><infon key="fpage">108</infon><infon key="lpage">117</infon><infon key="name_0">surname:Jinn;given-names:TL</infon><infon key="name_1">surname:Stone;given-names:JM</infon><infon key="name_2">surname:Walker;given-names:JC</infon><infon key="pub-id_doi">10.1101/gad.14.1.108</infon><infon key="pub-id_pmid">10640280</infon><infon key="section_type">REF</infon><infon key="source">Genes &amp; Development</infon><infon key="type">ref</infon><infon key="volume">14</infon><infon key="year">2000</infon><offset>50709</offset><text>HAESA, an Arabidopsis leucine-rich repeat receptor kinase, controls floral organ abscission</text></passage><passage><infon key="fpage">2577</infon><infon key="lpage">2637</infon><infon key="name_0">surname:Kabsch;given-names:W</infon><infon key="name_1">surname:Sander;given-names:C</infon><infon key="pub-id_doi">10.1002/bip.360221211</infon><infon key="pub-id_pmid">6667333</infon><infon key="section_type">REF</infon><infon key="source">Biopolymers</infon><infon key="type">ref</infon><infon key="volume">22</infon><infon key="year">1983</infon><offset>50801</offset><text>Dictionary of protein secondary structure: Pattern recognition of hydrogen-bonded and geometrical features</text></passage><passage><infon key="fpage">795</infon><infon key="lpage">800</infon><infon key="name_0">surname:Kabsch;given-names:W</infon><infon key="pub-id_doi">10.1107/S0021889893005588</infon><infon key="section_type">REF</infon><infon key="source">Journal of Applied Crystallography</infon><infon key="type">ref</infon><infon key="volume">26</infon><infon key="year">1993</infon><offset>50908</offset><text>Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants</text></passage><passage><infon key="fpage">845</infon><infon key="lpage">848</infon><infon key="name_0">surname:Kondo;given-names:T</infon><infon key="name_1">surname:Sawa;given-names:S</infon><infon key="name_2">surname:Kinoshita;given-names:A</infon><infon key="name_3">surname:Mizuno;given-names:S</infon><infon key="name_4">surname:Kakimoto;given-names:T</infon><infon key="name_5">surname:Fukuda;given-names:H</infon><infon key="name_6">surname:Sakagami;given-names:Y</infon><infon key="pub-id_doi">10.1126/science.1128439</infon><infon key="pub-id_pmid">16902141</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">313</infon><infon key="year">2006</infon><offset>51021</offset><text>A plant peptide encoded by CLV3 identified by in situ MALDI-TOF MS analysis</text></passage><passage><infon key="fpage">2</infon><infon key="name_0">surname:Lease;given-names:KA</infon><infon key="name_1">surname:Cho;given-names:SK</infon><infon key="name_2">surname:Walker;given-names:JC</infon><infon key="pub-id_doi">10.1186/1746-4811-2-2</infon><infon key="pub-id_pmid">16483376</infon><infon key="section_type">REF</infon><infon key="source">Plant Methods</infon><infon key="type">ref</infon><infon key="volume">2</infon><infon key="year">2006</infon><offset>51097</offset><text>A petal breakstrength meter for Arabidopsis abscission studies</text></passage><passage><infon key="fpage">817</infon><infon key="lpage">828</infon><infon key="name_0">surname:Lewis;given-names:MW</infon><infon key="name_1">surname:Leslie;given-names:ME</infon><infon key="name_2">surname:Fulcher;given-names:EH</infon><infon key="name_3">surname:Darnielle;given-names:L</infon><infon key="name_4">surname:Healy;given-names:PN</infon><infon key="name_5">surname:Youn;given-names:JY</infon><infon key="name_6">surname:Liljegren;given-names:SJ</infon><infon key="pub-id_doi">10.1111/j.1365-313X.2010.04194.x</infon><infon key="pub-id_pmid">20230490</infon><infon key="section_type">REF</infon><infon key="source">The Plant Journal</infon><infon key="type">ref</infon><infon key="volume">62</infon><infon key="year">2010</infon><offset>51160</offset><text>The SERK1 receptor-like kinase regulates organ separation in Arabidopsis flowers</text></passage><passage><infon key="fpage">402</infon><infon key="lpage">408</infon><infon key="name_0">surname:Livak;given-names:KJ</infon><infon key="name_1">surname:Schmittgen;given-names:TD</infon><infon key="pub-id_doi">10.1006/meth.2001.1262</infon><infon key="pub-id_pmid">11846609</infon><infon key="section_type">REF</infon><infon key="source">Methods</infon><infon key="type">ref</infon><infon key="volume">25</infon><infon key="year">2001</infon><offset>51241</offset><text>Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method</text></passage><passage><infon key="fpage">1065</infon><infon key="lpage">1067</infon><infon key="name_0">surname:Matsuzaki;given-names:Y</infon><infon key="name_1">surname:Ogawa-Ohnishi;given-names:M</infon><infon key="name_2">surname:Mori;given-names:A</infon><infon key="name_3">surname:Matsubayashi;given-names:Y</infon><infon key="pub-id_doi">10.1126/science.1191132</infon><infon key="pub-id_pmid">20798316</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">329</infon><infon key="year">2010</infon><offset>51352</offset><text>Secreted peptide signals required for maintenance of root stem cell niche in Arabidopsis</text></passage><passage><infon key="fpage">658</infon><infon key="lpage">674</infon><infon key="name_0">surname:McCoy;given-names:AJ</infon><infon key="name_1">surname:Grosse-Kunstleve;given-names:RW</infon><infon key="name_2">surname:Adams;given-names:PD</infon><infon key="name_3">surname:Winn;given-names:MD</infon><infon key="name_4">surname:Storoni;given-names:LC</infon><infon key="name_5">surname:Read;given-names:RJ</infon><infon key="pub-id_doi">10.1107/S0021889807021206</infon><infon key="pub-id_pmid">19461840</infon><infon key="section_type">REF</infon><infon key="source">Journal of Applied Crystallography</infon><infon key="type">ref</infon><infon key="volume">40</infon><infon key="year">2007</infon><offset>51441</offset><text>Phaser crystallographic software</text></passage><passage><infon key="fpage">2361</infon><infon key="lpage">2372</infon><infon key="name_0">surname:Meng;given-names:X</infon><infon key="name_1">surname:Chen;given-names:X</infon><infon key="name_2">surname:Mang;given-names:H</infon><infon key="name_3">surname:Liu;given-names:C</infon><infon key="name_4">surname:Yu;given-names:X</infon><infon key="name_5">surname:Gao;given-names:X</infon><infon key="name_6">surname:Torii;given-names:KU</infon><infon key="name_7">surname:He;given-names:P</infon><infon key="name_8">surname:Shan;given-names:L</infon><infon key="pub-id_doi">10.1016/j.cub.2015.07.068</infon><infon key="pub-id_pmid">26320950</infon><infon key="section_type">REF</infon><infon key="source">Current Biology</infon><infon key="type">ref</infon><infon key="volume">25</infon><infon key="year">2015</infon><offset>51474</offset><text>Differential function of Arabidopsis SERK family receptor-like kinases in stomatal patterning</text></passage><passage><infon key="fpage">1330</infon><infon key="lpage">1338</infon><infon key="name_0">surname:Meng;given-names:X</infon><infon key="name_1">surname:Zhou;given-names:J</infon><infon key="name_2">surname:Tang;given-names:J</infon><infon key="name_3">surname:Li;given-names:B</infon><infon key="name_4">surname:de Oliveira;given-names:MVV</infon><infon key="name_5">surname:Chai;given-names:J</infon><infon key="name_6">surname:He;given-names:P</infon><infon key="name_7">surname:Shan;given-names:L</infon><infon key="pub-id_doi">10.1016/j.celrep.2016.01.023</infon><infon key="pub-id_pmid">26854226</infon><infon key="section_type">REF</infon><infon key="source">Cell Reports</infon><infon key="type">ref</infon><infon key="volume">14</infon><infon key="year">2016</infon><offset>51568</offset><text>Ligand-induced receptor-like kinase complex regulates floral organ abscission in Arabidopsis</text></passage><passage><infon key="fpage">240</infon><infon key="lpage">255</infon><infon key="name_0">surname:Murshudov;given-names:GN</infon><infon key="name_1">surname:Vagin;given-names:AA</infon><infon key="name_2">surname:Dodson;given-names:EJ</infon><infon key="pub-id_doi">10.1107/S0907444996012255</infon><infon key="pub-id_pmid">15299926</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallographica Section D Biological Crystallography</infon><infon key="type">ref</infon><infon key="volume">53</infon><infon key="year">1997</infon><offset>51661</offset><text>Refinement of macromolecular structures by the maximum-likelihood method</text></passage><passage><infon key="fpage">4413</infon><infon key="lpage">4419</infon><infon key="name_0">surname:Muto;given-names:T</infon><infon key="name_1">surname:Todoroki;given-names:Y</infon><infon key="pub-id_doi">10.1016/j.bmc.2013.04.048</infon><infon key="pub-id_pmid">23673217</infon><infon key="section_type">REF</infon><infon key="source">Bioorganic &amp; Medicinal Chemistry</infon><infon key="type">ref</infon><infon key="volume">21</infon><infon key="year">2013</infon><offset>51734</offset><text>Brassinolide-2,3-acetonide: A brassinolide-induced rice lamina joint inclination antagonist</text></passage><passage><infon key="fpage">1251</infon><infon key="lpage">1263</infon><infon key="name_0">surname:Niederhuth;given-names:CE</infon><infon key="name_1">surname:Cho;given-names:SK</infon><infon key="name_2">surname:Seitz;given-names:K</infon><infon key="name_3">surname:Walker;given-names:JC</infon><infon key="pub-id_doi">10.1111/jipb.12116</infon><infon key="pub-id_pmid">24138310</infon><infon key="section_type">REF</infon><infon key="source">Journal of Integrative Plant Biology</infon><infon key="type">ref</infon><infon key="volume">55</infon><infon key="year">2013</infon><offset>51826</offset><text>Letting go is never easy: Abscission and receptor-like protein kinases</text></passage><passage><infon key="elocation-id">e15075</infon><infon key="name_0">surname:Niederhuth;given-names:CE</infon><infon key="name_1">surname:Patharkar;given-names:OR</infon><infon key="name_2">surname:Walker;given-names:JC</infon><infon key="pub-id_doi">10.1186/1471-2164-14-37</infon><infon key="section_type">REF</infon><infon key="source">BMC Genomics</infon><infon key="type">ref</infon><infon key="volume">14</infon><infon key="year">2013</infon><offset>51897</offset><text>Transcriptional profiling of the Arabidopsis abscission mutant hae hsl2 by RNA-Seq</text></passage><passage><infon key="fpage">294</infon><infon key="name_0">surname:Ogawa;given-names:M</infon><infon key="name_1">surname:Shinohara;given-names:H</infon><infon key="name_2">surname:Sakagami;given-names:Y</infon><infon key="name_3">surname:Matsubayashi;given-names:Y</infon><infon key="pub-id_doi">10.1126/science.1150083</infon><infon key="pub-id_pmid">18202283</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">319</infon><infon key="year">2008</infon><offset>51980</offset><text>Arabidopsis CLV3 peptide directly binds CLV1 ectodomain</text></passage><passage><infon key="fpage">578</infon><infon key="lpage">580</infon><infon key="name_0">surname:Ohyama;given-names:K</infon><infon key="name_1">surname:Shinohara;given-names:H</infon><infon key="name_2">surname:Ogawa-Ohnishi;given-names:M</infon><infon key="name_3">surname:Matsubayashi;given-names:Y</infon><infon key="pub-id_doi">10.1038/nchembio.182</infon><infon key="pub-id_pmid">19525968</infon><infon key="section_type">REF</infon><infon key="source">Nature Chemical Biology</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2009</infon><offset>52036</offset><text>A glycopeptide regulating stem cell fate in Arabidopsis thaliana</text></passage><passage><infon key="section_type">REF</infon><infon key="source">R Foundation for Statistical Computing</infon><infon key="type">ref</infon><infon key="year">2014</infon><offset>52101</offset></passage><passage><infon key="fpage">709</infon><infon key="lpage">714</infon><infon key="name_0">surname:Salaj;given-names:J</infon><infon key="name_1">surname:von Recklinghausen;given-names:IR</infon><infon key="name_2">surname:Hecht;given-names:V</infon><infon key="name_3">surname:de Vries;given-names:SC</infon><infon key="name_4">surname:Schel;given-names:JH</infon><infon key="name_5">surname:van Lammeren;given-names:AA</infon><infon key="pub-id_doi">10.1016/j.plaphy.2008.04.011</infon><infon key="pub-id_pmid">18515128</infon><infon key="section_type">REF</infon><infon key="source">Plant Physiology and Biochemistry</infon><infon key="type">ref</infon><infon key="volume">46</infon><infon key="year">2008</infon><offset>52102</offset><text>AtSERK1 expression precedes and coincides with early somatic embryogenesis in Arabidopsis thaliana</text></passage><passage><infon key="fpage">305</infon><infon key="lpage">320</infon><infon key="name_0">surname:Sanner;given-names:MF</infon><infon key="name_1">surname:Olson;given-names:AJ</infon><infon key="name_2">surname:Spehner;given-names:JC</infon><infon key="pub-id_doi">10.1002/(SICI)1097-0282(199603)38:3&amp;amp;lt;305::AID-BIP4&amp;amp;gt;3.0.CO;2-Y</infon><infon key="pub-id_pmid">8906967</infon><infon key="section_type">REF</infon><infon key="source">Biopolymers</infon><infon key="type">ref</infon><infon key="volume">38</infon><infon key="year">1996</infon><offset>52201</offset><text>Reduced surface: An efficient way to compute molecular surfaces</text></passage><passage><infon key="fpage">889</infon><infon key="lpage">892</infon><infon key="name_0">surname:Santiago;given-names:J</infon><infon key="name_1">surname:Henzler;given-names:C</infon><infon key="name_2">surname:Hothorn;given-names:M</infon><infon key="pub-id_doi">10.1126/science.1242468</infon><infon key="pub-id_pmid">23929946</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">341</infon><infon key="year">2013</infon><offset>52265</offset><text>Molecular mechanism for plant steroid receptor activation by somatic embryogenesis co-receptor kinases</text></passage><passage><infon key="fpage">41263</infon><infon key="lpage">41269</infon><infon key="name_0">surname:Shah;given-names:K</infon><infon key="name_1">surname:Vervoort;given-names:J</infon><infon key="name_2">surname:de Vries;given-names:SC</infon><infon key="pub-id_doi">10.1074/jbc.M102381200</infon><infon key="pub-id_pmid">11509554</infon><infon key="section_type">REF</infon><infon key="source">The Journal of Biological Chemistry</infon><infon key="type">ref</infon><infon key="volume">276</infon><infon key="year">2001</infon><offset>52368</offset><text>Role of threonines in the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 activation loop in phosphorylation</text></passage><passage><infon key="fpage">112</infon><infon key="lpage">122</infon><infon key="name_0">surname:Sheldrick;given-names:GM</infon><infon key="pub-id_doi">10.1107/S0108767307043930</infon><infon key="pub-id_pmid">18156677</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallographica Section A Foundations of Crystallography</infon><infon key="type">ref</infon><infon key="volume">64</infon><infon key="year">2008</infon><offset>52490</offset><text>A short history of SHELX</text></passage><passage><infon key="fpage">845</infon><infon key="lpage">854</infon><infon key="name_0">surname:Shinohara;given-names:H</infon><infon key="name_1">surname:Moriyama;given-names:Y</infon><infon key="name_2">surname:Ohyama;given-names:K</infon><infon key="name_3">surname:Matsubayashi;given-names:Y</infon><infon key="pub-id_doi">10.1111/j.1365-313X.2012.04934.x</infon><infon key="pub-id_pmid">22321211</infon><infon key="section_type">REF</infon><infon key="source">The Plant Journal</infon><infon key="type">ref</infon><infon key="volume">70</infon><infon key="year">2012</infon><offset>52515</offset><text>Biochemical mapping of a ligand-binding domain within Arabidopsis BAM1 reveals diversified ligand recognition mechanisms of plant LRR-RKs</text></passage><passage><infon key="fpage">1467</infon><infon key="lpage">1476</infon><infon key="name_0">surname:Stenvik;given-names:GE</infon><infon key="name_1">surname:Butenko;given-names:MA</infon><infon key="name_2">surname:Urbanowicz;given-names:BR</infon><infon key="name_3">surname:Rose;given-names:JK</infon><infon key="name_4">surname:Aalen;given-names:RB</infon><infon key="pub-id_doi">10.1105/tpc.106.042036</infon><infon key="pub-id_pmid">16679455</infon><infon key="section_type">REF</infon><infon key="source">The Plant Cell</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">2006</infon><offset>52653</offset><text>Overexpression of INFLORESCENCE DEFICIENT IN ABSCISSION activates cell separation in vestigial abscission zones in Arabidopsis</text></passage><passage><infon key="fpage">1805</infon><infon key="lpage">1817</infon><infon key="name_0">surname:Stenvik;given-names:GE</infon><infon key="name_1">surname:Tandstad;given-names:NM</infon><infon key="name_2">surname:Guo;given-names:Y</infon><infon key="name_3">surname:Shi;given-names:CL</infon><infon key="name_4">surname:Kristiansen;given-names:W</infon><infon key="name_5">surname:Holmgren;given-names:A</infon><infon key="name_6">surname:Clark;given-names:SE</infon><infon key="name_7">surname:Aalen;given-names:RB</infon><infon key="name_8">surname:Butenko;given-names:MA</infon><infon key="pub-id_doi">10.1105/tpc.108.059139</infon><infon key="pub-id_pmid">18660431</infon><infon key="section_type">REF</infon><infon key="source">The Plant Cell</infon><infon key="type">ref</infon><infon key="volume">20</infon><infon key="year">2008</infon><offset>52780</offset><text>The EPIP peptide of INFLORESCENCE DEFICIENT IN ABSCISSION is sufficient to induce abscission in Arabidopsis through the receptor-like kinases HAESA and HAESA-LIKE2</text></passage><passage><infon key="fpage">1326</infon><infon key="lpage">1329</infon><infon key="name_0">surname:Sun;given-names:Y</infon><infon key="name_1">surname:Han;given-names:Z</infon><infon key="name_2">surname:Tang;given-names:J</infon><infon key="name_3">surname:Hu;given-names:Z</infon><infon key="name_4">surname:Chai;given-names:C</infon><infon key="name_5">surname:Zhou;given-names:B</infon><infon key="name_6">surname:Chai;given-names:J</infon><infon key="pub-id_doi">10.1038/cr.2013.131</infon><infon key="pub-id_pmid">24126715</infon><infon key="section_type">REF</infon><infon key="source">Cell Research</infon><infon key="type">ref</infon><infon key="volume">23</infon><infon key="year">2013</infon><offset>52944</offset><text>Structure reveals that BAK1 as a co-receptor recognizes the BRI1-bound brassinolide</text></passage><passage><infon key="fpage">110</infon><infon key="lpage">120</infon><infon key="name_0">surname:Tang;given-names:J</infon><infon key="name_1">surname:Han;given-names:Z</infon><infon key="name_2">surname:Sun;given-names:Y</infon><infon key="name_3">surname:Zhang;given-names:H</infon><infon key="name_4">surname:Gong;given-names:X</infon><infon key="name_5">surname:Chai;given-names:J</infon><infon key="pub-id_doi">10.1038/cr.2014.161</infon><infon key="pub-id_pmid">25475059</infon><infon key="section_type">REF</infon><infon key="source">Cell Research</infon><infon key="type">ref</infon><infon key="volume">25</infon><infon key="year">2015</infon><offset>53028</offset><text>Structural basis for recognition of an endogenous peptide by the plant receptor kinase PEPR1</text></passage><passage><infon key="elocation-id">e15075</infon><infon key="name_0">surname:Taylor;given-names:I</infon><infon key="name_1">surname:Wang;given-names:Y</infon><infon key="name_2">surname:Seitz;given-names:K</infon><infon key="name_3">surname:Baer;given-names:J</infon><infon key="name_4">surname:Bennewitz;given-names:S</infon><infon key="name_5">surname:Mooney;given-names:BP</infon><infon key="name_6">surname:Walker;given-names:JC</infon><infon key="pub-id_doi">10.1371/journal.pone.0147203</infon><infon key="section_type">REF</infon><infon key="source">PLOS ONE</infon><infon key="type">ref</infon><infon key="volume">11</infon><infon key="year">2016</infon><offset>53121</offset><text>Analysis of phosphorylation of the receptor-like protein kinase HAESA during arabidopsis floral abscission</text></passage><passage><infon key="fpage">61</infon><infon key="lpage">69</infon><infon key="name_0">surname:Terwilliger;given-names:TC</infon><infon key="name_1">surname:Grosse-Kunstleve;given-names:RW</infon><infon key="name_2">surname:Afonine;given-names:PV</infon><infon key="name_3">surname:Moriarty;given-names:NW</infon><infon key="name_4">surname:Zwart;given-names:PH</infon><infon key="name_5">surname:Hung;given-names:L-W</infon><infon key="name_6">surname:Read;given-names:RJ</infon><infon key="name_7">surname:Adams;given-names:PD</infon><infon key="pub-id_doi">10.1107/S090744490705024X</infon><infon key="pub-id_pmid">18094468</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallographica Section D Biological Crystallography</infon><infon key="type">ref</infon><infon key="volume">64</infon><infon key="year">2008</infon><offset>53228</offset><text>Iterative model building, structure refinement and density modification with the PHENIX autobuild wizard</text></passage><passage><infon key="fpage">265</infon><infon key="lpage">268</infon><infon key="name_0">surname:Wang;given-names:J</infon><infon key="name_1">surname:Li;given-names:H</infon><infon key="name_2">surname:Han;given-names:Z</infon><infon key="name_3">surname:Zhang;given-names:H</infon><infon key="name_4">surname:Wang;given-names:T</infon><infon key="name_5">surname:Lin;given-names:G</infon><infon key="name_6">surname:Chang;given-names:J</infon><infon key="name_7">surname:Yang;given-names:W</infon><infon key="name_8">surname:Chai;given-names:J</infon><infon key="pub-id_doi">10.1038/nature14858</infon><infon key="pub-id_pmid">26308901</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">525</infon><infon key="year">2015</infon><offset>53333</offset><text>Allosteric receptor activation by the plant peptide hormone phytosulfokine</text></passage><passage><infon key="section_type">REF</infon><infon key="type">paragraph</infon><offset>53408</offset><text>10.7554/eLife.15075.017</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">title</infon><offset>53432</offset><text>Decision letter</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>53448</offset><text>Zhang</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>53454</offset><text>Mingjie</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>53462</offset><text>In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>53746</offset><text>Thank you for submitting your article &quot;Mechanistic insight into a peptide hormone signaling complex mediating floral organ abscission&quot; to eLife for consideration by eLife. Your article has been favorably evaluated by John Kuyiyan (Senior editor) and three reviewers, one of whom, (Mingjie Zhang) is a member of our Board of Reviewing Editors.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>54089</offset><text>The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. As you will see that the required revisions are essentially clarifications and some additional analysis of existing data in nature.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>54372</offset><text>Summary:</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>54381</offset><text>In this work, Hothorn and colleagues investigated the structural basis governing the recognition of peptide hormone IDA during plant floral abscission process. Through an array of complex structures, supplemented with biochemical and genetic experiments, the authors uncovered the IDA recognition mechanism by a co-receptor (HAESA and SERK1) detection mechanism. The structures also reveal the specific recognition mechanism of the 12-residue IDA core peptide sequence by the co-receptors, and suggest that this 12-residue IDA sequence is likely to be the mature peptide hormone functioning in plants. The comparison of the structures of the HAESA/SERK1/IDA complex and the previously determined BRA1/SERK1/brassinolide complex by the same group also suggests a co-receptor pairing mechanism for various plant hormones. The story gives detailed and novel mechanistic insights in the perception of IDA during floral abscission, and is convincing and worthy to be considered for publication in eLife with the following revisions.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>55409</offset><text>Key issues which need to be addressed:</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>55448</offset><text>1) As the results reported here rest largely on the interpretation of the structural data, the following points need to be addressed by the authors. i) Temperature factors (Wilson B and residual) are unusually high for the reported resolution. Are there portions of the structure that exhibit more disorder or are these high temperature factors throughout the structure? Are there potential problems with radiation damage due to the high multiplicity? ii) A simulated annealing omit map figure should be provided as the peptides all exhibit very high temperature factors. iii) How many side chains were trimmed due to poor electron density? If this is a significant percentage, it should be noted.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>56146</offset><text>2) A further discussion comparing the mechanisms of BRI1-BL-SERK1 and HAESA-IDA-SERK1 would be helpful as a structural comparison is presented. As BR1 binds its ligand with high affinity, the low affinity of HAESA for IDA could be further discussed.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>56396</offset><text>3) Are the results here applicable to the related receptor HSL2? Are the residues that interact with SERK1 and IDA conserved in HSL2?</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>56530</offset><text>4) The authors show that N-terminal extension of the peptide does not impact on binding efficiency, but what would happen if the peptide was extended at the C-terminal end, at the suggested cleavage site? Would cleavage be required for recognition? A brief discussion on this point may help.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>56822</offset><text>5) Figure 3C: Could the authors comment on the difference between the blue (SERK1 vs. HAESA-IDA) and the black (IDA vs. HAESA-SERK1) line? SERK1 vs. HAESA-IDA gives a Kd of 75 nM, yet IDA vs. SERK1-HAESA gives a Kd of 350 nM. In the text the authors keep referring to the 75 nM Kd, but not the 350 nM Kd. Is it really fair to say the Kd is 75 nM?</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>57169</offset><text>6) In the Discussion, can the authors comment further on the discrepancy between their study and the study of Lewis et al. (Plant J, 2010) concerning the role of SERK1 in floral abscission? Similarly, could the authors comment on the fact that the LRR-RLP EVD/SOBIR seems to be a negative regulator of the HAESA/HSL2 pathway (Leslie et al., Development, 2010), which seems puzzling given that EVD/SOBIR function is normally restricted to LRR-RLPs (Gust &amp; Felix, Curr Op Plant Biol, 2014).</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>57658</offset><text>7) Given that the central hydroxyproline in IDA is of such crucial importance for binding, isn't it surprising that IDAΔ69N does not bind to HAESA at all? Wouldn't it be expected that the remaining part of the peptide still binds to HAESA?</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>57902</offset><text>8) Figure 1—figure supplement 2: How is it possible that charged amino acids are involved in hydrophobic interactions?</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>58023</offset><text>9) Are the distances shown in the graphical representation of the structures proportional? It would seem that some of the aromatic rings could cause steric hindrance.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>58190</offset><text>10) Why did the authors decide to express HAESA and SERK1 without signal peptide? Would it make a difference for binding of IDA, if they leave the SP on?</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>58344</offset><text>11) Figure 7: Are the homologous regions also the active parts of these peptides? And could the authors display amino acids numbers on either side of the fragments?</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>58509</offset><text>12) Have the authors ever measured dissociation of the peptide from the complex? And in this regard, to what does &quot;highly stable receptor – co-receptor complex&quot; refer/compare to?</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>58690</offset><text>13) Figures 3A and 5C require statistical analyses.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>58742</offset><text>10.7554/eLife.15075.018</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">title</infon><offset>58766</offset><text>Author response</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>58782</offset><text> 1) As the results reported here rest largely on the interpretation of the structural data, the following points need to be addressed by the authors. i) Temperature factors (Wilson B and residual) are unusually high for the reported resolution. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>59028</offset><text>We thank the reviewer(s) for pointing out this issue: Indeed our mean B-values deviate substantially from the expected mean B-values (resolution 1.75 – 2.00 A, B(Wilson) ~ 27.0 over 5,510 structures in the PDB; Pavel Afonine, personal communication). We would like to note that due to the many N-glycosylation sites dispersed over the HAESA LRR domain (shown in Figure 1—figure supplement 1D), we find relatively few crystal contacts in our P3121 crystal form, which may rationalize our high B-values. We have reanalyzed our space group assignment (using the CCP4 program ZANUDA) and checked for any signs of problems during data collection (ice rings, multiple crystal lattices, splitting, using the programs XDS and XDSSTAT), as well as for twinning and pseudosymmetry (using phenix.xtriage). No such problems appear to exist, our structures refine very well and our refined B-values are in good agreement with our Wilson B-factors (see Table 2). Thus, the high B-values appear to represent an intrinsic property of our crystals and are not the result of a poor data collection strategy or inappropriate crystallographic analysis.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>60165</offset><text>Are there portions of the structure that exhibit more disorder or are these high temperature factors throughout the structure? </text></passage><passage><infon key="file">elife-15075-resp-fig1.jpg</infon><infon key="id">fig8</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>60293</offset><text>Cα trace of the HAESA LRR domain and IDA peptide colored according to B-factor from low (60.9, in blue) to high (134.7, in red).</text></passage><passage><infon key="file">elife-15075-resp-fig1.jpg</infon><infon key="id">fig8</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>60426</offset><text>Mean B-value is 79.5.</text></passage><passage><infon key="file">elife-15075-resp-fig1.jpg</infon><infon key="id">fig8</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>60448</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.15075.015</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>60495</offset><text>Yes. As shown in revised Figure 4B, the Cterminal LRRs of HAESA in contact with SERK1 in our complex structure appear to be somewhat flexible. Author response image 1. illustrates that the B-values are significantly higher in the C-terminal part of the HAESA LRR domain (with the Cterminal capping domain being the most flexible), while both the N-terminal LRRs of HAESA (with exception of the LRR N-terminal capping domain) and the IDA peptide appear better ordered in our P3121 crystals form.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>60990</offset><text>Are there potential problems with radiation damage due to the high multiplicity? </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>61072</offset><text>No. Data were collected at SLS beamline PXIII equipped with a Dectris Pilatus 2M-F detector. We perform all our data collections at very low dose and high multiplicity of measurement, which at this beam-line produces similar results compared to exposing the crystal at higher dose for a smaller angular range. We collected 360 deg with 0.1 deg slices and obtained a Wilson B-value of 80, with no sign of radiation damage in our data processing (subroutine COLSPOT in XDS over all frames). To test the reviewer's hypothesis we cut the data after 90 deg (when completeness approaches 100%) and we obtained a Wilson B-value of 78 and a refined mean B-value of around 75. These value do not significantly differ from our presented 360 deg data set and thus it is unlikely that radiation damage produces these high B-values. Again, they rather appear to be an intrinsic property of our crystals.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>61963</offset><text>ii) A simulated annealing omit map figure should be provided as the peptides all exhibit very high temperature factors.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>62083</offset><text>Thank you for this suggestion. In our first submission, we presented 2Fo-Fc omit electron density maps for our HAESA-IDA/IDL complex structures. As suggested, we now present simulated annealing omit maps in revised Figure 2A, B, C. The maps were generated like this: phenix.composite_omit_map *.pdb *.mtz *.cif nproc=8 anneal=True We would like to note that our peptide are well ordered in our structures, and their B-values match the B-values of their interacting LRR surface (compare Author response image 1).</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>62595</offset><text>iii) How many side chains were trimmed due to poor electron density? If this is a significant percentage, it should be noted. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>62722</offset><text>Here are the requested numbers (trimmed residues out of total residues in asymmetric unit, percentage):</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>62826</offset><text>HAESA apo: 7 out of 595 (1%)</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>62855</offset><text>HAESA IDA: 6 out of 597 + 12 (1%)</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>62889</offset><text>HAESA IDL1: 6 out of 597 + 12 (1%)</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>62924</offset><text>HAESA – IDA – SERK1: 5 out of 594 + 12 + 185 (0.6%)</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>62980</offset><text>We have included a statement in the Methods section that reads: “Amino acids whose side-chain position could not be modeled with confidence were truncated to alanine (0.6 – 1% of total residues)[…]”</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>63187</offset><text> 2) A further discussion comparing the mechanisms of BRI1-BL-SERK1 and HAESA-IDA-SERK1 would be helpful as a structural comparison is presented. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>63333</offset><text>We have expanded our discussion of the HAESA – SERK1 and BRI1 – SERK1 interfaces. We now specify the SERK1 residues in contact with both receptors and the SERK1 residues unique to HAESA/IDA sensing. We also comment on the very different ligand binding modes in HAESA and BRI1 and specify that different LRR segments contribute to the formation of the respective steroid and peptide hormone binding pockets. We feel however that an in-depth comparison of the interacting surfaces is beyond the scope of this report and partially redundant with our earlier work (Santiago et al., Science, 2013). In our opinion, such an analysis seems more appropriate for a review on the subject, which we are currently preparing. Our revised Discussion now reads: “Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor (Santiago, Henzler, and Hothorn 2013), reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A).[…] These residues are not involved in the sensing of the steroid hormone brassinolide (Santiago, Henzler, and Hothorn 2013). In both cases however, the co-receptor completes the hormone binding pocket.”</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>64732</offset><text>As BR1 binds its ligand with high affinity, the low affinity of HAESA for IDA could be further discussed. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>64839</offset><text>High affinity brassinosteroid binding to BRI1 was previously shown using BRI1-enriched plant extracts and radiolabeled brassinolide (Wang et al., Nature410:380-383, 2001). We now know that co-immunoprecipitations of BRI1 from Arabidopsis contain SERK proteins (compare for example Jaillais et al., PNAS, 2011) and thus the reported binding constants likely correspond to steroid binding to BRI1-SERK complexes, not to BRI1 alone. We would thus prefer not to compare the binding affinities for brassinosteroid and peptide hormone ligands at this point.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>65391</offset><text>3) Are the results here applicable to the related receptor HSL2? Are the residues that interact with SERK1 and IDA conserved in HSL2? </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>65526</offset><text>Yes. We present a structure-based sequence alignment of AtHAESA and AtHSL2, as well as other HAESA-type receptors from different plant species in Figure 1—figure supplement 3. In the peptide binding surface, 17 out of 26 contributing amino-acids are conserved among AtHAESA and AtHSL2. 13 out of 19 interacting residues in the HAESA – SERK1 complex are also present in AtHSL2. We feel that this is strong conservation given that the AtHAESA and AtHSL2 ectodomains share 45% overall sequence identity. We have included a statement in our manuscript that reads: “Indeed, we find many of the residues contributing to the formation of the IDA binding surface in HAESA to be conserved in HSL2 and other HAESA-type receptors in different plant species (Figure 1—figure supplement 3).”</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>66315</offset><text>4) The authors show that N-terminal extension of the peptide does not impact on binding efficiency, but what would happen if the peptide was extended at the C-terminal end, at the suggested cleavage site? </text></passage><passage><infon key="file">elife-15075-resp-fig2.jpg</infon><infon key="id">fig9</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>66521</offset><text>Isothermal titration calorimetry thermograph of the C-terminally extended IDA-SFVN peptide (200 μM) titrated into a solution containing 20 μM of the purified HAESA ectodomain.</text></passage><passage><infon key="file">elife-15075-resp-fig2.jpg</infon><infon key="id">fig9</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>66699</offset><text>No detectable binding is observed.</text></passage><passage><infon key="file">elife-15075-resp-fig2.jpg</infon><infon key="id">fig9</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>66734</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.15075.016</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>66781</offset><text>Thank you for suggesting this experiment. We synthesized a C-terminally extended version of the IDA peptide (IDA-SFVN with sequence YPIPPSA-Hyp- SKRHN SFVN) and performed quantitative binding assays by ITC. As shown in Author response image 2, we cannot observe any detectable binding of this C-terminally extended peptide to the HAESA ectodomain, consistent with our crystallographic models that suggest that HAESA specifically senses an active IDA 12mer. We have incorporated this new result in Figure 2D. We have included a new statement in the manuscript that reads: “The COO- group of Asn69IDA is in direct contact with Arg407HAESA and Arg409HAESA and HAESA cannot bind a C-terminally extended IDA-SFVN peptide (Figures 1D, F, 2D).”</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>67523</offset><text>Would cleavage be required for recognition? A brief discussion on this point may help. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>67611</offset><text>Yes. We have modified our manuscript accordingly: “This suggests that the conserved Asn69 may constitute the very C-terminus of the mature IDA peptide in planta and that active IDA is generated by proteolytic processing from a longer pre-protein (Stenvik et al. 2008).“</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>67885</offset><text>5) Figure 3C: Could the authors comment on the difference between the blue (SERK1 vs. HAESA-IDA) and the black (IDA vs. HAESA-SERK1) line? SERK1 vs. HAESA-IDA gives a Kd of 75 nM, yet IDA vs. SERK1-HAESA gives a Kd of 350 nM. In the text the authors keep referring to the 75 nM Kd, but not the 350 nM Kd. Is it really fair to say the Kd is 75 nM?</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>68232</offset><text>Yes and No. These are two different experiments. We first measured (by ITC) the binding affinity of IDA (in the syringe) binding to a protein solution containing equimolar ratios of HAESA and SERK1 (Kdis 350 nM in this case). Next, we titrated a concentrated SERK1 solution (in the syringe) into a solution of HAESA containing IDA in 10fold molar excess (Kdin this case is 75 nM). Given that the experimental conditions (protein and peptide concentrations and molar ratios between the components) are very different, we feel that the Kd 's obtained by these experiments are in good agreement (4.5 fold difference vs. a 60-260 fold difference when compared to the isolated HAESA ectodomain). Nevertheless, we addressed the reviewer's concern by modifying our manuscript which now states: “In this case, there was no detectable interaction between receptor and co-receptor, while in the presence of IDA, SERK1 strongly binds HAESA with a dissociation constant in the mid-nanomolar range. (Figure 3C).”</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>69236</offset><text>6) In the Discussion, can the authors comment further on the discrepancy between their study and the study of Lewis et al. (Plant J, 2010) concerning the role of SERK1 in floral abscission? </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>69427</offset><text>The process of floral organ abscission in Arabidopsis is divided into distinct steps where a gradual loosening of the cell wall between abscising cells can be measured as a reduction in petal breakstrength (Bleecker and Patterson, 1997). During floral abscission in wild-type plants a significant drop in breakstrength occurs shortly before the petals drop (shown in Figure 3A in our manuscript). Previously reported negative regulators of abscission, such as the transcription factor KNAT1, have an earlier reduction in breakstrength, indicative of early cell wall remodeling (Shi et al. 2011). Our results show that serk1 mutant plants, contrary to knat1 mutants and wild type, have a delay in cell wall loosening and organ separation (Figure 3A) thus positively regulating organ separation during abscission. The weaker phenotype when compared to haesa/hsl2 mutants is likely due to the redundant nature of other SERKs inthe abscission zone (recent work of Meng et al. 2016, cited in the Results and Discussion sections of our manuscript).</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>70470</offset><text>It has previously been reported that mutations in SERK1 can rescue the block in abscission in plants without the functional ADP-ribosylation factor GTPase-activating protein NEVERSHED (NEV) (Lewis et al. 2010). However, as a mutation in SERK1 is not capable of rescuing the ida mutant phenotype (Lewis et al. 2010) and revertant mutants capable of rescuing the abscission defect of ida do not complement nev, it has been suggested that NEV and IDA function in parallel pathways to promote cell separation (Liu 2013). Our work does not rule out a function for SERK1 in such a parallel pathway, we merely report SERK1 can ALSO act as a positive regulator of abscission by interacting with HAESA in an IDA-dependent manner. We do not observe negative regulation of floral abscission using our SERK1 mutant alleles. Based on the available evidence there is thus little to discuss and speculate about the different functions of SERK1 in abscission, as no molecular mechanism for the negative role of SERK1 in this pathway has been reported thus far. We feel that it is beyond the scope of our manuscript to clarify the different roles of SERK1 in the Arabidopsis abscission zone.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>71645</offset><text>Similarly, could the authors comment on the fact that the LRR-RLP EVD/SOBIR seems to be a negative regulator of the HAESA/HSL2 pathway (Leslie et al., Development, 2010), which seems puzzling given that EVD/SOBIR function is normally restricted to LRR-RLPs (Gust &amp; Felix, Curr Op Plant Biol, 2014).</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>71944</offset><text>We did attempt to express and purify the EVR/SOBIR extracellular domain, but in our hands the protein is not properly secreted and hence unfolded. We thus could not further investigate the potential mechanism of EVR/SOBIR in the HAESA pathway.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>72188</offset><text>7) Given that the central hydroxyproline in IDA is of such crucial importance for binding, isn't it surprising that IDAΔ69N does not bind to HAESA at all? Wouldn't it be expected that the remaining part of the peptide still binds to HAESA? </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>72433</offset><text>We thank the reviewers for pointing this out to us. Indeed, we find several structural and sequence features in IDA peptide to be important determinants for HAESA binding, namely the correct size of the peptide, the presence of a central Hyp residue and an intact C-terminal Arg-His-Asn motif that is buried in the structure. In the revised manuscript we now provide new experiments (binding of a C-terminal extended IDA peptide to HAESA) that clarifies this point (summarized in revised Figure 2D). We have revised our statement in the Discussion accordingly: “The central Hyp residue in IDA is found buried in the HAESA peptide binding surfaceand thus this post-translational modification may regulateIDA bioactivity.”</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>73158</offset><text>8) Figure 1—figure supplement 2: How is it possible that charged amino acids are involved in hydrophobic interactions? </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>73280</offset><text>We apologize for this confusing statement. It now reads: “A N-terminal Pro-rich motif in IDA makes contacts LRRs 2-6 of the receptor(Figure 1D, Figure 1—figure supplement 2A-C).”</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>73465</offset><text>9) Are the distances shown in the graphical representation of the structures proportional? It would seem that some of the aromatic rings could cause steric hindrance. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>73633</offset><text>No. The graphical representation are proportional and e.g. Trp218 in the back of the binding pocket is not producing steric clashes with the peptide with the closest distance being 4.5 A.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>73821</offset><text>10) Why did the authors decide to express HAESA and SERK1 without signal peptide? Would it make a difference for binding of IDA, if they leave the SP on? </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>73976</offset><text>No. We did express both the HAESA and SERK1 ectodomains fused to the signal peptide of human azurocidin, which provides very efficient secretion of LRR proteins in insect cells (Olczak &amp; Olczak, Anal. Biochem., 2006) (see Methods, subsection “Protein Expression and Purification“). Both the native signal peptides for SERK1 and HAESA as well as the azurocidin signal peptide are being recognized and cleaved by the Trichoplusia ni signal peptidase. This results, just like in planta, in a mature receptor/coreceptor ectodomain starting with the first α-helix of the N-terminal capping domain (residues 20 and 24, respectively). Thus, there is no reason to believe that the signal peptide would play a role in IDA sensing. Using our system, we cannot produce HAESA and/or SERK1 ectodomains with an intact signal peptide, as this would impair folding and proper secretion of the recombinant proteins.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>74880</offset><text>11) Figure 7: Are the homologous regions also the active parts of these peptides? </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>74963</offset><text>Yes. We have included three additional references in the Discussion section of our manuscript, which report the bioactive regions of CLV3/CLE, RGF and PEP peptides shown in Figure 7. The revised section now reads: “Importantly, this motif can also be found in other peptide hormone families (Kondo et al. 2006; Matsuzaki et al. 2010; Tang et al. 2015)(Figure 7). Among these are the CLE peptides regulating stem cell maintenance in the shoot and the root (Clark, Running, and Meyerowitz 1995). It is interesting to note, that CLEs in their mature form are also hydroxyprolinated dodecamers, which bind to a surface area in the BARELY ANY MERISTEM 1 receptor that would correspond to part of the IDA binding cleft in HAESA (Kondo et al. 2006;Ogawa et al. 2008; Shinohara et al. 2012).”</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>75752</offset><text>And could the authors display amino acids numbers on either side of the fragments? </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>75836</offset><text>Yes. We have now included the residues number of each peptide in Figure 7.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>75911</offset><text>12) Have the authors ever measured dissociation of the peptide from the complex? </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>75993</offset><text>No. We have not performed any biochemical experiment that would allow us to quantify the dissociation of the peptide from the ternary complex. In qualitative terms it is however of note that HAESA-IDA-SERK1 complexes do not dissociate in size exclusion chromatography experiments, even when the peptide is not provided in excess or supplied in the running buffer.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>76357</offset><text>And in this regard, to what does &quot;highly stable receptor – co-receptor complex&quot; refer/compare to? </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>76458</offset><text>The reviewers are correct, we should not claim that the complex is 'highly stable' if we have not quantified the dissociation rate. The revised sentence reads: “This ligand-induced formation of a receptor – co-receptor complex may allow the HAESA and SERK1 kinase domains to efficiently trans-phosphorylate and activate each other in the cytoplasm.”</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>76814</offset><text>13) Figures 3A and 5C require statistical analyses.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>76866</offset><text>Thank you for pointing this out to us. The statistical analysis of the petal break-strength assays shown in Figures 3A and 5C has been carried out by Prof. Ludwig A. Hothorn, Institute for Biostatistics, University of Hannover, Germany, whom we have added as an author on our manuscript:</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>77154</offset><text>Statistical analysis for Figure 3A: The statistical analysis is described in the figure legend of Figure 3A; statistical significant changes are indicated by a * in the Figure itself. The revised figure legend reads: “Petal break-strength assays measure the force (expressed in gram equivalents) required to remove the petals from the flower of serk mutant plants compared to haesa/hsl2 mutant and Col-0 wild-type flowers. […] Petal break was found significantly increased in almost all positions (indicated with a *) for haesa/hsl2 and serk1-1 mutant plants with respect to the Col-0 control. Calculations were performed in R (R Core Team 2014) (version 3.2.3).” The two new references have been added to the Reference section of the manuscript.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>77907</offset><text>We have changed our Results section accordingly: “Our experiments suggest that among the SERK family members, SERK1 is a positive regulator offloral abscission. We found that the force required to remove the petals of serk1-1 mutants is significantlyhigher than that needed for wild-type plants, as previously observed for haesa/hsl2 mutants (Stenvik et al. 2008), and that floral abscission is delayed in serk1-1 (Figure 3A). The serk2-2, serk3-1, serk4-1 and serk5-1 mutant lines (Albrecht et al. 2008) showed a petal break-strength profile not significantly differentfrom wild-type plants.”</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>78505</offset><text>Statistical analysis for Figure 5C: The statistical analysis is described in the figure legend of Figure 5C; statistical significant changes are indicated by * and # symbols in the Figure itself. The revised figure legend reads:”Quantitative petal break assay for Col-0 wild-type flowers and 35S::IDA wild-type and 35S::IDA K66A/R67A mutant flowers. […] Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c).”</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>79041</offset><text>We have changed our Results section accordingly: “We overexpressed full-length wild-type IDA or this Lys66IDA/Arg67IDA → Ala double-mutant to similar levels in Col-0 Arabidopsis plants (Figure 5D). […] Comparison of 35S::IDA wild-type and mutant plants further indicates that mutation of Lys66IDA/Arg67IDA→ Ala may cause a weak dominant negative effect (Figure 5C-E).”</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>79420</offset><text>Following the suggestions from for example Nuzzo (Nature506:150-152, 2014) and Trafirmow and Marks (Basic and Applied Social Psychology37:1-2, 2015), we decided not to report p-values.</text></passage></document></collection>
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+<collection><source>PMC</source><date>20201216</date><key>pmc.key</key><document><id>4848761</id><infon key="license">CC BY</infon><passage><infon key="alt-title">Jerome C Nwachukwu et al</infon><infon key="article-id_doi">10.15252/msb.20156701</infon><infon key="article-id_pmc">4848761</infon><infon key="article-id_pmid">27107013</infon><infon key="article-id_publisher-id">MSB156701</infon><infon key="elocation-id">864</infon><infon key="issue">4</infon><infon key="kwd">Breast cancer Chemical biology Crystal structure Nuclear receptor Signal transduction Chemical Biology Structural Biology Transcription</infon><infon key="license">This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</infon><infon key="name_0">surname:Nwachukwu;given-names:Jerome C</infon><infon key="name_1">surname:Srinivasan;given-names:Sathish</infon><infon key="name_10">surname:Carlson;given-names:Kathryn E</infon><infon key="name_11">surname:Josan;given-names:Jatinder S</infon><infon key="name_12">surname:Elemento;given-names:Olivier</infon><infon key="name_13">surname:Katzenellenbogen;given-names:John A</infon><infon key="name_14">surname:Zhou;given-names:Hai‐Bing</infon><infon key="name_15">surname:Nettles;given-names:Kendall W</infon><infon key="name_2">surname:Zheng;given-names:Yangfan</infon><infon key="name_3">surname:Wang;given-names:Song</infon><infon key="name_4">surname:Min;given-names:Jian</infon><infon key="name_5">surname:Dong;given-names:Chune</infon><infon key="name_6">surname:Liao;given-names:Zongquan</infon><infon key="name_7">surname:Nowak;given-names:Jason</infon><infon key="name_8">surname:Wright;given-names:Nicholas J</infon><infon key="name_9">surname:Houtman;given-names:René</infon><infon key="notes">
+
+. () : 864
+</infon><infon key="section_type">TITLE</infon><infon key="source">Mol Syst Biol</infon><infon key="title">Subject Categories</infon><infon key="type">front</infon><infon key="volume">12</infon><infon key="year">2016</infon><offset>0</offset><text>Predictive features of ligand‐specific signaling through the estrogen receptor</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>81</offset><text>Abstract</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>90</offset><text>Some estrogen receptor‐α (ERα)‐targeted breast cancer therapies such as tamoxifen have tissue‐selective or cell‐specific activities, while others have similar activities in different cell types. To identify biophysical determinants of cell‐specific signaling and breast cancer cell proliferation, we synthesized 241 ERα ligands based on 19 chemical scaffolds, and compared ligand response using quantitative bioassays for canonical ERα activities and X‐ray crystallography. Ligands that regulate the dynamics and stability of the coactivator‐binding site in the C‐terminal ligand‐binding domain, called activation function‐2 (AF‐2), showed similar activity profiles in different cell types. Such ligands induced breast cancer cell proliferation in a manner that was predicted by the canonical recruitment of the coactivators NCOA1/2/3 and induction of the GREB1 proliferative gene. For some ligand series, a single inter‐atomic distance in the ligand‐binding domain predicted their proliferative effects. In contrast, the N‐terminal coactivator‐binding site, activation function‐1 (AF‐1), determined cell‐specific signaling induced by ligands that used alternate mechanisms to control cell proliferation. Thus, incorporating systems structural analyses with quantitative chemical biology reveals how ligands can achieve distinct allosteric signaling outcomes through ERα.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1503</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1516</offset><text>Many drugs are small‐molecule ligands of allosteric signaling proteins, including G protein‐coupled receptors (GPCRs) and nuclear receptors such as ERα. These receptors regulate distinct phenotypic outcomes (i.e., observable characteristics of cells and tissues, such as cell proliferation or the inflammatory response) in a ligand‐dependent manner. Small‐molecule ligands control receptor activity by modulating recruitment of effector enzymes to distal regions of the receptor, relative to the ligand‐binding site. Some of these ligands achieve selectivity for a subset of tissue‐ or pathway‐specific signaling outcomes, which is called selective modulation, functional selectivity, or biased signaling, through structural mechanisms that are poorly understood (Frolik et al, 1996; Nettles &amp; Greene, 2005; Overington et al, 2006; Katritch et al, 2012; Wisler et al, 2014). For example, selective estrogen receptor modulators (SERMs) such as tamoxifen (Nolvadex®; AstraZeneca) or raloxifene (Evista®; Eli Lilly) (Fig 1A) block the ERα‐mediated proliferative effects of the native estrogen, 17β‐estradiol (E2), on breast cancer cells, but promote beneficial estrogenic effects on bone mineral density and adverse estrogenic effects such as uterine proliferation, fatty liver, or stroke (Frolik et al, 1996; Fisher et al, 1998; McDonnell et al, 2002; Jordan, 2003).</text></passage><passage><infon key="file">MSB-12-864-g002.jpg</infon><infon key="id">msb156701-fig-0001</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>2912</offset><text>Allosteric control of ERα activity</text></passage><passage><infon key="file">MSB-12-864-g002.jpg</infon><infon key="id">msb156701-fig-0001</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>2951</offset><text>Chemical structures of some common ERα ligands. BSC, basic side chain. E2‐rings are numbered A‐D. The E‐ring is the common site of attachment for BSC found in many SERMS.</text></passage><passage><infon key="file">MSB-12-864-g002.jpg</infon><infon key="id">msb156701-fig-0001</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>3129</offset><text>ERα domain organization lettered, A‐F. DBD, DNA‐binding domain; LBD, ligand‐binding domain; AF, activation function</text></passage><passage><infon key="file">MSB-12-864-g002.jpg</infon><infon key="id">msb156701-fig-0001</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>3252</offset><text>Schematic illustration of the canonical ERα signaling pathway.</text></passage><passage><infon key="file">MSB-12-864-g002.jpg</infon><infon key="id">msb156701-fig-0001</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>3319</offset><text>Linear causality model for ERα‐mediated cell proliferation.</text></passage><passage><infon key="file">MSB-12-864-g002.jpg</infon><infon key="id">msb156701-fig-0001</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>3383</offset><text>Branched causality model for ERα‐mediated cell proliferation.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3449</offset><text>ERα contains structurally conserved globular domains of the nuclear receptor superfamily, including a DNA‐binding domain (DBD) that is connected by a flexible hinge region to the ligand‐binding domain (LBD), as well as unstructured AB and F domains at its amino and carboxyl termini, respectively (Fig 1B). The LBD contains a ligand‐dependent coactivator‐binding site called activation function‐2 (AF‐2). However, the agonist activity of SERMs derives from activation function‐1 (AF‐1)—a coactivator recruitment site located in the AB domain (Berry et al, 1990; Shang &amp; Brown, 2002; Abot et al, 2013).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4073</offset><text>AF‐1 and AF‐2 bind distinct but overlapping sets of coregulators (Webb et al, 1998; Endoh et al, 1999; Delage‐Mourroux et al, 2000; Yi et al, 2015). AF‐2 binds the signature LxxLL motif peptides of coactivators such as NCOA1/2/3 (also known as SRC‐1/2/3). AF‐1 binds a separate surface on these coactivators (Webb et al, 1998; Yi et al, 2015). Yet, it is unknown how different ERα ligands control AF‐1 through the LBD, and whether this inter‐domain communication is required for cell‐specific signaling or anti‐proliferative responses.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4636</offset><text>In the canonical model of the ERα signaling pathway (Fig 1C), E2‐bound ERα forms a homodimer that binds DNA at estrogen‐response elements (EREs), recruits NCOA1/2/3 (Metivier et al, 2003; Johnson &amp; O'Malley, 2012), and activates the GREB1 gene, which is required for proliferation of ERα‐positive breast cancer cells (Ghosh et al, 2000; Rae et al, 2005; Deschenes et al, 2007; Liu et al, 2012; Srinivasan et al, 2013). However, ERα‐mediated proliferative responses vary in a ligand‐dependent manner (Srinivasan et al, 2013); thus, it is not known whether this canonical model is widely applicable across diverse ERα ligands.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5284</offset><text>Our long‐term goal is to be able to predict proliferative or anti‐proliferative activity of a ligand in different tissues from its crystal structure by identifying different structural perturbations that lead to specific signaling outcomes. The simplest response model for ligand‐specific proliferative effects is a linear causality model, where the degree of NCOA1/2/3 recruitment determines GREB1 expression, which in turn drives ligand‐specific cell proliferation (Fig 1D). Alternatively, a more complicated branched causality model could explain ligand‐specific proliferative responses (Fig 1E). In this signaling model, multiple coregulator binding events and target genes (Won Jeong et al, 2012; Nwachukwu et al, 2014), LBD conformation, nucleocytoplasmic shuttling, the occupancy and dynamics of DNA binding, and other biophysical features could contribute independently to cell proliferation (Lickwar et al, 2012).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6222</offset><text>To test these signaling models, we profiled a diverse library of ERα ligands using systems biology approaches to X‐ray crystallography and chemical biology (Srinivasan et al, 2013), including a series of quantitative bioassays for ERα function that were statistically robust and reproducible, based on the Z’‐statistic (Fig EV1A and B; see Materials and Methods). We also determined the structures of 76 distinct ERα LBD complexes bound to different ligand types, which allowed us to understand how diverse ligand scaffolds distort the active conformation of the ERα LBD. Our findings here indicate that specific structural perturbations can be tied to ligand‐selective domain usage and signaling patterns, thus providing a framework for structure‐based design of improved breast cancer therapeutics, and understanding the different phenotypic effects of environmental estrogens.</text></passage><passage><infon key="file">MSB-12-864-g003.jpg</infon><infon key="id">msb156701-fig-0001ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>7118</offset><text>High‐throughput screens for ERα ligand profiling</text></passage><passage><infon key="file">MSB-12-864-g003.jpg</infon><infon key="id">msb156701-fig-0001ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>7171</offset><text>Summary of ligand screening assays used to measure ER‐mediated activities. ERE, estrogen‐response element; Luc, luciferase reporter gene; M2H, mammalian 2‐hybrid; UAS, upstream‐activating sequence.</text></passage><passage><infon key="file">MSB-12-864-g003.jpg</infon><infon key="id">msb156701-fig-0001ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>7377</offset><text>Controls for screening assays described in panel (A), above. Error bars indicate mean ± SEM, n = 3.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>7478</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>7486</offset><text>Strength of AF‐1 signaling does not determine cell‐specific signaling</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7560</offset><text>To compare ERα signaling induced by diverse ligand types, we synthesized and assayed a library of 241 ERα ligands containing 19 distinct molecular scaffolds. These include 15 indirect modulator series, which lack a SERM‐like side chain and modulate coactivator binding indirectly from the ligand‐binding pocket (Fig 2A–E; Dataset EV1) (Zheng et al, 2012) (Zhu et al, 2012) (Muthyala et al, 2003; Seo et al, 2006) (Srinivasan et al, 2013) (Wang et al, 2012) (Liao et al, 2014) (Min et al, 2013). We also generated four direct modulator series with side chains designed to directly dislocate h12 and thereby completely occlude the AF‐2 surface (Fig 2C and E; Dataset EV1) (Kieser et al, 2010). Ligand profiling using our quantitative bioassays revealed a wide range of ligand‐induced GREB1 expression, reporter gene activities, ERα‐coactivator interactions, and proliferative effects on MCF‐7 breast cancer cells (Figs EV1 and EV2A–J). This wide variance enabled us to probe specific features of ERα signaling using ligand class analyses, and identify signaling patterns shared by specific ligand series or scaffolds.</text></passage><passage><infon key="file">MSB-12-864-g004.jpg</infon><infon key="id">msb156701-fig-0002</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>8708</offset><text>Classes of compounds in the ERα ligand library</text></passage><passage><infon key="file">MSB-12-864-g004.jpg</infon><infon key="id">msb156701-fig-0002</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>8759</offset><text>Structure of the E2‐bound ERα LBD in complex with an NCOA2 peptide of (PDB 1GWR).</text></passage><passage><infon key="file">MSB-12-864-g004.jpg</infon><infon key="id">msb156701-fig-0002</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>8845</offset><text>Structural details of the ERα LBD bound to the indicated ligands. Unlike E2 (PDB 1GWR), TAM is a direct modulator with a BSC that dislocates h12 to block the NCOA2‐binding site (PDB 3ERT). OBHS is an indirect modulator that dislocates the h11 C‐terminus to destabilize the h11–h12 interface (PDB 4ZN9).</text></passage><passage><infon key="file">MSB-12-864-g004.jpg</infon><infon key="id">msb156701-fig-0002</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>9155</offset><text>The ERα ligand library contains 241 ligands representing 15 indirect modulator scaffolds, plus 4 direct modulator scaffolds. The number of compounds per scaffold is shown in parentheses (see Dataset EV1 for individual compound information and Appendix Supplementary Methods for synthetic protocols).</text></passage><passage><infon key="file">MSB-12-864-g005.jpg</infon><infon key="id">msb156701-fig-0002ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>9459</offset><text>ERα ligands induced a range of agonist activity profiles</text></passage><passage><infon key="file">MSB-12-864-g005.jpg</infon><infon key="id">msb156701-fig-0002ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>9520</offset><text>Screening data from individual ligands are shown, grouped by scaffold. Each data point represents the activity of a distinct compound. Error bars indicate the class average (mean) ± range. *Direct modulator.</text></passage><passage><infon key="file">MSB-12-864-g005.jpg</infon><infon key="id">msb156701-fig-0002ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>9731</offset><text>
+Source data are available online for this figure.
+</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9783</offset><text>We first asked whether direct modulation of the receptor with an extended side chain is required for cell‐specific signaling. To this end, we compared the average ligand‐induced GREB1 mRNA levels in MCF‐7 cells and 3×ERE‐Luc reporter gene activity in Ishikawa endometrial cancer cells (E‐Luc) or in HepG2 cells transfected with wild‐type ERα (L‐Luc ERα‐WT) (Figs 3A and EV2A–C). Direct modulators showed significant differences in average activity between cell types except OBHS‐ASC analogs, which had similar low agonist activities in the three cell types. The other direct modulators had low agonist activity in Ishikawa cells, no or inverse agonist activity in MCF‐7 cells, and more variable activity in HepG2 liver cells. While it was known that direct modulators such as tamoxifen drive cell‐specific signaling, these experiments reveal that indirect modulators also drive cell‐specific signaling, since eight of fourteen classes showed significant differences in average activity (Figs 3A and EV2A–C).</text></passage><passage><infon key="file">MSB-12-864-g006.jpg</infon><infon key="id">msb156701-fig-0003</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>10824</offset><text>Ligand‐specific signaling underlies ERα‐mediated cell proliferation</text></passage><passage><infon key="file">MSB-12-864-g006.jpg</infon><infon key="id">msb156701-fig-0003</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>10897</offset><text>(A) Ligand‐specific ERα activities in HepG2, Ishikawa and MCF‐7 cells. The ligand‐induced L‐Luc ERα‐WT and E‐Luc activities and GREB1 mRNA levels are shown by scaffold (mean + SD). (B) Ligand class analysis of the L‐Luc ERα‐WT and ERα‐ΔAB activities in HepG2 cells. Significant sensitivity to AB domain deletion was determined by Student's t‐test (n = number of ligands per scaffold in Fig 2). The average activities of ligands classes are shown (mean + SEM).</text></passage><passage><infon key="file">MSB-12-864-g006.jpg</infon><infon key="id">msb156701-fig-0003</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>11389</offset><text>Correlation and regression analyses in a large test set. The r
+2 values are plotted as a heat map. In cluster 1, the first three comparisons (rows) showed significant positive correlations (F‐test for nonzero slope, P ≤ 0.05). In cluster 2, only one of these comparisons revealed a significant positive correlation, while none was significant in cluster 3. +, statistically significant correlations gained by deletion of the AB or F domains. −, significant correlations lost upon deletion of AB or F domains.</text></passage><passage><infon key="file">MSB-12-864-g006.jpg</infon><infon key="id">msb156701-fig-0003</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>11906</offset><text>
+Source data are available online for this figure.
+</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11958</offset><text>Tamoxifen depends on AF‐1 for its cell‐specific activity (Sakamoto et al, 2002); therefore, we asked whether cell‐specific signaling observed here is due to a similar dependence on AF‐1 for activity (Fig EV1). To test this idea, we compared the average L‐Luc activities of each scaffold in HepG2 cells co‐transfected with wild‐type ERα or with ERα lacking the AB domain (Figs 1B and EV1). While E2 showed similar L‐Luc ERα‐WT and ERα‐ΔAB activities, tamoxifen showed complete loss of activity without the AB domain (Fig EV1B). Deletion of the AB domain significantly reduced the average L‐Luc activities of 14 scaffolds (Student's t‐test, P ≤ 0.05) (Fig 3B). These “AF‐1‐sensitive” activities were exhibited by both direct and indirect modulators, and were not limited to scaffolds that showed cell‐specific signaling (Fig 3A and B). Thus, the strength of AF‐1 signaling does not determine cell‐specific signaling.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>12926</offset><text>Identifying cell‐specific signaling clusters in ERα ligand classes</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12997</offset><text>As another approach to identifying cell‐specific signaling, we determined the degree of correlation between ligand‐induced activities in the different cell types. Here, we compared ligands within each class (Fig 3C), instead of comparing average activities (Fig 3A and B). For each ligand class or scaffold, we calculated the Pearson's correlation coefficient, r, for pairwise comparison of activity profiles in breast (GREB1), liver (L‐Luc), and endometrial cells (E‐Luc). The value of r ranges from −1 to 1, and it defines the extent to which the data fit a straight line when compounds show similar agonist/antagonist activity profiles between cell types (Fig EV3A). We also calculated the coefficient of determination, r 2, which describes the percentage of variance in a dependent variable such as proliferation that can be predicted by an independent variable such as GREB1 expression. We present both calculations as r 2 to readily compare signaling specificities using a heat map on which the red–yellow palette indicates significant positive correlations (P ≤ 0.05, F‐test for nonzero slope), while the blue palette denotes negative correlations (Fig 3C–F).</text></passage><passage><infon key="file">MSB-12-864-g007.jpg</infon><infon key="id">msb156701-fig-0003ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>14188</offset><text>The side chain of OBHS‐BSC analogs induces cell‐specific signaling</text></passage><passage><infon key="file">MSB-12-864-g007.jpg</infon><infon key="id">msb156701-fig-0003ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>14259</offset><text>Correlation analysis of OBHS versus OBHS‐BSC activity across cell types.</text></passage><passage><infon key="file">MSB-12-864-g007.jpg</infon><infon key="id">msb156701-fig-0003ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>14334</offset><text>Correlation analysis of L‐Luc ERα‐ΔAB activity versus endogenous ERα activity of OBHS analogs. In panel (D), L‐Luc ERα‐WT activity from panel (B) is shown for comparison.</text></passage><passage><infon key="file">MSB-12-864-g007.jpg</infon><infon key="id">msb156701-fig-0003ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>14521</offset><text>Correlation analysis of L‐Luc ERα‐ΔF activity versus endogenous ERα activities of OBHS analogs.</text></passage><passage><infon key="file">MSB-12-864-g007.jpg</infon><infon key="id">msb156701-fig-0003ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>14629</offset><text>Correlation analysis of MCF‐7 cell proliferation versus NCOA2/3 recruitment or GREB1 levels observed in response to (G) OBHS‐N and (H) OBHS‐BSC analogs.</text></passage><passage><infon key="file">MSB-12-864-g007.jpg</infon><infon key="id">msb156701-fig-0003ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>14788</offset><text>
+Data information: In each panel, a data point indicates the activity of a distinct compound.Source data are available online for this figure.
+</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14932</offset><text>This analysis revealed diverse signaling specificities that we grouped into three clusters. Scaffolds in cluster 1 exhibited strongly correlated GREB1 levels, E‐Luc and L‐Luc activity profiles across the three cell types (Fig 3C lanes 1–4), suggesting these ligands use similar ERα signaling pathways in the breast, endometrial, and liver cell types. This cluster includes WAY‐C, OBHS, OBHS‐N, and triaryl‐ethylene analogs, all of which are indirect modulators. Cluster 2 contains scaffolds with activities that were positively correlated in only two of the three cell types, indicating cell‐specific signaling (Fig 3C lanes 5–12). This cluster includes two classes of direct modulators (cyclofenil‐ASC and WAY dimer), and six classes of indirect modulators (2,5‐DTP, 3,4‐DTP, S‐OBHS‐2 and S‐OBHS‐3, furan, and WAY‐D). In this cluster, the correlated activities varied by scaffold. For example, 3,4‐DTP, furan, and S‐OBHS‐2 drove positively correlated GREB1 levels and E‐Luc but not L‐Luc ERα‐WT activity (Fig 3C lanes 5–7). In contrast, WAY dimer and WAY‐D analogs drove positively correlated GREB1 levels and L‐Luc ERα‐WT but not E‐Luc activity (Fig 3C lanes 8 and 9). The last set of scaffolds, cluster 3, displayed cell‐specific activities that were not correlated in any of the three cell types (Fig 3C lanes 13–19). This cluster includes two direct modulator scaffolds (OBHS‐ASC and OBHS‐BSC), and five indirect modulator scaffolds (A‐CD, cyclofenil, 3,4‐DTPD, imine, and imidazopyridine).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16503</offset><text>These results suggest that addition of an extended side chain to an ERα ligand scaffold is sufficient to induce cell‐specific signaling, where the relative activity profiles of the individual ligands change between cell types. This is demonstrated by directly comparing the signaling specificities of matched OBHS (indirect modulator, cluster 1) and OBHS‐BSC analogs (direct modulator, cluster 3), which differ only in the basic side chain (Fig 2E). The activities of OBHS analogs were positively correlated across the three cell types, but the side chain of OBHS‐BSC analogs was sufficient to abolish these correlations (Figs 3C lanes 1 and 19, and EV3A–C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17173</offset><text>The indirect modulator scaffolds in clusters 2 and 3 showed cell‐specific signaling patterns without the extended side chain typically viewed as the primary chemical and structural mechanism driving cell‐specific activity. Many of these scaffolds drove similar average activities of the ligand class in the different cell types (Fig 3A), but the individual ligands in each class had different cell‐specific activities (Fig EV2A–C). Thus, examining the correlated patterns of ERα activity within each scaffold demonstrates that an extended side chain is not required for cell‐specific signaling.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>17781</offset><text>Modulation of signaling specificity by AF‐1</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17827</offset><text>To evaluate the role of AF‐1 and the F domain in ERα signaling specificity, we compared activity of truncated ERα constructs in HepG2 liver cells with endogenous ERα activity in the other cell types. The positive correlation between the L‐Luc and E‐Luc activities or GREB1 levels induced by scaffolds in cluster 1 was generally retained without the AB domain, or the F domain (Fig 3D lanes 1–4). This demonstrates that the signaling specificities underlying these positive correlations are not modified by AF‐1. OBHS analogs showed an average L‐Luc ERα‐ΔAB activity of 3.2% ± 3 (mean + SEM) relative to E2. Despite this nearly complete lack of activity, the pattern of L‐Luc ERα‐ΔAB activity was still highly correlated with the E‐Luc activity and GREB1 expression (Fig EV3D and E), demonstrating that very small AF‐2 activities can be amplified by AF‐1 to produce robust signals. Similarly, deletion of the F domain did not abolish correlations between the L‐Luc and E‐Luc or GREB1 levels induced by OBHS analogs (Fig EV3F). These similar patterns of ligand activity in the wild‐type and deletion mutants suggest that AF‐1 and the F domain purely amplify the AF‐2 activities of ligands in cluster 1.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19077</offset><text>In contrast, AF‐1 was a determinant of signaling specificity for scaffolds in cluster 2. Deletion of the AB or F domain altered correlations for six of the eight scaffolds in this cluster (2,5‐DTP, 3,4‐DTP, S‐OBHS‐3, WAY‐D, WAY dimer, and cyclofenil‐ASC) (Fig 3D lanes 5–12). Comparing Fig 3C and D, the + and − signs indicate where the deletion mutant assays led to a gain or loss of statically significant correlation, respectively. Thus, in cluster 2, AF‐1 substantially modulated the specificity of ligands with cell‐specific activity (Fig 3D lanes 5–12). For ligands in cluster 3, we could not eliminate a role for AF‐1 in determining signaling specificity, since this cluster lacked positively correlated activity profiles (Fig 3C), and deletion of the AB or F domain rarely induced such correlations (Fig 3D), except for A‐CD and OBHS‐ASC analogs, where deletion of the AB domain or F domain led to positive correlations with E‐Luc activity and/or GREB1 levels (Fig 3D lanes 13 and 18). Thus, ligands in cluster 2 rely on AF‐1 for both activity (Fig 3B) and signaling specificity (Fig 3D). As discussed below, this cell specificity derives from alternate coactivator preferences.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>20306</offset><text>Ligand‐specific control of GREB1 expression</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20352</offset><text>To determine whether ligand classes control expression of native ERα target genes through the canonical linear signaling pathway, we performed pairwise linear regression analyses using ERα–NCOA1/2/3 interactions in M2H assay as independent predictors of GREB1 expression (the dependent variable) (Figs EV1 and EV2A, F–H). In cluster 1, the recruitment of NCOA1 and NCOA2 was highest for WAY‐C, followed by triaryl‐ethylene, OBHS‐N, and OBHS series, while for NCOA3, OBHS‐N compounds induced the most recruitment and OBHS ligands were inverse agonists (Fig EV2F–H). The average induction of GREB1 by cluster 1 ligands showed greater variance, with a range between ~25 and ~75% for OBHS and a range from full agonist to inverse agonist for the others in cluster 1 (Fig EV2A). GREB1 levels induced by OBHS analogs were determined by recruitment of NCOA1 but not NCOA2/3 (Fig 3E lane 1), suggesting that there may be alternate or preferential use of these coactivators by different classes. However, in cluster 1, NCOA1/2/3 recruitment generally predicted GREB1 levels (Fig 3E lanes 1–4), consistent with the canonical signaling model (Fig 1D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21517</offset><text>For clusters 2 and 3, GREB1 activity was generally not predicted by NCOA1/2/3 recruitment. Direct modulators showed low NCOA1/2/3 recruitment (Fig EV2F–H), but only OBHS‐ASC analogs had NCOA2 recruitment profiles that predicted a full range of effects on GREB1 levels (Figs 3E lanes 9, 11, 18–19, and EV2A). The indirect modulators in clusters 2 and 3 stimulated NCOA1/2/3 recruitment and GREB1 expression with substantial variance (Figs 3A and EV2F–H). However, ligand‐induced GREB1 levels were generally not determined by NCOA1/2/3 recruitment (Fig 3E lanes 5–19), consistent with an alternate causality model (Fig 1E). Out of 11 indirect modulator series in cluster 2 or 3, only the S‐OBHS‐3 class had NCOA1/2/3 recruitment profiles that predicted GREB1 levels (Fig 3E lane 12). These results suggest that compounds that show cell‐specific signaling do not activate GREB1, or use coactivators other than NCOA1/2/3 to control GREB1 expression (Fig 1E).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>22497</offset><text>Ligand‐specific control of cell proliferation</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22545</offset><text>To determine mechanisms for ligand‐dependent control of breast cancer cell proliferation, we performed linear regression analyses across the 19 scaffolds using MCF‐7 cell proliferation as the dependent variable, and the other activities as independent variables (Fig 3F). In cluster 1, E‐Luc and L‐Luc activities, NCOA1/2/3 recruitment, and GREB1 levels generally predicted the proliferative response (Fig 3F lanes 2–4). With the OBHS‐N compounds, NCOA3 and GREB1 showed near perfect prediction of proliferation (Fig EV3G), with unexplained variance similar to the noise in the assays. The lack of significant predictors for OBHS analogs (Fig 3F lane 1) reflects their small range of proliferative effects on MCF‐7 cells (Fig EV2I). The significant correlations with GREB1 expression and NCOA1/2/3 recruitment observed in this cluster are consistent with the canonical signaling model (Fig 1D), where NCOA1/2/3 recruitment determines GREB1 expression, which then drives proliferation.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23550</offset><text>Ligands in cluster 2 and cluster 3 showed a wide range of proliferative effects on MCF‐7 cells (Fig EV2I). Despite this phenotypic variance, proliferation was not generally predicted by correlated NCOA1/2/3 recruitment and GREB1 induction (Figs 3F lanes 5–19, and EV3H). Out of 15 ligand series in these clusters, only 2,5‐DTP analogs induced a proliferative response that was predicted by GREB1 levels, which were not determined by NCOA1/2/3 recruitment (Fig 3E and F lane 10). 3,4‐DTP, cyclofenil, 3,4‐DTPD, and imidazopyridine analogs had NCOA1/3 recruitment profiles that predicted their proliferative effects, without determining GREB1 levels (Fig 3E and F, lanes 5 and 14–16). Similarly, S‐OBHS‐3, cyclofenil‐ASC, and OBHS‐ASC had positively correlated NCOA1/2/3 recruitment and GREB1 levels, but none of these activities determined their proliferative effects (Fig 3E and F lanes 11–12 and 18). For ligands that show cell‐specific signaling, ERα‐mediated recruitment of other coregulators and activation of other target genes likely determine their proliferative effects on MCF‐7 cells.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>24678</offset><text>NCOA3 occupancy at GREB1 did not predict the proliferative response</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24746</offset><text>We also questioned whether promoter occupancy by coactivators is statistically robust and reproducible for ligand class analysis using a chromatin immunoprecipitation (ChIP)‐based quantitative assay, and whether it has a better predictive power than the M2H assay. ERα and NCOA3 cycle on and off the GREB1 promoter (Nwachukwu et al, 2014). Therefore, we first performed a time‐course study, and found that E2 and the WAY‐C analog, AAPII‐151‐4, induced recruitment of NCOA3 to the GREB1 promoter in a temporal cycle that peaked after 45 min in MCF‐7 cells (Fig 4A). At this time point, other WAY‐C analogs also induced recruitment of NCOA3 at this site to varying degrees (Fig 4B). The Z’ for this assay was 0.6, showing statistical robustness (see Materials and Methods). We prepared biological replicates with different cell passage numbers and separately prepared samples, which showed r 2 of 0.81, demonstrating high reproducibility (Fig 4C).</text></passage><passage><infon key="file">MSB-12-864-g008.jpg</infon><infon key="id">msb156701-fig-0004</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>25714</offset><text>
+NCOA3 occupancy at GREB1 is statistically robust but does not predict transcriptional activity</text></passage><passage><infon key="file">MSB-12-864-g008.jpg</infon><infon key="id">msb156701-fig-0004</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>25810</offset><text>Kinetic ChIP assay examining recruitment of NCOA3 to the GREB1 gene in MCF‐7 cells stimulated with E2 or the indicated WAY‐C analog. The average of duplicate experiments (mean ± SEM) is shown.</text></passage><passage><infon key="file">MSB-12-864-g008.jpg</infon><infon key="id">msb156701-fig-0004</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>26010</offset><text>NCOA3 occupancy at GREB1 was compared by ChIP assay 45 min after stimulation with vehicle, E2, or the WAY‐C analogs. In panel (B), the average recruitment of two biological replicates are shown as mean + SEM, and the Z‐score is indicated. In panel (C), correlation analysis was performed for two biological replicates.</text></passage><passage><infon key="file">MSB-12-864-g008.jpg</infon><infon key="id">msb156701-fig-0004</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>26336</offset><text>Linear regression analyses comparing the ability of NCOA3 recruitment, measured by ChIP or M2H, to predict other agonist activities of WAY‐C analogs. *Significant positive correlation (F‐test for nonzero slope, P‐value).</text></passage><passage><infon key="file">MSB-12-864-g008.jpg</infon><infon key="id">msb156701-fig-0004</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>26563</offset><text>
+Source data are available online for this figure.
+</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>26615</offset><text>The M2H assay for NCOA3 recruitment broadly correlated with the other assays, and was predictive for GREB1 expression and cell proliferation (Fig 3E). However, the ChIP assays for WAY‐C‐induced recruitment of NCOA3 to the GREB1 promoter did not correlate with any of the other WAY‐C activity profiles (Fig 4D), although the positive correlation between ChIP assays and NCOA3 recruitment via M2H assay showed a trend toward significance with r 2 = 0.36 and P = 0.09 (F‐test for nonzero slope). Thus, the simplified coactivator‐binding assay showed much greater predictive power than the ChIP assay for ligand‐specific effects on GREB1 expression and cell proliferation.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>27302</offset><text>ERβ activity is not an independent predictor of cell‐specific activity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>27376</offset><text>One difference between MCF‐7 breast cancer cells and Ishikawa endometrial cancer cells is the contribution of ERβ to estrogenic response, as Ishikawa cells may express ERβ (Bhat &amp; Pezzuto, 2001). When overexpressed in MCF‐7 cells, ERβ alters E2‐induced expression of only a subset of ERα‐target genes (Wu et al, 2011), raising the possibility that ligand‐induced ERβ activity may contribute to E‐Luc activities, and thus underlie the lack of correlation between the E‐Luc and L‐Luc ERα‐WT activities or GREB1 levels induced by cell‐specific modulators in cluster 2 and cluster 3 (Fig 3C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>27993</offset><text>To test this idea, we determined the L‐Luc ERβ activity profiles of the ligands (Fig EV1). All direct modulator and two indirect modulator scaffolds (OBHS and S‐OBHS‐3) lacked ERβ agonist activity. However, the other ligands showed a range of ERβ activities (Fig EV2J). For most scaffolds, L‐Luc ERβ and E‐Luc activities were not correlated, except for 2,5‐DTP and cyclofenil analogs, which showed moderate but significant correlations (Fig EV4A). Nevertheless, the E‐Luc activities of both 2,5‐DTP and cyclofenil analogs were better predicted by their L‐Luc ERα‐WT than L‐Luc ERβ activities (Fig EV4A and B). Thus, ERβ activity was not an independent determinant of the observed activity profiles.</text></passage><passage><infon key="file">MSB-12-864-g009.jpg</infon><infon key="id">msb156701-fig-0004ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>28725</offset><text>ERβ activity is not an independent predictor of E‐Luc activity</text></passage><passage><infon key="file">MSB-12-864-g009.jpg</infon><infon key="id">msb156701-fig-0004ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28791</offset><text>ERβ activity in HepG2 cells rarely correlates with E‐Luc activity.</text></passage><passage><infon key="file">MSB-12-864-g009.jpg</infon><infon key="id">msb156701-fig-0004ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28861</offset><text>ERα activity of 2,5‐DTP and cyclofenil analogs correlates with E‐Luc activity.</text></passage><passage><infon key="file">MSB-12-864-g009.jpg</infon><infon key="id">msb156701-fig-0004ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28945</offset><text>
+Data information: The r
+2 and P values for the indicated correlations are shown in both panels. *Significant positive correlation (F‐test for nonzero slope, P‐value)</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>29116</offset><text>Structural features of consistent signaling across cell types</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>29178</offset><text>To overcome barriers to crystallization of ERα LBD complexes, we developed a conformation‐trapping X‐ray crystallography approach using the ERα‐Y537S mutation (Nettles et al, 2008; Bruning et al, 2010; Srinivasan et al, 2013). To further validate this approach, we solved the structure of the ERα‐Y537S LBD in complex with diethylstilbestrol (DES), which bound identically in the wild‐type and ERα‐Y537S LBDs, demonstrating again that this surface mutation stabilizes h12 dynamics to facilitate crystallization without changing ligand binding (Appendix Fig S1A and B) (Nettles et al, 2008; Bruning et al, 2010; Delfosse et al, 2012). Using this approach, we solved 76 ERα LBD structures in the active conformation and bound to ligands studied here (Appendix Fig S1C). Eleven of these structures have been published, while 65 are new, including the DES‐bound ERα‐Y537S LBD. We present 57 of these new structures here (Dataset EV2), while the remaining eight new structures bound to OBHS‐N analogs will be published elsewhere (S. Srinivasan et al, in preparation). Examining many closely related structures allows us to visualize subtle structural differences, in effect using X‐ray crystallography as a systems biology tool.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>30436</offset><text>The indirect modulator scaffolds in cluster 1 did not show cell‐specific signaling (Fig 3C), but shared common structural perturbations that we designed to modulate h12 dynamics. Based on our original OBHS structure, the OBHS, OBHS‐N, and triaryl‐ethylene compounds were modified with h11‐directed pendant groups (Zheng et al, 2012; Zhu et al, 2012; Liao et al, 2014). Superposing the LBDs based on the class of bound ligands provides an ensemble view of the structural variance and clarifies what part of the ligand‐binding pocket is differentially perturbed or targeted.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>31023</offset><text>The 24 structures containing OBHS, OBHS‐N, or triaryl‐ethylene analogs showed structural diversity in the same part of the scaffolds (Figs 5A and EV5A), and the same region of the LBD—the C‐terminal end of h11 (Figs 5B and C, and EV5B), which in turn nudges h12 (Fig 5C and D). We observed that the OBHS‐N analogs displaced h11 along a vector away from Leu354 in a region of h3 that is unaffected by the ligands, and toward the dimer interface. For the triaryl‐ethylene analogs, the displacement of h11 was in a perpendicular direction, away from Ile424 in h8 and toward h12. Remarkably, these individual inter‐atomic distances showed a ligand class‐specific ability to significantly predict proliferative effects (Fig 5E and F), demonstrating the feasibility of developing a minimal set of activity predictors from crystal structures.</text></passage><passage><infon key="file">MSB-12-864-g010.jpg</infon><infon key="id">msb156701-fig-0005</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>31879</offset><text>Structural determinants of consistent signaling</text></passage><passage><infon key="file">MSB-12-864-g010.jpg</infon><infon key="id">msb156701-fig-0005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>31927</offset><text>Structure‐class analysis of triaryl‐ethylene analogs. Triaryl‐ethylene analogs bound to the superposed crystal structures of the ERα LBD are shown. Arrows indicate chemical variance in the orientation of the different h11‐directed ligand side groups (PDB 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF and 5DP0).</text></passage><passage><infon key="file">MSB-12-864-g010.jpg</infon><infon key="id">msb156701-fig-0005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>32255</offset><text>Triaryl‐ethylene analogs induce variance of ERα conformations at the C‐terminal region of h11. Panel (B) shows the crystal structure of a triaryl‐ethylene analog‐bound ERα LBD (PDB 5DLR). The h11–h12 interface (circled) includes the C‐terminal part of h11. This region was expanded in panel (C), where the 10 triaryl‐ethylene analog‐bound ERα LBD structures (see Datasets EV1 and EV2) were superposed to show variations in the h11 C‐terminus (PDB 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF, and 5DP0).</text></passage><passage><infon key="file">MSB-12-864-g010.jpg</infon><infon key="id">msb156701-fig-0005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>32789</offset><text>ERα LBDs in complex with diethylstilbestrol (DES) or a triaryl‐ethylene analog were superposed to show that the ligand‐induced difference in h11 conformation is transmitted to the C‐terminus of h12 (PDB 4ZN7, 5DMC).</text></passage><passage><infon key="file">MSB-12-864-g010.jpg</infon><infon key="id">msb156701-fig-0005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>33012</offset><text>Inter‐atomic distances predict the proliferative effects of specific ligand series. Ile424–His524 distance measured in the crystal structures correlates with the proliferative effect of triaryl‐ethylene analogs in MCF‐7 cells. In contrast, the Leu354–Leu525 distance correlates with the proliferative effects of OBHS‐N analogs in MCF‐7 cells.</text></passage><passage><infon key="file">MSB-12-864-g010.jpg</infon><infon key="id">msb156701-fig-0005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>33369</offset><text>Structure‐class analysis of WAY‐C analogs. WAY‐C side groups subtly nudge h12 Leu540. ERα LBD structures bound to 4 distinct WAY‐C analogs were superposed (PDB 4 IU7, 4IV4, 4IVW, 4IW6) (see Datasets EV1 and EV2).</text></passage><passage><infon key="file">MSB-12-864-g010.jpg</infon><infon key="id">msb156701-fig-0005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>33592</offset><text>
+Source data are available online for this figure.
+</text></passage><passage><infon key="file">MSB-12-864-g011.jpg</infon><infon key="id">msb156701-fig-0005ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>33644</offset><text>Structure‐class analysis of indirect modulators</text></passage><passage><infon key="file">MSB-12-864-g011.jpg</infon><infon key="id">msb156701-fig-0005ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>33694</offset><text>Structure‐class analysis of indirect modulators in cluster 1. Crystal structures of the ERα LBD bound to OBHS and OBHS‐N analogs were superposed. The bound ligands are shown in panel (A). Arrows indicate chemical variance in the orientation of the different h11‐directed ligand side groups. Panel (B) shows the ligand‐induced conformational variation at the C‐terminal region of h11 (OBHS: PDB 4ZN9, 4ZNH, 4ZNS, 4ZNT, 4ZNU, 4ZNV, and 4ZNW; OBHS‐N: PDB 4ZUB, 4ZUC, 4ZWH, 4ZWK, 5BNU, 5BP6, 5BPR, and 5BQ4).</text></passage><passage><infon key="file">MSB-12-864-g011.jpg</infon><infon key="id">msb156701-fig-0005ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>34213</offset><text>Structure‐class analysis of indirect modulators in clusters 2 and 3. Crystal structures of the ERα LBD bound to ligands with cell‐specific activities were superposed. The bound ligands are shown, and arrows indicate considerable variation in the orientation of the different h3‐, h8‐, h11‐, or h12‐directed ligand side groups.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>34553</offset><text>As visualized in four LBD structures (Srinivasan et al, 2013), WAY‐C analogs were designed with small substitutions that slightly nudge h12 Leu540, without exiting the ligand‐binding pocket (Fig 5G and H). Therefore, changing h12 dynamics maintains the canonical signaling pathway defined by E2 (Fig 1D) to support AF‐2‐driven signaling and recruit NCOA1/2/3 for GREB1‐stimulated proliferation.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>34961</offset><text>Ligands with cell‐specific activity alter the shape of the AF‐2 surface</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>35037</offset><text>Direct modulators like tamoxifen drive AF‐1‐dependent cell‐specific activity by completely occluding AF‐2, but it is not known how indirect modulators produce cell‐specific ERα activity. Therefore, we examined another 50 LBD structures containing ligands in clusters 2 and 3. These structures demonstrated that cell‐specific activity derived from altering the shape of the AF‐2 surface without an extended side chain.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>35470</offset><text>Ligands in cluster 2 and cluster 3 showed conformational heterogeneity in parts of the scaffold that were directed toward multiple regions of the receptor including h3, h8, h11, h12, and/or the β‐sheets (Fig EV5C–G). For instance, S‐OBHS‐2 and S‐OBHS‐3 analogs (Fig 2) had similar ERα activity profiles in the different cell types (Fig EV2A–C), but the 2‐ versus 3‐methyl substituted phenol rings altered the correlated signaling patterns in different cell types (Fig 3B lanes 7 and 12). Structurally, the 2‐ versus 3‐methyl substitutions changed the binding position of the A‐ and E‐ring phenols by 1.0 Å and 2.2 Å, respectively (Fig EV5C). This difference in ligand positioning altered the AF‐2 surface via a shift in the N‐terminus of h12, which directly contacts the coactivator. This effect is evident in a single structure due to its 1 Å magnitude (Fig 6A and B). The shifts in h12 residues Asp538 and Leu539 led to rotation of the coactivator peptide (Fig 6C). Thus, cell‐specific activity can stem from perturbation of the AF‐2 surface without an extended side chain, which presumably alters the receptor–coregulator interaction profile.</text></passage><passage><infon key="file">MSB-12-864-g012.jpg</infon><infon key="id">msb156701-fig-0006</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>36665</offset><text>Structural correlates of cell‐specific signaling</text></passage><passage><infon key="file">MSB-12-864-g012.jpg</infon><infon key="id">msb156701-fig-0006</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>36716</offset><text>S‐OBHS‐2/3 analogs subtly distort the AF‐2 surface. Panel (A) shows the crystal structure of an S‐OBHS‐3‐bound ERα LBD (PDB 5DUH). The h3–h12 interface (circled) at AF‐2 (pink) was expanded in panels (B, C). The S‐OBHS‐2/3‐bound ERα LBDs were superposed to show shifts in h3 (panel B) and the NCOA2 peptide docked at the AF‐2 surface (panel C).</text></passage><passage><infon key="file">MSB-12-864-g012.jpg</infon><infon key="id">msb156701-fig-0006</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>37088</offset><text>Crystal structures show that 2,5‐DTP analogs shift h3 and h11 further apart compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DRM, 5DRJ). The 2F
+o‐F
+c electron density map and F
+o‐F
+c difference map of a 2,5‐DTP‐bound structure (PDB 5DRJ) were contoured at 1.0 sigma and ± 3.0 sigma, respectively.</text></passage><passage><infon key="file">MSB-12-864-g012.jpg</infon><infon key="id">msb156701-fig-0006</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>37398</offset><text>Average (mean + SEM) α‐carbon distance measured from h3 Thr347 to h11 Leu525 of A‐CD‐, 2,5‐DTP‐, and 3,4‐DTPD‐bound ERα LBDs. *Two‐tailed Student's t‐test, P = 0.002 (PDB A‐CD: 5DI7, 5DID, 5DIE, 5DIG, and 4PPS; 2,5‐DTP: 4IWC, 5DRM, and 5DRJ; 3,4‐DTPD: 5DTV and 5DU5).</text></passage><passage><infon key="file">MSB-12-864-g012.jpg</infon><infon key="id">msb156701-fig-0006</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>37696</offset><text>Crystal structures show that a 3,4‐DTPD analog shifts h3 (F) and the NCOA2 (G) peptide compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DTV).</text></passage><passage><infon key="file">MSB-12-864-g012.jpg</infon><infon key="id">msb156701-fig-0006</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>37841</offset><text>Hierarchical clustering of ligand‐specific binding of 154 interacting peptides to the ERα LBD was performed in triplicate by MARCoNI analysis.</text></passage><passage><infon key="file">MSB-12-864-g012.jpg</infon><infon key="id">msb156701-fig-0006</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>37988</offset><text>
+Source data are available online for this figure.
+</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>38040</offset><text>The 2,5‐DTP analogs showed perturbation of h11, as well as h3, which forms part of the AF‐2 surface. These compounds bind the LBD in an unusual fashion because they have a phenol‐to‐phenol length of ~12 Å, which is longer than steroids and other prototypical ERα agonists that are ~10 Å in length. One phenol pushed further toward h3 (Fig 6D), while the other phenol pushed toward the C‐terminus of h11 to a greater extent than A‐CD‐ring estrogens (Nwachukwu et al, 2014), which are close structural analogs of E2 that lack a B‐ring (Fig 2). To quantify this difference, we compared the distance between α‐carbons at h3 Thr347 and h11 Leu525 in the set of structures containing 2,5‐DTP analogs (n = 3) or A‐CD‐ring analogs (n = 5) (Fig 6E). We observed a difference of 0.4 Å that was significant (two‐tailed Student's t‐test, P = 0.002) due to the very tight clustering of the 2,5‐DTP‐induced LBD conformation. The shifts in h3 suggest these compounds are positioned to alter coregulator preferences.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>39088</offset><text>The 2,5‐DTP and 3,4‐DTP scaffolds are isomeric, but with aryl groups at obtuse and acute angles, respectively (Fig 2). The crystal structure of ERα in complex with a 3,4‐DTP is unknown; however, we solved two crystal structures of ERα bound to 3,4‐DTPD analogs and one structure containing a furan ligand—all of which have a 3,4‐diaryl configuration (Fig 2; Datasets EV1 and EV2). In these structures, the A‐ring mimetic of the 3,4‐DTPD scaffold bound h3 Glu353 as expected, but the other phenol wrapped around h3 to form a hydrogen bond with Thr347, indicating a change in binding epitopes in the ERα ligand‐binding pocket (Fig 6F). The 3,4‐DTPD analogs also induced a shift in h3 positioning, which translated again into a shift in the bound coactivator peptide (Fig 6F). Therefore, these indirect modulators, including S‐OBHS‐2, S‐OBHS‐3, 2,5‐DTP, and 3,4‐DTPD analogs—all of which show cell‐specific activity profiles—induced shifts in h3 and h12 that were transmitted to the coactivator peptide via an altered AF‐2 surface.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>40164</offset><text>To test whether the AF‐2 surface shows changes in shape in solution, we used the microarray assay for real‐time coregulator–nuclear receptor interaction (MARCoNI) analysis (Aarts et al, 2013). Here, the ligand‐dependent interactions of the ERα LBD with over 150 distinct LxxLL motif peptides were assayed to define structural fingerprints for the AF‐2 surface, in a manner similar to the use of phage display peptides as structural probes (Connor et al, 2001). Despite the similar average activities of these ligand classes (Fig 3A and B), 2,5‐DTP and 3,4‐DTP analogs displayed remarkably different peptide recruitment patterns (Fig 6H), consistent with the structural analyses.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>40862</offset><text>Hierarchical clustering revealed that many of the 2,5‐DTP analogs recapitulated most of the peptide recruitment and dismissal patterns observed with E2 (Fig 6H). However, there was a unique cluster of peptides that were recruited by E2 but not the 2,5‐DTP analogs. In contrast, 3,4‐DTP analogs dismissed most of the peptides from the AF‐2 surface (Fig 6H). Thus, the isomeric attachment of diaryl groups to the thiophene core changed the AF‐2 surface from inside the ligand‐binding pocket, as predicted by the crystal structures. Together, these findings suggest that without an extended side chain, cell‐specific activity stems from different coregulator recruitment profiles, due to unique ligand‐induced conformations of the AF‐2 surface, in addition to differential usage of AF‐1. Indirect modulators in cluster 1 avoid this by perturbing the h11–h12 interface, and modulating the dynamics of h12 without changing the shape of AF‐2 when stabilized.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>41841</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>41852</offset><text>Our goal was to identify a minimal set of predictors that would link specific structural perturbations to ERα signaling pathways that control cell‐specific signaling and proliferation. We found a very strong set of predictors, where ligands in cluster 1, defined by similar signaling across cell types, showed indirect modulation of h12 dynamics via the h11–12 interface or slight contact with h12. This perturbation determined proliferation that correlated strongly with AF‐2 activity, recruitment of NCOA1/2/3 family members, and induction of the GREB1 gene, consistent with the canonical ERα signaling pathway (Fig 1D). For ligands in cluster 1, deletion of AF‐1 reduced activity to varying degrees, but did not change the underlying signaling patterns established through AF‐2. In contrast, an extended side chain designed to directly reposition h12 and completely disrupt the AF‐2 surface results in cell‐specific signaling. This was demonstrated with direct modulators in clusters 2 and 3. Cluster 2 was defined by ligand classes that showed correlated activities in two of the three cell types tested, while ligand classes in cluster 3 did not show correlated activities among any of the three cell types. Compared to cluster 1, the structural rules are less clear in clusters 2 and 3, but a number of indirect modulator classes perturbed the LBD conformation at the intersection of h3, the h12 N‐terminus, and the AF‐2 surface. Ligands in these classes altered the shape of AF‐2 to affect coregulator preferences. For direct and indirect modulators in cluster 2 or 3, the canonical ERα signaling pathway involving recruitment of NCOA1/2/3 and induction of GREB1 did not generally predict their proliferative effects, indicating an alternate causal model (Fig 1E).</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>43650</offset><text>These principles outlined above provide a structural basis for how the ligand–receptor interface leads to different signaling specificities through AF‐1 and AF‐2. It is noteworthy that regulation of h12 dynamics indirectly through h11 can virtually abolish AF‐2 activity, and yet still drive robust transcriptional activity through AF‐1, as demonstrated with the OBHS series. This finding can be explained by the fact that NCOA1/2/3 contain distinct binding sites for interaction with AF‐1 and AF‐2 (McInerney et al, 1996; Webb et al, 1998), which allows ligands to nucleate ERα–NCOA1/2/3 interaction through AF‐2, and reinforce this interaction with additional binding to AF‐1. Completely blocking AF‐2 with an extended side chain or altering the shape of AF‐2 changes the preference away from NCOA1/2/3 for determining GREB1 levels and proliferation of breast cancer cells. AF‐2 blockade also allows AF‐1 to function independently, which is important since AF‐1 drives tissue‐selective effects in vivo. This was demonstrated with AF‐1 knockout mice that show E2‐dependent vascular protection, but not uterine proliferation, thus highlighting the role of AF‐1 in tissue‐selective or cell‐specific signaling (Billon‐Gales et al, 2009; Abot et al, 2013).</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>44954</offset><text>One current limitation to our approach is the identification of statistical variables that predict ligand‐specific activity. Here, we examined many LBD structures and tested several variables that were not predictive, including ERβ activity, the strength of AF‐1 signaling, and NCOA3 occupancy at the GREB1 gene. Similarly, we visualized structures to identify patterns. There are many systems biology approaches that could contribute to the unbiased identification of predictive variables for statistical modeling. For example, phage display was used to identify the androgen receptor interactome, which was cloned into an M2H library and used to identify clusters of ligand‐selective interactions (Norris et al, 2009). Also, we have used siRNA screening to identify a number of coregulators required for ERα‐mediated repression of the IL‐6 gene (Nwachukwu et al, 2014). However, the use of larger datasets to identify such predictor variables has its own limitations, one of the major ones being the probability of false positives from multiple hypothesis testing. If we calculated inter‐atomic distance matrices containing 4,000 atoms per structure × 76 ligand–receptor complexes, we would have 3 × 105 predictions. One way to address this issue is to use the cross‐validation concept, where hypotheses are generated on training sets of ligands and tested with another set of ligands.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>46367</offset><text>Based on this work, we propose several testable hypotheses for drug discovery. We have identified atomic vectors for the OBHS‐N and triaryl‐ethylene classes that predict ligand response (Fig 5E and F). These ligands in cluster 1 drive consistent, canonical signaling across cell types, which is desirable for generating full antagonists. Indeed, the most anti‐proliferative compound in the OBHS‐N series had a fulvestrant‐like profile across a battery of assays (S. Srinivasan et al, in preparation). Secondly, our finding that WAY‐C compounds do not rely of AF‐1 for signaling efficacy may derive from the slight contacts with h12 observed in crystal structures (Figs 3B and 5H), unlike other compounds in cluster 1 that dislocate h11 and rely on AF‐1 for signaling efficacy (Figs 3B and 5C, and EV5B). Thirdly, we found ligands that achieved cell‐specific activity without a prototypical extended side chain. Some of these ligands altered the shape of the AF‐2 surface by perturbing the h3–h12 interface, thus providing a route to new SERM‐like activity profiles by combining indirect and direct modulation of receptor structure. Incorporation of statistical approaches to understand relationships between structure and signaling variables moves us toward predictive models for complex ERα‐mediated responses such as in vivo uterine proliferation or tumor growth, and more generally toward structure‐based design for other allosteric drug targets including GPCRs and other nuclear receptors.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>47895</offset><text>Materials and Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>47917</offset><text>Statistical analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>47938</offset><text>Correlation and linear regression analyses were performed using GraphPad Prism software. For correlation analysis, the degree to which two datasets vary together was calculated with the Pearson correlation coefficient (r). However, we reported r 2 rather than r, to facilitate comparison with the linear regression results for which we calculated and reported r 2 (Fig 3C–F). Significance for r 2 was determined using the F‐test for nonzero slope. High‐throughput assays were considered statistically robust if they show Z’ &gt; 0.5, where Z’ = 1 − (3(σp+σn)/|μp−μn|), for the mean (σ) and standard deviations (μ) of the positive and negative controls (Fig EV1A and B).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>48631</offset><text>ERα ligand library</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>48654</offset><text>The library of compounds examined includes both previously reported (Srinivasan et al, 2013) and newly synthesized compound series (see Dataset EV1 for individual compound information, and Appendix Supplementary Methods for synthetic protocols).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>48902</offset><text>Luciferase reporter assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>48929</offset><text>Cells were transfected with FugeneHD reagent (Roche Applied Sciences, Indianapolis, IN) in 384‐well plates. After 24 h, cells were stimulated with 10 μM compounds dispensed using a 100‐nl pintool Biomeck NXP workstation (Beckman Coulter Inc.). Luciferase activity was measured 24 h later (see Appendix Supplementary Methods for more details).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>49281</offset><text>Mammalian 2‐hybrid (M2H) assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>49315</offset><text>HEK293T cells were transfected with 5× UAS‐luciferase reporter, and wild‐type ERα‐VP16 activation domain plus full‐length NCOA1/2/3‐GAL4 DBD fusion protein expression plasmids, using the TransIT‐LT1 transfection reagent (Mirus Bio LLC, Madison, WI). The next day, cells were stimulated with 10 μM compounds using a 100‐nl pintool Biomeck NXP workstation (Beckman Coulter Inc.). Luciferase activity was measured after 24 h (see Appendix Supplementary Methods for more details).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>49813</offset><text>Cell proliferation assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>49838</offset><text>MCF‐7 cells were plated on 384‐well plates in phenol red‐free media plus 10% FBS and stimulated with 10 μM compounds using 100‐nl pintool Biomeck NXP workstation (Beckman Coulter Inc.). Cell numbers determined 1 week later (see Appendix Supplementary Methods for more details).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>50128</offset><text>Quantitative RT–PCR</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>50150</offset><text>MCF‐7 cells were steroid‐deprived and stimulated with compounds for 24 h. Total RNA was extracted and reverse‐transcribed. The cDNA was analyzed using TaqMan Gene Expression Master Mix (Life Technologies, Grand Island, NY), GREB1 and GAPDH (control) primers, and hybridization probes (see Appendix Supplementary Methods for more details).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>50497</offset><text>MARCoNI coregulator‐interaction profiling</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>50541</offset><text>This assay was performed as previously described with the ERα LBD, 10 μM compounds, and a PamChiP peptide microarray (PamGene International) containing 154 unique coregulator peptides (Aarts et al, 2013) (see Appendix Supplementary Methods for more details).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>50805</offset><text>Protein production and X‐ray crystallography</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>50852</offset><text>ERα protein was produced as previously described (Bruning et al, 2010). New ERα LBD structures (see Dataset EV2 for data collection and refinement statistics) were solved by molecular replacement using PHENIX (Adams et al, 2010), refined using ExCoR as previously described (Nwachukwu et al, 2013), and COOT (Emsley &amp; Cowtan, 2004) for ligand‐docking and rebuilding.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>51227</offset><text>Data availability</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>51245</offset><text>Crystal structures analyzed in this study include the following: 1GWR (Warnmark et al, 2002), 3ERD and 3ERT (Shiau et al, 1998), 4ZN9 (Zheng et al, 2012), 4IWC, 4 IU7, 4IV4, 4IVW, 4IW6, 4IUI, 4IV2, 4IVY and 4IW8 (Srinivasan et al, 2013), and 4PPS (Nwachukwu et al, 2014). New crystal structures analyzed in this study were deposited in the RCSB protein data bank (http://www.pdb.org): 4ZN7, 4ZNH, 4ZNS, 4ZNT, 4ZNU, 4ZNV, 4ZNW, 5DI7, 5DID, 5DIE, 5DIG, 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF, 5DP0, 5DRM, 5DRJ, 5DTV, 5DU5, 5DUE, 5DUG, 5DUH, 5DXK, 5DXM, 5DXP, 5DXQ, 5DXR, 5EHJ, 5DY8, 5DYB, 5DYD, 5DZ0, 5DZ1, 5DZ3, 5DZH, 5DZI, 5E0W, 5E0X, 5E14, 5E15, 5E19, 5E1C, 5DVS, 5DVV, 5DWE, 5DWG, 5DWI, 5DWJ, 5EGV, 5EI1, 5EIT.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">title_1</infon><offset>51978</offset><text>Author contributions</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">paragraph</infon><offset>51999</offset><text>JCN and SS contributed equally to this work. JCN and SS designed and performed experiments and wrote the manuscript; YZ, KEC, SW, JM, CD, ZL, VC, JN, NJW, JSJ, and RH performed experiments; HBZ designed experiments; and JAK and KWN designed experiments and wrote the manuscript.</text></passage><passage><infon key="section_type">COMP_INT</infon><infon key="type">title_1</infon><offset>52278</offset><text>Conflict of Interest</text></passage><passage><infon key="section_type">COMP_INT</infon><infon key="type">paragraph</infon><offset>52299</offset><text>The authors declare that they have no conflict of interest.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>52359</offset><text>Supporting information</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>52382</offset><text>References</text></passage><passage><infon key="fpage">336</infon><infon key="lpage">346</infon><infon key="pub-id_pmid">23383871</infon><infon key="section_type">REF</infon><infon key="source">Chem Res Toxicol</infon><infon key="type">ref</infon><infon key="volume">26</infon><infon key="year">2013</infon><offset>52393</offset><text>Robust array‐based coregulator binding assay predicting ERalpha‐agonist potency and generating binding profiles reflecting ligand structure</text></passage><passage><infon key="fpage">2222</infon><infon key="lpage">2233</infon><infon key="pub-id_pmid">23580568</infon><infon key="section_type">REF</infon><infon key="source">Endocrinology</infon><infon key="type">ref</infon><infon key="volume">154</infon><infon key="year">2013</infon><offset>52537</offset><text>The AF‐1 activation function of estrogen receptor alpha is necessary and sufficient for uterine epithelial cell proliferation in vivo</text></passage><passage><infon key="fpage">213</infon><infon key="lpage">221</infon><infon key="pub-id_pmid">20124702</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>52674</offset><text>PHENIX: a comprehensive Python‐based system for macromolecular structure solution</text></passage><passage><infon key="fpage">2811</infon><infon key="lpage">2818</infon><infon key="pub-id_pmid">2118104</infon><infon key="section_type">REF</infon><infon key="source">EMBO J</infon><infon key="type">ref</infon><infon key="volume">9</infon><infon key="year">1990</infon><offset>52758</offset><text>Role of the two activating domains of the oestrogen receptor in the cell‐type and promoter‐context dependent agonistic activity of the anti‐oestrogen 4‐hydroxytamoxifen</text></passage><passage><infon key="fpage">6137</infon><infon key="lpage">6144</infon><infon key="pub-id_pmid">11507064</infon><infon key="section_type">REF</infon><infon key="source">Cancer Res</infon><infon key="type">ref</infon><infon key="volume">61</infon><infon key="year">2001</infon><offset>52935</offset><text>Resveratrol exhibits cytostatic and antiestrogenic properties with human endometrial adenocarcinoma (Ishikawa) cells</text></passage><passage><infon key="fpage">2053</infon><infon key="lpage">2058</infon><infon key="pub-id_pmid">19188600</infon><infon key="section_type">REF</infon><infon key="source">Proc Natl Acad Sci USA</infon><infon key="type">ref</infon><infon key="volume">106</infon><infon key="year">2009</infon><offset>53052</offset><text>The transactivating function 1 of estrogen receptor alpha is dispensable for the vasculoprotective actions of 17beta‐estradiol</text></passage><passage><infon key="fpage">837</infon><infon key="lpage">843</infon><infon key="pub-id_pmid">20924370</infon><infon key="section_type">REF</infon><infon key="source">Nat Chem Biol</infon><infon key="type">ref</infon><infon key="volume">6</infon><infon key="year">2010</infon><offset>53181</offset><text>Coupling of receptor conformation and ligand orientation determine graded activity</text></passage><passage><infon key="fpage">2917</infon><infon key="lpage">2922</infon><infon key="pub-id_pmid">11306468</infon><infon key="section_type">REF</infon><infon key="source">Cancer Res</infon><infon key="type">ref</infon><infon key="volume">61</infon><infon key="year">2001</infon><offset>53264</offset><text>Circumventing tamoxifen resistance in breast cancers using antiestrogens that induce unique conformational changes in the estrogen receptor</text></passage><passage><infon key="fpage">35848</infon><infon key="lpage">35856</infon><infon key="pub-id_pmid">10960470</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">275</infon><infon key="year">2000</infon><offset>53404</offset><text>Analysis of estrogen receptor interaction with a repressor of estrogen receptor activity (REA) and the regulation of estrogen receptor transcriptional activity by REA</text></passage><passage><infon key="fpage">14930</infon><infon key="lpage">14935</infon><infon key="pub-id_pmid">22927406</infon><infon key="section_type">REF</infon><infon key="source">Proc Natl Acad Sci USA</infon><infon key="type">ref</infon><infon key="volume">109</infon><infon key="year">2012</infon><offset>53571</offset><text>Structural and mechanistic insights into bisphenols action provide guidelines for risk assessment and discovery of bisphenol A substitutes</text></passage><passage><infon key="fpage">17335</infon><infon key="lpage">17339</infon><infon key="pub-id_pmid">17463000</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">282</infon><infon key="year">2007</infon><offset>53710</offset><text>Regulation of GREB1 transcription by estrogen receptor alpha through a multipartite enhancer spread over 20 kb of upstream flanking sequences</text></passage><passage><infon key="fpage">2126</infon><infon key="lpage">2132</infon><infon key="pub-id_pmid">15572765</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">60</infon><infon key="year">2004</infon><offset>53853</offset><text>Coot: model‐building tools for molecular graphics</text></passage><passage><infon key="fpage">5363</infon><infon key="lpage">5372</infon><infon key="pub-id_pmid">10409727</infon><infon key="section_type">REF</infon><infon key="source">Mol Cell Biol</infon><infon key="type">ref</infon><infon key="volume">19</infon><infon key="year">1999</infon><offset>53905</offset><text>Purification and identification of p68 RNA helicase acting as a transcriptional coactivator specific for the activation function 1 of human estrogen receptor alpha</text></passage><passage><infon key="fpage">1371</infon><infon key="lpage">1388</infon><infon key="pub-id_pmid">9747868</infon><infon key="section_type">REF</infon><infon key="source">J Natl Cancer Inst</infon><infon key="type">ref</infon><infon key="volume">90</infon><infon key="year">1998</infon><offset>54069</offset><text>Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P‐1 Study</text></passage><passage><infon key="fpage">621</infon><infon key="lpage">627</infon><infon key="pub-id_pmid">8806005</infon><infon key="section_type">REF</infon><infon key="source">Bone</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">1996</infon><offset>54190</offset><text>Time‐dependent changes in biochemical bone markers and serum cholesterol in ovariectomized rats: effects of raloxifene HCl, tamoxifen, estrogen, and alendronate</text></passage><passage><infon key="fpage">6367</infon><infon key="lpage">6375</infon><infon key="pub-id_pmid">11103799</infon><infon key="section_type">REF</infon><infon key="source">Cancer Res</infon><infon key="type">ref</infon><infon key="volume">60</infon><infon key="year">2000</infon><offset>54353</offset><text>PDZK1 and GREB1 are estrogen‐regulated genes expressed in hormone‐responsive breast cancer</text></passage><passage><infon key="fpage">430</infon><infon key="lpage">439</infon><infon key="pub-id_pmid">21664237</infon><infon key="section_type">REF</infon><infon key="source">Mol Cell Endocrinol</infon><infon key="type">ref</infon><infon key="volume">348</infon><infon key="year">2012</infon><offset>54448</offset><text>Steroid receptor coactivators 1, 2, and 3: critical regulators of nuclear receptor activity and steroid receptor modulator (SRM)‐based cancer therapy</text></passage><passage><infon key="fpage">883</infon><infon key="lpage">908</infon><infon key="pub-id_pmid">12620065</infon><infon key="section_type">REF</infon><infon key="source">J Med Chem</infon><infon key="type">ref</infon><infon key="volume">46</infon><infon key="year">2003</infon><offset>54600</offset><text>Antiestrogens and selective estrogen receptor modulators as multifunctional medicines. 1. Receptor interactions</text></passage><passage><infon key="fpage">17</infon><infon key="lpage">27</infon><infon key="pub-id_pmid">22032986</infon><infon key="section_type">REF</infon><infon key="source">Trends Pharmacol Sci</infon><infon key="type">ref</infon><infon key="volume">33</infon><infon key="year">2012</infon><offset>54712</offset><text>Diversity and modularity of G protein‐coupled receptor structures</text></passage><passage><infon key="fpage">3320</infon><infon key="lpage">3329</infon><infon key="pub-id_pmid">20334372</infon><infon key="section_type">REF</infon><infon key="source">J Med Chem</infon><infon key="type">ref</infon><infon key="volume">53</infon><infon key="year">2010</infon><offset>54780</offset><text>Characterization of the pharmacophore properties of novel selective estrogen receptor downregulators (SERDs)</text></passage><passage><infon key="fpage">3532</infon><infon key="lpage">3545</infon><infon key="pub-id_pmid">24708493</infon><infon key="section_type">REF</infon><infon key="source">J Med Chem</infon><infon key="type">ref</infon><infon key="volume">57</infon><infon key="year">2014</infon><offset>54889</offset><text>Triaryl‐substituted Schiff bases are high‐affinity subtype‐selective ligands for the estrogen receptor</text></passage><passage><infon key="fpage">251</infon><infon key="lpage">255</infon><infon key="pub-id_pmid">22498630</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">484</infon><infon key="year">2012</infon><offset>54998</offset><text>Genome‐wide protein‐DNA binding dynamics suggest a molecular clutch for transcription factor function</text></passage><passage><infon key="fpage">e46410</infon><infon key="pub-id_pmid">23056300</infon><infon key="section_type">REF</infon><infon key="source">PLoS ONE</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2012</infon><offset>55104</offset><text>GREB1 functions as a growth promoter and is modulated by IL6/STAT3 in breast cancer</text></passage><passage><infon key="fpage">295</infon><infon key="lpage">316</infon><infon key="pub-id_pmid">12017549</infon><infon key="section_type">REF</infon><infon key="source">Recent Prog Horm Res</infon><infon key="type">ref</infon><infon key="volume">57</infon><infon key="year">2002</infon><offset>55188</offset><text>Definition of the molecular and cellular mechanisms underlying the tissue‐selective agonist/antagonist activities of selective estrogen receptor modulators</text></passage><passage><infon key="fpage">10069</infon><infon key="lpage">10073</infon><infon key="pub-id_pmid">8816752</infon><infon key="section_type">REF</infon><infon key="source">Proc Natl Acad Sci USA</infon><infon key="type">ref</infon><infon key="volume">93</infon><infon key="year">1996</infon><offset>55346</offset><text>Analysis of estrogen receptor transcriptional enhancement by a nuclear hormone receptor coactivator</text></passage><passage><infon key="fpage">751</infon><infon key="lpage">763</infon><infon key="pub-id_pmid">14675539</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">115</infon><infon key="year">2003</infon><offset>55446</offset><text>Estrogen receptor‐alpha directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter</text></passage><passage><infon key="fpage">3346</infon><infon key="lpage">3366</infon><infon key="pub-id_pmid">23586645</infon><infon key="section_type">REF</infon><infon key="source">J Med Chem</infon><infon key="type">ref</infon><infon key="volume">56</infon><infon key="year">2013</infon><offset>55571</offset><text>Thiophene‐core estrogen receptor ligands having superagonist activity</text></passage><passage><infon key="fpage">1589</infon><infon key="lpage">1602</infon><infon key="pub-id_pmid">12699377</infon><infon key="section_type">REF</infon><infon key="source">J Med Chem</infon><infon key="type">ref</infon><infon key="volume">46</infon><infon key="year">2003</infon><offset>55643</offset><text>Bridged bicyclic cores containing a 1,1‐diarylethylene motif are high‐affinity subtype‐selective ligands for the estrogen receptor</text></passage><passage><infon key="fpage">309</infon><infon key="lpage">333</infon><infon key="pub-id_pmid">15709961</infon><infon key="section_type">REF</infon><infon key="source">Annu Rev Physiol</infon><infon key="type">ref</infon><infon key="volume">67</infon><infon key="year">2005</infon><offset>55780</offset><text>Ligand control of coregulator recruitment to nuclear receptors</text></passage><passage><infon key="fpage">241</infon><infon key="lpage">247</infon><infon key="pub-id_pmid">18344977</infon><infon key="section_type">REF</infon><infon key="source">Nat Chem Biol</infon><infon key="type">ref</infon><infon key="volume">4</infon><infon key="year">2008</infon><offset>55843</offset><text>NFκB selectivity of estrogen receptor ligands revealed by comparative crystallographic analyses</text></passage><passage><infon key="fpage">452</infon><infon key="lpage">460</infon><infon key="pub-id_pmid">19389631</infon><infon key="section_type">REF</infon><infon key="source">Chem Biol</infon><infon key="type">ref</infon><infon key="volume">16</infon><infon key="year">2009</infon><offset>55943</offset><text>Differential presentation of protein interaction surfaces on the androgen receptor defines the pharmacological actions of bound ligands</text></passage><passage><infon key="fpage">1923</infon><infon key="lpage">1930</infon><infon key="pub-id_pmid">24076406</infon><infon key="section_type">REF</infon><infon key="source">Structure</infon><infon key="type">ref</infon><infon key="volume">21</infon><infon key="year">2013</infon><offset>56079</offset><text>Improved crystallographic structures using extensive combinatorial refinement</text></passage><passage><infon key="fpage">e02057</infon><infon key="pub-id_pmid">24771768</infon><infon key="section_type">REF</infon><infon key="source">eLife</infon><infon key="type">ref</infon><infon key="volume">3</infon><infon key="year">2014</infon><offset>56157</offset><text>Resveratrol modulates the inflammatory response via an estrogen receptor‐signal integration network</text></passage><passage><infon key="fpage">993</infon><infon key="lpage">996</infon><infon key="pub-id_pmid">17139284</infon><infon key="section_type">REF</infon><infon key="source">Nat Rev Drug Discov</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2006</infon><offset>56259</offset><text>How many drug targets are there?</text></passage><passage><infon key="fpage">141</infon><infon key="lpage">149</infon><infon key="pub-id_pmid">15986123</infon><infon key="section_type">REF</infon><infon key="source">Breast Cancer Res Treat</infon><infon key="type">ref</infon><infon key="volume">92</infon><infon key="year">2005</infon><offset>56292</offset><text>GREB 1 is a critical regulator of hormone dependent breast cancer growth</text></passage><passage><infon key="fpage">93</infon><infon key="lpage">104</infon><infon key="pub-id_pmid">12088871</infon><infon key="section_type">REF</infon><infon key="source">Mol Cell Endocrinol</infon><infon key="type">ref</infon><infon key="volume">192</infon><infon key="year">2002</infon><offset>56365</offset><text>Estrogen receptor‐mediated effects of tamoxifen on human endometrial cancer cells</text></passage><passage><infon key="fpage">2496</infon><infon key="lpage">2511</infon><infon key="pub-id_pmid">16610793</infon><infon key="section_type">REF</infon><infon key="source">J Med Chem</infon><infon key="type">ref</infon><infon key="volume">49</infon><infon key="year">2006</infon><offset>56449</offset><text>Fluorine‐substituted cyclofenil derivatives as estrogen receptor ligands: synthesis and structure‐affinity relationship study of potential positron emission tomography agents for imaging estrogen receptors in breast cancer</text></passage><passage><infon key="fpage">2465</infon><infon key="lpage">2468</infon><infon key="pub-id_pmid">11923541</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">295</infon><infon key="year">2002</infon><offset>56676</offset><text>Molecular determinants for the tissue specificity of SERMs</text></passage><passage><infon key="fpage">927</infon><infon key="lpage">937</infon><infon key="pub-id_pmid">9875847</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">95</infon><infon key="year">1998</infon><offset>56735</offset><text>The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen</text></passage><passage><infon key="fpage">326</infon><infon key="lpage">332</infon><infon key="pub-id_pmid">23524984</infon><infon key="section_type">REF</infon><infon key="source">Nat Chem Biol</infon><infon key="type">ref</infon><infon key="volume">9</infon><infon key="year">2013</infon><offset>56853</offset><text>Ligand‐binding dynamics rewire cellular signaling via estrogen receptor‐alpha</text></passage><passage><infon key="fpage">2324</infon><infon key="lpage">2341</infon><infon key="pub-id_pmid">22283328</infon><infon key="section_type">REF</infon><infon key="source">J Med Chem</infon><infon key="type">ref</infon><infon key="volume">55</infon><infon key="year">2012</infon><offset>56935</offset><text>Identification and structure‐activity relationships of a novel series of estrogen receptor ligands based on 7‐thiabicyclo[2.2.1]hept‐2‐ene‐7‐oxide</text></passage><passage><infon key="fpage">21862</infon><infon key="lpage">21868</infon><infon key="pub-id_pmid">11937504</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">277</infon><infon key="year">2002</infon><offset>57094</offset><text>Interaction of transcriptional intermediary factor 2 nuclear receptor box peptides with the coactivator binding site of estrogen receptor alpha</text></passage><passage><infon key="fpage">1605</infon><infon key="lpage">1618</infon><infon key="pub-id_pmid">9773983</infon><infon key="section_type">REF</infon><infon key="source">Mol Endocrinol</infon><infon key="type">ref</infon><infon key="volume">12</infon><infon key="year">1998</infon><offset>57238</offset><text>Estrogen receptor activation function 1 works by binding p160 coactivator proteins</text></passage><passage><infon key="fpage">18</infon><infon key="lpage">24</infon><infon key="pub-id_pmid">24680426</infon><infon key="section_type">REF</infon><infon key="source">Curr Opin Cell Biol</infon><infon key="type">ref</infon><infon key="volume">27</infon><infon key="year">2014</infon><offset>57321</offset><text>Recent developments in biased agonism</text></passage><passage><infon key="fpage">955</infon><infon key="lpage">966</infon><infon key="pub-id_pmid">22543272</infon><infon key="section_type">REF</infon><infon key="source">Mol Endocrinol</infon><infon key="type">ref</infon><infon key="volume">26</infon><infon key="year">2012</infon><offset>57359</offset><text>Gene‐specific patterns of coregulator requirements by estrogen receptor‐alpha in breast cancer cells</text></passage><passage><infon key="fpage">R27</infon><infon key="pub-id_pmid">21392396</infon><infon key="section_type">REF</infon><infon key="source">Breast Cancer Res</infon><infon key="type">ref</infon><infon key="volume">13</infon><infon key="year">2011</infon><offset>57464</offset><text>Estrogen receptor‐beta sensitizes breast cancer cells to the anti‐estrogenic actions of endoxifen</text></passage><passage><infon key="fpage">1047</infon><infon key="lpage">1058</infon><infon key="pub-id_pmid">25728767</infon><infon key="section_type">REF</infon><infon key="source">Mol Cell</infon><infon key="type">ref</infon><infon key="volume">57</infon><infon key="year">2015</infon><offset>57566</offset><text>Structure of a biologically active estrogen receptor‐coactivator complex on DNA</text></passage><passage><infon key="fpage">1094</infon><infon key="lpage">1100</infon><infon key="pub-id_pmid">22517684</infon><infon key="section_type">REF</infon><infon key="source">ChemMedChem</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2012</infon><offset>57648</offset><text>Development of selective estrogen receptor modulator (SERM)‐like activity through an indirect mechanism of estrogen receptor antagonism: defining the binding mode of 7‐oxabicyclo[2.2.1]hept‐5‐ene scaffold core ligands</text></passage><passage><infon key="fpage">8692</infon><infon key="lpage">8700</infon><infon key="pub-id_pmid">23033157</infon><infon key="section_type">REF</infon><infon key="source">Org Biomol Chem</infon><infon key="type">ref</infon><infon key="volume">10</infon><infon key="year">2012</infon><offset>57874</offset><text>Bicyclic core estrogens as full antagonists: synthesis, biological evaluation and structure‐activity relationships of estrogen receptor ligands based on bridged oxabicyclic core arylsulfonamides</text></passage></document></collection>
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+<collection><source>PMC</source><date>20201222</date><key>pmc.key</key><document><id>4850273</id><infon key="license">CC BY-NC-SA</infon><passage><infon key="alt-title">Xyloglucan Recognition by Gut Bacteria Tauzin et al.</infon><infon key="article-id_doi">10.1128/mBio.02134-15</infon><infon key="article-id_pmc">4850273</infon><infon key="article-id_pmid">27118585</infon><infon key="article-id_publisher-id">mBio02134-15</infon><infon key="elocation-id">e02134-15</infon><infon key="issue">2</infon><infon key="license">This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.</infon><infon key="name_0">surname:Tauzin;given-names:Alexandra S.</infon><infon key="name_1">surname:Kwiatkowski;given-names:Kurt J.</infon><infon key="name_2">surname:Orlovsky;given-names:Nicole I.</infon><infon key="name_3">surname:Smith;given-names:Christopher J.</infon><infon key="name_4">surname:Creagh;given-names:A. Louise</infon><infon key="name_5">surname:Haynes;given-names:Charles A.</infon><infon key="name_6">surname:Wawrzak;given-names:Zdzislaw</infon><infon key="name_7">surname:Brumer;given-names:Harry</infon><infon key="name_8">surname:Koropatkin;given-names:Nicole M.</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">7</infon><infon key="year">2016</infon><offset>0</offset><text>Molecular Dissection of Xyloglucan Recognition in a Prominent Human Gut Symbiont</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>81</offset><text>ABSTRACT</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>90</offset><text>Polysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as “dietary fiber.” However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. In particular, the molecular basis of complex polysaccharide recognition, an essential prerequisite to hydrolysis by cell surface glycosidases and subsequent metabolism, is generally poorly understood. Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus, which are integral to growth on this key dietary vegetable polysaccharide. Biochemical analysis reveals that these outer membrane-anchored proteins are in fact exquisitely specific for the highly branched xyloglucan (XyG) polysaccharide. The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the β-glucan backbone. The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain. Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>1947</offset><text>IMPORTANCE</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1958</offset><text>The Bacteroidetes are dominant bacteria in the human gut that are responsible for the digestion of the complex polysaccharides that constitute “dietary fiber.” Although this symbiotic relationship has been appreciated for decades, little is currently known about how Bacteroidetes seek out and bind plant cell wall polysaccharides as a necessary first step in their metabolism. Here, we provide the first biochemical, crystallographic, and genetic insight into how two surface glycan-binding proteins from the complex Bacteroides ovatus xyloglucan utilization locus (XyGUL) enable recognition and uptake of this ubiquitous vegetable polysaccharide. Our combined analysis illuminates new fundamental aspects of complex polysaccharide recognition, cleavage, and import at the Bacteroidetes cell surface that may facilitate the development of prebiotics to target this phylum of gut bacteria.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>2852</offset><text>INTRODUCTION</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2865</offset><text>The human gut microbiota influences the course of human development and health, playing key roles in immune stimulation, intestinal cell proliferation, and metabolic balance. This microbial community is largely bacterial, with the Bacteroidetes, Firmicutes, and Actinobacteria comprising the dominant phyla. The ability to acquire energy from carbohydrates of dietary or host origin is central to the adaptation of human gut bacterial species to their niche. More importantly, this makes diet a tractable way to manipulate the abundance and metabolic output of the microbiota toward improved human health. However, there is a paucity of data regarding how the vast array of complex carbohydrate structures are selectively recognized and imported by members of the microbiota, a critical process that enables these organisms to thrive in the competitive gut environment. The human gut bacteria Bacteroidetes share a profound capacity for dietary glycan degradation, with many species containing &gt;250 predicted carbohydrate-active enzymes (CAZymes), compared to 50 to 100 within many Firmicutes and only 17 in the human genome devoted toward carbohydrate utilization. A remarkable feature of the Bacteroidetes is the packaging of genes for carbohydrate catabolism into discrete polysaccharide utilization loci (PUL), which are transcriptionally regulated by specific substrate signatures. The archetypal PUL-encoded system is the starch utilization system (Sus) (Fig. 1B) of Bacteroides thetaiotaomicron. The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. The importance of PUL as a successful evolutionary strategy is underscored by the observation that Bacteroidetes such as B. thetaiotaomicron and Bacteroides ovatus devote ~18% of their genomes to these systems. Moving beyond seminal genomic and transcriptomic analyses, the current state-of-the-art PUL characterization involves combined reverse-genetic, biochemical, and structural studies to illuminate the molecular details of PUL function.</text></passage><passage><infon key="file">mbo0021627940001.jpg</infon><infon key="id">fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>5194</offset><text>Xyloglucan and the Bacteroides ovatus xyloglucan utilization locus (XyGUL). (A) Representative structures of common xyloglucans using the Consortium for Functional Glycomics Symbol Nomenclature (http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml). Cleavage sites for BoXyGUL glycosidases (GHs) are indicated for solanaceous xyloglucan. (B) BtSus and BoXyGUL. (C) Localization of BoXyGUL-encoded proteins in cellular membranes and concerted modes of action in the degradation of xyloglucans to monosaccharides. The location of SGBP-A/B is presented in this work; the location of GH5 has been empirically determined, and the enzymes have been placed based upon their predicted cellular location.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5909</offset><text>We recently reported the detailed molecular characterization of a PUL that confers the ability of the human gut commensal B. ovatus ATCC 8483 to grow on a prominent family of plant cell wall glycans, the xyloglucans (XyG). XyG variants (Fig. 1A) constitute up to 25% of the dry weight of common vegetables. Analogous to the Sus locus, the xyloglucan utilization locus (XyGUL) encodes a cohort of carbohydrate-binding, -hydrolyzing, and -importing proteins (Fig. 1B and C). The number of glycoside hydrolases (GHs) encoded by the XyGUL is, however, more expansive than that by the Sus locus (Fig. 1B), which reflects the greater complexity of glycosidic linkages found in XyG vis-à-vis starch. Whereas our previous study focused on the characterization of the linkage specificity of these GHs, a key outstanding question regarding this locus is how XyG recognition is mediated at the cell surface.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6811</offset><text>In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. Bacteroidetes PUL ubiquitously encode homologs of SusC and SusD, as well as proteins whose genes are immediately downstream of susD, akin to susE/F, and these are typically annotated as “putative lipoproteins”. The genes coding for these proteins, sometimes referred to as “susE/F positioned,” display products with a wide variation in amino acid sequence and which have little or no homology to other PUL-encoded proteins or known carbohydrate-binding proteins. As the Sus SGBPs remain the only structurally characterized cohort to date, we therefore wondered whether such glycan binding and function are extended to other PUL that target more complex and heterogeneous polysaccharides, such as XyG.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>8045</offset><text>We describe here the detailed functional and structural characterization of the noncatalytic SGBPs encoded by Bacova_02651 and Bacova_02650 of the XyGUL, here referred to as SGBP-A and SGBP-B, to elucidate their molecular roles in carbohydrate acquisition in vivo. Combined biochemical, structural, and reverse-genetic approaches clearly illuminate the distinct, yet complementary, functions that these two proteins play in XyG recognition as it impacts the physiology of B. ovatus. These data extend our current understanding of the Sus-like glycan uptake paradigm within the Bacteroidetes and reveals how the complex dietary polysaccharide xyloglucan is recognized at the cell surface.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>8734</offset><text>RESULTS AND DISCUSSION</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8757</offset><text>SGBP-A and SGBP-B are cell-surface-localized, xyloglucan-specific binding proteins.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8841</offset><text>SGBP-A, encoded by the XyGUL locus tag Bacova_02651 (Fig. 1B), shares 26% amino acid sequence identity (40% similarity) with its homolog, B. thetaiotaomicron SusD, and similar homology with the SusD-like proteins encoded within syntenic XyGUL identified in our earlier work. In contrast, SGBP-B, encoded by locus tag Bacova_02650, displays little sequence similarity to the products of similarly positioned genes in syntenic XyGUL nor to any other gene product among the diversity of Bacteroidetes PUL. Whereas sequence similarity among SusC/SusD homolog pairs often serves as a hallmark for PUL identification, the sequence similarities of downstream genes encoding SGBPs are generally too low to allow reliable bioinformatic classification of their products into protein families, let alone prediction of function. Hence, there is a critical need for the elucidation of detailed structure-function relationships among PUL SGBPs, in light of the manifold glycan structures in nature.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9828</offset><text>Immunofluorescence of formaldehyde-fixed, nonpermeabilized cells grown in minimal medium with XyG as the sole carbon source to induce XyGUL expression, reveals that both SGBP-A and SGBP-B are presented on the cell surface by N-terminal lipidation, as predicted by signal peptide analysis with SignalP (Fig. 2). Here, the SGBPs very likely work in concert with the cell-surface-localized endo-xyloglucanase B. ovatus GH5 (BoGH5) to recruit and cleave XyG for subsequent periplasmic import via the SusC-like TBDT of the XyGUL (Fig. 1B and C).</text></passage><passage><infon key="file">mbo0021627940002.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>10372</offset><text>SGBP-A and SGBP-B visualized by immunofluorescence. Formalin-fixed, nonpermeabilized B. ovatus cells were grown in minimal medium plus XyG, probed with custom rabbit antibodies to SGBP-A or SGBP-B, and then stained with Alexa Fluor 488 goat anti-rabbit IgG. (A) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-A. (B) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-B. (C) Bright-field image of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. (D) FITC images of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. Cells lacking SGBP-A (ΔSGBP-A) do not grow on XyG and therefore could not be tested in parallel.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11064</offset><text>In our initial study focused on the functional characterization of the glycoside hydrolases of the XyGUL, we reported preliminary affinity PAGE and isothermal titration calorimetry (ITC) data indicating that both SGBP-A and SGBP-B are competent xyloglucan-binding proteins (affinity constant [Ka] values of 3.74 × 105 M−1 and 4.98 × 104 M−1, respectively [23]). Additional affinity PAGE analysis (Fig. 3) demonstrates that SGBP-A also has moderate affinity for the artificial soluble cellulose derivative hydroxyethyl cellulose [HEC; a β(1 → 4)-glucan] and limited affinity for mixed-linkage β(1→3)/β(1→4)-glucan (MLG) and glucomannan (GM; mixed glucosyl and mannosyl backbone), which together indicate general binding to polysaccharide backbone residues and major contributions from side-chain recognition. In contrast, SGBP-B bound to HEC more weakly than SGBP-A and did not bind to MLG or GM. Neither SGBP recognized galactomannan (GGM), starch, carboxymethylcellulose, or mucin (see Fig. S1 in the supplemental material). Together, these results highlight the high specificities of SGBP-A and SGBP-B for XyG, which is concordant with their association with XyG-specific GHs in the XyGUL, as well as transcriptomic analysis indicating that B. ovatus has discrete PUL for MLG, GM, and GGM (11). Notably, the absence of carbohydrate-binding modules in the GHs encoded by the XyGUL implies that noncatalytic recognition of xyloglucan is mediated entirely by SGBP-A and -B.</text></passage><passage><infon key="file">mbo0021627940003.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>12557</offset><text>SGBP-A and SGBP-B preferentially bind xyloglucan. Affinity electrophoresis (10% acrylamide) of SGBP-A and SGBP-B with BSA as a control protein. All samples were loaded on the same gel next to the BSA controls; thin black lines indicate where intervening lanes were removed from the final image for both space and clarity. The percentage of polysaccharide incorporated into each native gel is displayed.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12960</offset><text>The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig. 1A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig. 1A; n = 2). ITC demonstrates that SGBP-A binds to XyG polysaccharide and XyGO2 (based on a Glc8 backbone) with essentially equal affinities, while no binding of XyGO1 (Glc4 backbone) was detectable (Table 1; see Fig. S2 and S3 in the supplemental material). Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. Together, these data clearly suggest that polysaccharide binding of both SGBPs is fulfilled by a dimer of the minimal repeat, corresponding to XyGO2 (cf. Fig. 1A). The observation by affinity PAGE that these proteins specifically recognize XyG is further substantiated by their lack of binding for the undecorated oligosaccharide cellotetraose (Table 1; see Fig. S3). Furthermore, SGBP-A binds cellohexaose with ~770-fold weaker affinity than XyG, while SGBP-B displays no detectable binding to this linear hexasaccharide. To provide molecular-level insight into how the XyGUL SGBPs equip B. ovatus to specifically harvest XyG from the gut environment, we performed X-ray crystallography analysis of both SGBP-A and SGPB-B in oligosaccharide-complex forms.</text></passage><passage><infon key="file">tab1.xml</infon><infon key="id">tab1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>14796</offset><text>Summary of thermodynamic parameters for wild-type SGBP-A and SGBP-B obtained by isothermal titration calorimetry at 25°Ca</text></passage><passage><infon key="file">tab1.xml</infon><infon key="id">tab1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;colgroup span=&quot;1&quot;&gt;&lt;col span=&quot;1&quot;/&gt;&lt;col span=&quot;1&quot;/&gt;&lt;col span=&quot;1&quot;/&gt;&lt;col span=&quot;1&quot;/&gt;&lt;col span=&quot;1&quot;/&gt;&lt;col span=&quot;1&quot;/&gt;&lt;col span=&quot;1&quot;/&gt;&lt;col span=&quot;1&quot;/&gt;&lt;col span=&quot;1&quot;/&gt;&lt;/colgroup&gt;&lt;thead&gt;&lt;tr&gt;&lt;th rowspan=&quot;2&quot; colspan=&quot;1&quot;&gt;Carbohydrate&lt;/th&gt;&lt;th colspan=&quot;2&quot; rowspan=&quot;1&quot;&gt;&lt;italic&gt;K&lt;sub&gt;a&lt;/sub&gt;&lt;/italic&gt; (M&lt;sup&gt;−1&lt;/sup&gt;)&lt;hr/&gt;&lt;/th&gt;&lt;th colspan=&quot;2&quot; rowspan=&quot;1&quot;&gt;Δ&lt;italic&gt;G&lt;/italic&gt; (kcal ⋅ mol&lt;sup&gt;−1&lt;/sup&gt;)&lt;hr/&gt;&lt;/th&gt;&lt;th colspan=&quot;2&quot; rowspan=&quot;1&quot;&gt;Δ&lt;italic&gt;H&lt;/italic&gt; (kcal ⋅ mol&lt;sup&gt;−1&lt;/sup&gt;)&lt;hr/&gt;&lt;/th&gt;&lt;th colspan=&quot;2&quot; rowspan=&quot;1&quot;&gt;&lt;italic&gt;T&lt;/italic&gt;Δ&lt;italic&gt;S&lt;/italic&gt; (kcal ⋅ mol&lt;sup&gt;−1&lt;/sup&gt;)&lt;hr/&gt;&lt;/th&gt;&lt;/tr&gt;&lt;tr&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SGBP-A&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SGBP-B&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SGBP-A&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SGBP-B&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SGBP-A&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SGBP-B&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SGBP-A&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SGBP-B&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;XyG&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;ngtab1.2&quot;&gt;&lt;sup&gt;b&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;(4.4 ± 0.1) × 10&lt;sup&gt;5&lt;/sup&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;(5.7 ± 0.2) × 10&lt;sup&gt;4&lt;/sup&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−7.7&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−6.5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−14 ± 3&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−14 ± 2&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−6.5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−7.6&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;XyGO&lt;sub&gt;2&lt;/sub&gt;&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;ngtab1.3&quot;&gt;&lt;sup&gt;c&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.0 × 10&lt;sup&gt;5&lt;/sup&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.0 × 10&lt;sup&gt;4&lt;/sup&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−7.5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−5.9&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−17.2&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−17.6&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−9.7&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−11.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;XyGO&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;ngtab1.4&quot;&gt;&lt;sup&gt;d&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;(2.4 ± 0.1) × 10&lt;sup&gt;3&lt;/sup&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−4.6&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−4.4 ± 0.2&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Cellohexaose&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;568.0 ± 291.0&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−3.8&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−16 ± 8&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−12.7&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Cellotetraose&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>14919</offset><text>Carbohydrate	Ka (M−1)	ΔG (kcal ⋅ mol−1)	ΔH (kcal ⋅ mol−1)	TΔS (kcal ⋅ mol−1)	 	SGBP-A	SGBP-B	SGBP-A	SGBP-B	SGBP-A	SGBP-B	SGBP-A	SGBP-B	 	XyGb	(4.4 ± 0.1) × 105	(5.7 ± 0.2) × 104	−7.7	−6.5	−14 ± 3	−14 ± 2	−6.5	−7.6	 	XyGO2c	3.0 × 105	2.0 × 104	−7.5	−5.9	−17.2	−17.6	−9.7	−11.7	 	XyGO1	NBd	(2.4 ± 0.1) × 103	NB	−4.6	NB	−4.4 ± 0.2	NB	0.2	 	Cellohexaose	568.0 ± 291.0	NB	−3.8	NB	−16 ± 8	NB	−12.7	NB	 	Cellotetraose	NB	NB	NB	NB	NB	NB	NB	NB	 	</text></passage><passage><infon key="file">tab1.xml</infon><infon key="id">tab1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>15427</offset><text>Shown are average values ± standard errors from two independent titrations, unless otherwise indicated.</text></passage><passage><infon key="file">tab1.xml</infon><infon key="id">tab1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>15532</offset><text>Binding thermodynamics for XyG based on the concentration of the binding unit, XyGO2.</text></passage><passage><infon key="file">tab1.xml</infon><infon key="id">tab1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>15618</offset><text>Values from a single titration.</text></passage><passage><infon key="file">tab1.xml</infon><infon key="id">tab1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>15650</offset><text>NB, no binding observed.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>15675</offset><text>SGBP-A is a SusD homolog with an extensive glycan-binding platform.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15743</offset><text>As anticipated by sequence similarity, the high-resolution tertiary structure of apo-SGBP-A (1.36 Å, Rwork = 14.7%, Rfree = 17.4%, residues 28 to 546) (Table 2) displays the canonical “SusD-like” protein fold dominated by four tetratrico-peptide repeat (TPR) motifs that cradle the rest of the structure (Fig. 4A). Specifically, SGBP-A overlays B. thetaiotaomicron SusD (BtSusD) with a root mean square deviation (RMSD) value of 2.2 Å for 363 Cα pairs, which is notable given the 26% amino acid identity (40% similarity) between these homologs (Fig. 4C). Cocrystallization of SGBP-A with XyGO2 generated a substrate complex structure (2.3 Å, Rwork = 21.8%, Rfree = 24.8%, residues 36 to 546) (Fig. 4A and B; Table 2) that revealed the distinct binding-site architecture of the XyG binding protein. The SGBP-A:XyGO2 complex superimposes closely with the apo structure (RMSD of 0.6 Å) and demonstrates that no major conformational change occurs upon substrate binding; small deviations in the orientation of several surface loops are likely the result of differential crystal packing. It is particularly notable that although the location of the ligand-binding site is conserved between SGBP-A and SusD, that of SGBP-A displays an ~29-Å-long aromatic platform to accommodate the extended, linear XyG chain (see reference for a review of XyG secondary structure), versus the shorter, ~18-Å-long, site within SusD that complements the helical conformation of amylose (Fig. 4C and D).</text></passage><passage><infon key="file">mbo0021627940004.jpg</infon><infon key="id">fig4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>17245</offset><text>Molecular structure of SGBP-A (Bacova_02651). (A) Overlay of SGBP-A from the apo (rainbow) and XyGO2 (gray) structures. The apo structure is color ramped from blue to red. An omit map (2σ) for XyGO2 (orange and red sticks) is displayed. (B) Close-up view of the omit map as in panel A, rotated 90° clockwise. (C) Overlay of the Cα backbones of SGBP-A (black) with XyGO2 (orange and red spheres) and BtSusD (blue) with maltoheptaose (pink and red spheres), highlighting the conservation of the glycan-binding site location. (D) Close-up of the SGBP-A (black and orange) and SusD (blue and pink) glycan-binding sites. The approximate length of each glycan-binding site is displayed, colored to match the protein structures. (E) Stereo view of the xyloglucan-binding site of SGBP-A, displaying all residues within 4 Å of the ligand. The backbone glucose residues are numbered from the nonreducing end; xylose residues are labeled X1 and X2. Potential hydrogen-bonding interactions are shown as dashed lines, and the distance is shown in angstroms.</text></passage><passage><infon key="file">tab2.xml</infon><infon key="id">tab2</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>18298</offset><text>X-ray data collection and refinement statistics</text></passage><passage><infon key="file">tab2.xml</infon><infon key="id">tab2</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;colgroup span=&quot;1&quot;&gt;&lt;col span=&quot;1&quot;/&gt;&lt;col span=&quot;1&quot;/&gt;&lt;col span=&quot;1&quot;/&gt;&lt;col span=&quot;1&quot;/&gt;&lt;col span=&quot;1&quot;/&gt;&lt;/colgroup&gt;&lt;thead&gt;&lt;tr&gt;&lt;th rowspan=&quot;2&quot; colspan=&quot;1&quot;&gt;Parameter&lt;/th&gt;&lt;th colspan=&quot;4&quot; rowspan=&quot;1&quot;&gt;Value(s) for&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;ngtab2.1&quot;&gt;&lt;sup&gt;a&lt;/sup&gt;&lt;/xref&gt;:&lt;hr/&gt;&lt;/th&gt;&lt;/tr&gt;&lt;tr&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SGBP-A &lt;italic&gt;apo&lt;/italic&gt;&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SGBP-A/XyGO&lt;sub&gt;2&lt;/sub&gt;&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SGBP-B/XyGO&lt;sub&gt;2&lt;/sub&gt;&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SGBP-B (CD)/XyGO&lt;sub&gt;2&lt;/sub&gt;&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PDB ID no.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5E75&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5E76&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5E7G&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5E7H&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution (Å)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;21.48–1.36 (1.409–1.36)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;56.13–2.3 (2.382–2.3)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;39.19–2.37 (2.455–2.37)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;30.69–1.57 (1.626–1.570)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Space group&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;P2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;I422&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;R32&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;P6&lt;sub&gt;1&lt;/sub&gt;22&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Unit cell dimensions, &lt;italic&gt;a&lt;/italic&gt;, &lt;italic&gt;b&lt;/italic&gt;, &lt;italic&gt;c&lt;/italic&gt; (Å)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;52.8, 81.4, 57.7; β = 107.85°&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;131.5, 131.5, 188&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;207.4, 207.4, 117.9&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;87.1, 87.1, 201.6&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;No. of reflections&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Total&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;355,272 (26,772)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1,068,014 (102,923)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;324,544 (32,355)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1,366,812 (129,645)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Unique&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99,136 (9,762)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;36,775 (3,625)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;39,362 (3,898)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;62,808 (6,068)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Multiplicity&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.6 (2.7)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;29.0 (28.4)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;8.2 (8.3)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;21.8 (21.4)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Completeness (%)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.71 (98.82)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.63 (99.42)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.96 (100.00)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;98.4 (96.98)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Mean &lt;italic&gt;I&lt;/italic&gt;/σ〈&lt;italic&gt;I&lt;/italic&gt;〉&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;15.57 (2.29)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;24.93 (6.71)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;20.98 (2.36)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;38.52 (5.03)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Wilson B-factor&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;11.91&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;31.14&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;43.91&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;17.86&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;merge&lt;/sub&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.04759 (0.4513)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1428 (0.7178)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.09159 (1.197)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.05559 (0.7748)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;CC&lt;sub&gt;1/2&lt;/sub&gt;&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;ngtab2.2&quot;&gt;&lt;sup&gt;b&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.999 (0.759)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.999 (0.982)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.999 (0.794)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.000 (0.933)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;CC*&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;ngtab2.3&quot;&gt;&lt;sup&gt;c&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.000 (0.929)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.000 (0.995)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.000 (0.941)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.000 (0.982)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;work&lt;/sub&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1468 (0.2597)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.2178 (0.2788)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1975 (0.3018)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1560 (0.2008)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1738 (0.2632)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.2482 (0.2978)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.2260 (0.3219)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1712 (0.2019)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;No. of non-hydrogen atoms&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    All&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4,562&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4,319&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3,678&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2,328&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Macromolecules&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4,079&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3,974&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3,425&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1,985&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Ligands&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;39&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;116&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;127&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;25&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Water&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;444&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;229&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;126&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;318&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;No. of protein residues&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;506&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;492&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;446&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;260&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;RMSD&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Bond length (Å)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.008&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.007&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.005&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.009&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Bond angle (°)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.15&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.96&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.87&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.18&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Ramachandran statistics&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Favored (%)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;98&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;95&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;97&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;98&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Outliers (%)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.41&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.23&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Clash score&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.13&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.86&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.27&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Avg B-factors&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    All&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;16.1&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;53.2&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;53&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;25.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Macromolecules&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;15.2&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;53.5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;52.5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;22.9&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Ligands&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;24.7&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;61&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;71.1&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;47&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Solvent&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;24.4&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;42.9&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;47.6&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;39&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>18346</offset><text>Parameter	Value(s) fora:	 	SGBP-A apo	SGBP-A/XyGO2	SGBP-B/XyGO2	SGBP-B (CD)/XyGO2	 	PDB ID no.	5E75	5E76	5E7G	5E7H	 	Resolution (Å)	21.48–1.36 (1.409–1.36)	56.13–2.3 (2.382–2.3)	39.19–2.37 (2.455–2.37)	30.69–1.57 (1.626–1.570)	 	Space group	P21	I422	R32	P6122	 	Unit cell dimensions, a, b, c (Å)	52.8, 81.4, 57.7; β = 107.85°	131.5, 131.5, 188	207.4, 207.4, 117.9	87.1, 87.1, 201.6	 	No. of reflections					 	    Total	355,272 (26,772)	1,068,014 (102,923)	324,544 (32,355)	1,366,812 (129,645)	 	    Unique	99,136 (9,762)	36,775 (3,625)	39,362 (3,898)	62,808 (6,068)	 	Multiplicity	3.6 (2.7)	29.0 (28.4)	8.2 (8.3)	21.8 (21.4)	 	Completeness (%)	99.71 (98.82)	99.63 (99.42)	99.96 (100.00)	98.4 (96.98)	 	Mean I/σ〈I〉	15.57 (2.29)	24.93 (6.71)	20.98 (2.36)	38.52 (5.03)	 	Wilson B-factor	11.91	31.14	43.91	17.86	 	Rmerge	0.04759 (0.4513)	0.1428 (0.7178)	0.09159 (1.197)	0.05559 (0.7748)	 	CC1/2b	0.999 (0.759)	0.999 (0.982)	0.999 (0.794)	1.000 (0.933)	 	CC*c	1.000 (0.929)	1.000 (0.995)	1.000 (0.941)	1.000 (0.982)	 	Rwork	0.1468 (0.2597)	0.2178 (0.2788)	0.1975 (0.3018)	0.1560 (0.2008)	 	Rfree	0.1738 (0.2632)	0.2482 (0.2978)	0.2260 (0.3219)	0.1712 (0.2019)	 	No. of non-hydrogen atoms					 	    All	4,562	4,319	3,678	2,328	 	    Macromolecules	4,079	3,974	3,425	1,985	 	    Ligands	39	116	127	25	 	    Water	444	229	126	318	 	No. of protein residues	506	492	446	260	 	RMSD					 	    Bond length (Å)	0.008	0.007	0.005	0.009	 	    Bond angle (°)	1.15	0.96	0.87	1.18	 	Ramachandran statistics					 	    Favored (%)	98	95	97	98	 	    Outliers (%)	0	0.41	0.23	0	 	    Clash score	0.5	2.13	0.86	1.27	 	Avg B-factors					 	    All	16.1	53.2	53	25.4	 	    Macromolecules	15.2	53.5	52.5	22.9	 	    Ligands	24.7	61	71.1	47	 	    Solvent	24.4	42.9	47.6	39	 	</text></passage><passage><infon key="file">tab2.xml</infon><infon key="id">tab2</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>20239</offset><text>Numbers in parentheses are for the highest-resolution shell.</text></passage><passage><infon key="file">tab2.xml</infon><infon key="id">tab2</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>20300</offset><text>CC1/2, Pearson correlation coefficient between the average intensities of each subset.</text></passage><passage><infon key="file">tab2.xml</infon><infon key="id">tab2</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>20387</offset><text>CC*, Pearson correlation coefficient for correlation between the observed data set and true signal.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20487</offset><text>Seven of the eight backbone glucosyl residues of XyGO2 could be convincingly modeled in the ligand electron density, and only two α(1→6)-linked xylosyl residues were observed (Fig. 4B; cf. Fig. 1). Indeed, the electron density for the ligand suggests some disorder, which may arise from multiple oligosaccharide orientations along the binding site. Three aromatic residues—W82, W283, W306—comprise the flat platform that stacks along the naturally twisted β-glucan backbone (Fig. 4E). The functional importance of this platform is underscored by the observation that the W82A W283A W306A mutant of SGBP-A, designated SGBP-A*, is completely devoid of XyG affinity (Table 3; see Fig. S4 in the supplemental material). Dissection of the individual contribution of these residues reveals that the W82A mutant displays a significant 4.9-fold decrease in the Ka value for XyG, while the W306A substitution completely abolishes XyG binding. Contrasting with the clear importance of these hydrophobic interactions, there are remarkably few hydrogen-bonding interactions with the ligand, which are provided by R65, N83, and S308, which are proximal to Glc5 and Glc3. Most surprising in light of the saccharide-binding data, however, was a lack of extensive recognition of the XyG side chains; only Y84 appeared to provide a hydrophobic interface for a xylosyl residue (Xyl1).</text></passage><passage><infon key="file">tab3.xml</infon><infon key="id">tab3</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>21867</offset><text>Summary of thermodynamic parameters for site-directed mutants of SGBP-A and SGBP-B obtained by ITC with XyG at 25°Ca</text></passage><passage><infon key="file">tab3.xml</infon><infon key="id">tab3</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;colgroup span=&quot;1&quot;&gt;&lt;col span=&quot;1&quot;/&gt;&lt;col span=&quot;1&quot;/&gt;&lt;col span=&quot;1&quot;/&gt;&lt;col span=&quot;1&quot;/&gt;&lt;col span=&quot;1&quot;/&gt;&lt;col span=&quot;1&quot;/&gt;&lt;/colgroup&gt;&lt;thead&gt;&lt;tr&gt;&lt;th rowspan=&quot;2&quot; colspan=&quot;1&quot;&gt;Protein name&lt;/th&gt;&lt;th colspan=&quot;2&quot; rowspan=&quot;1&quot;&gt;&lt;italic&gt;K&lt;sub&gt;a&lt;/sub&gt;&lt;/italic&gt;&lt;hr/&gt;&lt;/th&gt;&lt;th rowspan=&quot;2&quot; colspan=&quot;1&quot;&gt;Δ&lt;italic&gt;G&lt;/italic&gt; (kcal ⋅ mol&lt;sup&gt;−1&lt;/sup&gt;)&lt;/th&gt;&lt;th rowspan=&quot;2&quot; colspan=&quot;1&quot;&gt;Δ&lt;italic&gt;H&lt;/italic&gt; (kcal ⋅ mol&lt;sup&gt;−1&lt;/sup&gt;)&lt;/th&gt;&lt;th rowspan=&quot;2&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;T&lt;/italic&gt;Δ&lt;italic&gt;S&lt;/italic&gt; (kcal ⋅ mol&lt;sup&gt;−1&lt;/sup&gt;)&lt;/th&gt;&lt;/tr&gt;&lt;tr&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Fold change&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;ngtab3.2&quot;&gt;&lt;sup&gt;b&lt;/sup&gt;&lt;/xref&gt;&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;M&lt;sup&gt;−1&lt;/sup&gt;&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SGBP-A(W82A W283A W306A)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ND&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SGBP-A(W82A)&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;ngtab3.3&quot;&gt;&lt;sup&gt;c&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4.9&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;9.1 × 10&lt;sup&gt;4&lt;/sup&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−6.8&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−6.3&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SGBP-A(W306)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ND&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NB&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SGBP-B(230–489)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.7&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;(8.6 ± 0.20) × 10&lt;sup&gt;4&lt;/sup&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−6.7&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−14.9 ± 0.1&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−8.2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SGBP-B(Y363A)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;19.7&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;(2.9 ± 0.10) × 10&lt;sup&gt;3&lt;/sup&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−4.7&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−18.1 ± 0.1&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−13.3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SGBP-B(W364A)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ND&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Weak&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Weak&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Weak&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Weak&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SGBP-B(F414A)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.2&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;(1.80 ± 0.03) × 10&lt;sup&gt;4&lt;/sup&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−5.8&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−11.4 ± 0.1&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−5.6&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>21985</offset><text>Protein name	Ka	ΔG (kcal ⋅ mol−1)	ΔH (kcal ⋅ mol−1)	TΔS (kcal ⋅ mol−1)	 	Fold changeb	M−1	 	SGBP-A(W82A W283A W306A)	ND	NB	NB	NB	NB	 	SGBP-A(W82A)c	4.9	9.1 × 104	−6.8	−6.3	0.5	 	SGBP-A(W306)	ND	NB	NB	NB	NB	 	SGBP-B(230–489)	0.7	(8.6 ± 0.20) × 104	−6.7	−14.9 ± 0.1	−8.2	 	SGBP-B(Y363A)	19.7	(2.9 ± 0.10) × 103	−4.7	−18.1 ± 0.1	−13.3	 	SGBP-B(W364A)	ND	Weak	Weak	Weak	Weak	 	SGBP-B(F414A)	3.2	(1.80 ± 0.03) × 104	−5.8	−11.4 ± 0.1	−5.6	 	</text></passage><passage><infon key="file">tab3.xml</infon><infon key="id">tab3</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>22478</offset><text>Shown are average values ± standard deviations from two independent titrations, unless otherwise indicated. Binding thermodynamics are based on the concentration of the binding unit, XyGO2. Weak binding represents a Ka of &lt;500 M−1. ND, not determined; NB, no binding.</text></passage><passage><infon key="file">tab3.xml</infon><infon key="id">tab3</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>22749</offset><text>Ka fold change = Ka of wild-type protein/Ka of mutant protein for xyloglucan binding.</text></passage><passage><infon key="file">tab3.xml</infon><infon key="id">tab3</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>22835</offset><text>Values from a single titration.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>22867</offset><text>SGBP-B has a multimodular structure with a single, C-terminal glycan-binding domain.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22952</offset><text>The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37 Å, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table 2) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig. 5A). Domains A, B, and C display similar β-sandwich folds; domains B (residues 134 to 230) and C (residues 231 to 313) can be superimposed onto domain A (residues 34 to 133) with RMSDs of 1.1 and 1.2 Å, respectively, for 47 atom pairs (23% and 16% sequence identity, respectively). These domains also display similarity to the C-terminal β-sandwich domains of many GH13 enzymes, including the cyclodextrin glucanotransferase of Geobacillus stearothermophilus (Fig. 5B). Such domains are not typically involved in carbohydrate binding. Indeed, visual inspection of the SGBP-B structure, as well as individual production of the A and B domains and affinity PAGE analysis (see Fig. S5 in the supplemental material), indicates that these domains do not contribute to XyG capture. On the other hand, production of the fused domains C and D in tandem (SGBP-B residues 230 to 489) retains complete binding of xyloglucan in vitro, with the observed slight increase in affinity likely arising from a reduced potential for steric hindrance of the smaller protein construct during polysaccharide interactions (Table 3). While neither the full-length protein nor domain D displays structural homology to known XyG-binding proteins, the topology of SGBP-B resembles the xylan-binding protein Bacova_04391 (PDB 3ORJ) encoded within a xylan-targeting PUL of B. ovatus (Fig. 5C). The structure-based alignment of these proteins reveals 17% sequence identity, with a core RMSD of 3.6 Å for 253 aligned residues. While there is no substrate-complexed structure of Bacova_04391 available, the binding site is predicted to include W241 and Y404, which are proximal to the XyGO binding site in SGBP-B. However, the opposing, clamp-like arrangement of these residues in Bacova_04391 is clearly distinct from the planar surface arrangement of the residues that interact with XyG in SGBP-B (described below).</text></passage><passage><infon key="file">mbo0021627940005.jpg</infon><infon key="id">fig5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>25179</offset><text>Multimodular structure of SGBP-B (Bacova_02650). (A) Full-length structure of SGBP-B, color coded by domain as indicated. Prolines between domains are indicated as spheres. An omit map (2σ) for XyGO2 is displayed to highlight the location of the glycan-binding site. (B) Overlay of SGBP-B domains A, B, and C (colored as in panel A), with a C-terminal Ig-like domain of the G. stearothermophilus cyclodextrin glucanotransferase (PDB 1CYG [residues 375 to 493]) in green. (C) Cα overlay of SGBP-B (gray) and Bacova_04391 (PDB 3ORJ) (pink). (D) Close-up omit map for the XyGO2 ligand, contoured at 2σ. (E) Stereo view of the xyloglucan-binding site of SGBP-B, displaying all residues within 4 Å of the ligand. The backbone glucose residues are numbered from the nonreducing end, xylose residues are shown as X1, X2, and X3, potential hydrogen-bonding interactions are shown as dashed lines, and the distance is shown in angstroms.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>26120</offset><text>Inspection of the tertiary structure indicates that domains C and D are effectively inseparable, with a contact interface of 396 Å2. Domains A, B, and C do not pack against each other. Moreover, the five-residue linkers between these first three domains all feature a proline as the middle residue, suggesting significant conformational rigidity (Fig. 5A). Despite the lack of sequence and structural conservation, a similarly positioned proline joins the Ig-like domains of the xylan-binding Bacova_04391 and the starch-binding proteins SusE and SusF. We speculate that this is a biologically important adaptation that serves to project the glycan binding site of these proteins far from the membrane surface. Any mobility of SGBP-B on the surface of the cell (beyond lateral diffusion within the membrane) is likely imparted by the eight-residue linker that spans the predicted lipidated Cys (C28) and the first β-strand of domain A. Other outer membrane proteins from various Sus-like systems possess a similar 10- to 20-amino-acid flexible linker between the lipidated Cys that tethers the protein to the outside the cell and the first secondary structure element. Analogously, the outer membrane-anchored endo-xyloglucanase BoGH5 of the XyGUL contains a 100-amino-acid, all-β-strand, N-terminal module and flexible linker that imparts conformational flexibility and distances the catalytic module from the cell surface.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>27551</offset><text>XyG binds to domain D of SGBP-B at the concave interface of the top β-sheet, with binding mediated by loops connecting the β-strands. Six glucosyl residues, comprising the main chain, and three branching xylosyl residues of XyGO2 can be modeled in the density (Fig. 5D; cf. Fig. 1A). The backbone is flat, with less of the “twisted-ribbon” geometry observed in some cello- and xylogluco-oligosaccharides. The aromatic platform created by W330, W364, and Y363 spans four glucosyl residues, compared to the longer platform of SGBP-A, which supports six glucosyl residues (Fig. 5E). The Y363A site-directed mutant of SGBP-B displays a 20-fold decrease in the Ka for XyG, while the W364A mutant lacks XyG binding (Table 3; see Fig. S6 in the supplemental material). There are no additional contacts between the protein and the β-glucan backbone and surprisingly few interactions with the side-chain xylosyl residues, despite that fact that ITC data demonstrate that SGBP-B does not measurably bind the cellohexaose (Table 1). F414 stacks with the xylosyl residue of Glc3, while Q407 is positioned for hydrogen bonding with the O4 of xylosyl residue Xyl1. Surprisingly, an F414A mutant of SGBP-B displays only a mild 3-fold decrease in the Ka value for XyG, again suggesting that glycan recognition is primarily mediated via contact with the β-glucan backbone (Table 3; see Fig. S6). Additional residues surrounding the binding site, including Y369 and E412, may contribute to the recognition of more highly decorated XyG, but precisely how this is mediated is presently unclear. Hoping to achieve a higher-resolution view of the SGBP-B–xyloglucan interaction, we solved the crystal structure of the fused CD domains in complex with XyGO2 (1.57 Å, Rwork = 15.6%, Rfree = 17.1%, residues 230 to 489) (Table 2). The CD domains of the truncated and full-length proteins superimpose with a 0.4-Å RMSD of the Cα backbone, with no differences in the position of any of the glycan-binding residues (see Fig. S7A in the supplemental material). While density is observed for XyGO2, the ligand could not be unambiguously modeled into this density to achieve a reasonable fit between the X-ray data and the known stereochemistry of the sugar (see Fig. S7B and C). While this may occur for a number of reasons in crystal structures, it is likely that the poor ligand density even at higher resolution is due to movement or multiple orientations of the sugar averaged throughout the lattice.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>30050</offset><text>SGBP-A and SGBP-B have distinct, coordinated functions in vivo.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>30114</offset><text>The similarity of the glycan specificity of SGBP-A and SGBP-B presents an intriguing conundrum regarding their individual roles in XyG utilization by B. ovatus. To disentangle the functions of SGBP-A and SGBP-B in XyG recognition and uptake, we created individual in-frame deletion and complementation mutant strains of B. ovatus. In these growth experiments, overnight cultures of strains grown on minimal medium plus glucose were back-diluted 1:100-fold into minimal medium containing 5 mg/ml of the reported carbohydrate. Growth on glucose displayed the shortest lag time for each strain, and so lag times were normalized for each carbohydrate by subtracting the lag time of that strain in glucose (Fig. 6; see Fig. S8 in the supplemental material). A strain in which the entire XyGUL is deleted displays a lag of 24.5 h during growth on glucose compared to the isogenic parental wild-type (WT) Δtdk strain, for which exponential growth lags for 19.8 h (see Fig. S8D). It is unknown whether this is because cultures were not normalized by the starting optical density (OD) or viable cells or reflects a minor defect for glucose utilization. The former seems more likely as the growth rates are nearly identical for these strains on glucose and xylose. The ΔXyGUL and WT Δtdk strains display normalized lag times on xylose within experimental error, and curiously some of the mutant and complemented strains display a nominally shorter lag time on xylose than the WT Δtdk strain. Complementation of the ΔSGBP-A strain (ΔSGBP-A::SGBP-A) restores growth to wild-type rates on xyloglucan and XyGO1, yet the calculated rate of the complemented strain is ~72% that of the WT Δtdk strain on XyGO2; similar results were obtained for the SGBP-B complemented strain despite the fact that the growth curves do not appear much different (see Fig. S8C and F). The reason for this observation on XyGO2 is unclear, as the ΔSGBP-B mutant does not have a significantly different growth rate from the WT on XyGO2. Therefore, we limit our discussion to those mutants that displayed the most obvious defects in growth on particular substrates.</text></passage><passage><infon key="file">mbo0021627940006.jpg</infon><infon key="id">fig6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>32273</offset><text>Growth of select XyGUL mutants on xyloglucan and oligosaccharides. B. ovatus mutants were created in a thymidine kinase deletion (Δtdk) mutant as described previously. SGBP-A* denotes the Bacova_02651 (W82A W283A W306A) allele, and the GH9 gene is Bacova_02649. Growth was measured over time in minimal medium containing (A) XyG, (B) XyGO2, (C) XyGO1, (D) glucose, and (E) xylose. In panel F, the growth rate of each strain on the five carbon sources is displayed, and in panel G, the normalized lag time of each culture, relative to its growth on glucose, is displayed. Solid bars indicate conditions that are not statistically significant from the WT Δtdk cultures grown on the indicated carbohydrate, while open bars indicate a P value of &lt;0.005 compared to the WT Δtdk strain. Conditions denoted by the same letter (b, c, or d) are not statistically significant from each other but are significantly different from the condition labeled “a.” Complementation of ΔSGBP-A and ΔSBGP-B was performed by allelic exchange of the wild-type genes back into the genome for expression via the native promoter: these growth curves, quantified rates and lag times are displayed in Fig. S8 in the supplemental material.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>33503</offset><text>The ΔSGBP-A (ΔBacova_02651) strain (cf. Fig. 1B) was completely incapable of growth on XyG, XyGO1, and XyGO2, indicating that SGBP-A is essential for XyG utilization (Fig. 6). This result mirrors our previous data for the canonical Sus of B. thetaiotaomicron, which revealed that a homologous ΔsusD mutant is unable to grow on starch or malto-oligosaccharides, despite normal cell surface expression of all other PUL-encoded proteins. More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of ΔsusD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B. thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition. Similarly, the function of SGBP-A extends beyond glycan binding. Complementation of ΔSGBP-A with the SGBP-A* (W82A W283A W306A) variant, which does not bind XyG, supports growth on XyG and XyGOs (Fig. 6; ΔSGBP-A::SGBP-A*), with growth rates that are ~70% that of the WT. In previous studies, we observed that carbohydrate binding by SusD enhanced the sensitivity of the cells to limiting concentrations of malto-oligosaccharides by several orders of magnitude, such that the addition of 0.5 g/liter maltose was required to restore growth of the ΔsusD::SusD* strain on starch, which nonetheless occurred following an extended lag phase. In contrast, the ΔSGBP-A::SGBP-A* strain does not display an extended lag time on any of the xyloglucan substrates compared to the WT (Fig. 6). The specific glycan signal that upregulates BoXyGUL is currently unknown. From our present data, we cannot eliminate the possibility that the glycan binding by SGBP-A enhances transcriptional activation of the XyGUL. However, the modest rate defect displayed by the SGBP-A::SGBP-A* strain suggests that recognition of XyG and product import is somewhat less efficient in these cells.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>35481</offset><text>Intriguingly, the ΔSGBP-B strain (ΔBacova_02650) (cf. Fig. 1B) exhibited a minor growth defect on both XyG and XyGO2, with rates 84.6% and 93.9% that of the WT Δtdk strain. However, growth of the ΔSGBP-B strain on XyGO1 was 54.2% the rate of the parental strain, despite the fact that SGBP-B binds this substrate ca. 10-fold more weakly than XyGO2 and XyG (Fig. 6; Table 1). As such, the data suggest that SGBP-A can compensate for the loss of function of SGBP-B on longer oligo- and polysaccharides, while SGBP-B may adapt the cell to recognize smaller oligosaccharides efficiently. Indeed, a double mutant, consisting of a crippled SGBP-A and a deletion of SGBP-B (ΔSGBP-A::SGBP-A*/ΔSGBP-B), exhibits an extended lag time on both XyG and XyGO2, as well as XyGO1. Taken together, the data indicate that SGBP-A and SGBP-B functionally complement each other in the capture of XyG polysaccharide, while SGBP-B may allow B. ovatus to scavenge smaller XyGOs liberated by other gut commensals. This additional role of SGBP-B is especially notable in the context of studies on BtSusE and BtSusF (positioned similarly in the archetypal Sus locus) (Fig. 1B), for which growth defects on starch or malto-oligosaccharides have never been observed. Beyond SGBP-A and SGBP-B, we speculated that the catalytically feeble endo-xyloglucanase GH9, which is expendable for growth in the presence of GH5, might also play a role in glycan binding to the cell surface. However, combined deletion of the genes encoding GH9 (encoded by Bacova_02649) and SGBP-B does not exacerbate the growth defect on XyGO1 (Fig. 6; ΔSGBP-B/ΔGH9).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>37123</offset><text>The necessity of SGBP-B is elevated in the SGBP-A* strain, as the ΔSGBP-A::SGBP-A*/ ΔSGBP-B mutant displays an extended lag during growth on XyG and xylogluco-oligosaccharides, while growth rate differences are more subtle. The precise reason for this lag is unclear, but recapitulating our findings on the role of SusD in malto-oligosaccharide sensing in B. thetaiotaomicron, this extended lag may be due to inefficient import and thus sensing of xyloglucan in the environment in the absence of glycan binding by essential SGBPs. Our previous work demonstrates that B. ovatus cells grown in minimal medium plus glucose express low levels of the XyGUL transcript. Thus, in our experiments, we presume that each strain, initially grown in glucose, expresses low levels of the XyGUL transcript and thus low levels of the XyGUL-encoded surface proteins, including the vanguard GH5. Presumably without glycan binding by the SGBPs, the GH5 protein cannot efficiently process xyloglucan, and/or the lack of SGBP function prevents efficient capture and import of the processed oligosaccharides. It may then be that only after a sufficient amount of glycan is processed and imported by the cell is XyGUL upregulated and exponential growth on the glycan can begin. We hypothesize that during exponential growth the essential role of SGBP-A extends beyond glycan recognition, perhaps due to a critical interaction with the TBDT. In the BtSus, SusD and the TBDT SusC interact, and we speculate that this interaction is necessary for glycan uptake, as suggested by the fact that a ΔsusD mutant cannot grow on starch, but a ΔsusD::SusD* strain regains this ability if a transcriptional activator of the sus operon is supplied. Likewise, such cognate interactions between homologous protein pairs such as SGBP-A and its TBDT may underlie our observation that a ΔSGBP-A mutant cannot grow on xyloglucan. However, unlike the Sus, in which elimination of SusE and SusF does not affect growth on starch, SGBP-B appears to have a dedicated role in growth on small xylogluco-oligosaccharides.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>39216</offset><text>Conclusions.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>39229</offset><text>The ability of gut-adapted microorganisms to thrive in the gastrointestinal tract is critically dependent upon their ability to efficiently recognize, cleave, and import glycans. The human gut, in particular, is a densely packed ecosystem with hundreds of species, in which there is potential for both competition and synergy in the utilization of different substrates. Recent work has elucidated that Bacteroidetes cross-feed during growth on many glycans; the glycoside hydrolases expressed by one species liberate oligosaccharides for consumption by other members of the community. Thus, understanding glycan capture at the cell surface is fundamental to explaining, and eventually predicting, how the carbohydrate content of the diet shapes the gut community structure as well as its causative health effects. Here, we demonstrate that the surface glycan binding proteins encoded within the BoXyGUL play unique and essential roles in the acquisition of the ubiquitous and abundant vegetable polysaccharide xyloglucan. Yet, a number of questions remain regarding the molecular interplay of SGBPs with their cotranscribed cohort of glycoside hydrolases and TonB-dependent transporters.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>40417</offset><text>A particularly understudied aspect of glycan utilization is the mechanism of import via TBDTs (SusC homologs) (Fig. 1), which are ubiquitous and defining components of all PUL. PUL-encoded TBDTs in Bacteroidetes are larger than the well-characterized iron-targeting TBDTs from many Proteobacteria and are further distinguished as the only known glycan-importing TBDTs coexpressed with an SGBP. A direct interaction between the BtSusC TBDT and the SusD SGBP has been previously demonstrated, as has an interaction between the homologous components encoded by an N-glycan-scavenging PUL of Capnocytophaga canimorsus. Our observation here that the physical presence of the SusD homolog SGBP-A, independent of XyG-binding ability, is both necessary and sufficient for XyG utilization further supports a model of glycan import whereby the SusC-like TBDTs and the SusD-like SGBPs must be intimately associated to support glycan uptake (Fig. 1C). It is yet presently unclear whether this interaction is static or dynamic and to what extent the association of cognate TBDT/SGBPs is dependent upon the structure of the carbohydrate to be imported. On the other hand, there is clear evidence for independent TBDTs in Bacteroidetes that do not require SGBP association for activity. For example, it was recently demonstrated that expression of nanO, which encodes a SusC-like TBDT as part of a sialic-acid-targeting PUL from B. fragilis, restored growth on this monosaccharide in a mutant strain of E. coli. In this instance, coexpression of the susD-like gene nanU was not required, nor did the expression of the nanU gene enhance growth kinetics. Similarly, the deletion of BT1762 encoding a fructan-targeting SusD-like protein in B. thetaiotaomicron did not result in a dramatic loss of growth on fructans. Thus, the strict dependence on a SusD-like SGBP for glycan uptake in the Bacteroidetes may be variable and substrate dependent. Furthermore, considering the broader distribution of TBDTs in PUL lacking SGBPs (sometimes known as carbohydrate utilization containing TBDT [CUT] loci; see reference and reviewed in reference) across bacterial phyla, it appears that the intimate biophysical association of these substrate-transport and -binding proteins is the result of specific evolution within the Bacteroidetes.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>42733</offset><text>Equally intriguing is the observation that while SusD-like proteins such as SGBP-A share moderate primary and high tertiary structural conservation, the genes for the SGBPs encoded immediately downstream (Fig. 1B [sometimes referred to as “susE positioned”]) encode glycan-binding lipoproteins with little or no sequence or structural conservation, even among syntenic PUL that target the same polysaccharide. Such is the case for XyGUL from related Bacteroides species, which may encode either one or two of these predicted SGBPs, and these proteins vary considerably in length. The extremely low similarity of these SGBPs is striking in light of the moderate sequence conservation observed among homologous GHs in syntenic PUL. This, together with the observation that these SGBPs, as exemplified by BtSusE and BtSusF and the XyGUL SGBP-B of the present study, are expendable for polysaccharide growth, implies a high degree of evolutionary flexibility to enhance glycan capture at the cell surface. Because the intestinal ecosystem is a dense consortium of bacteria that must compete for their nutrients, these multimodular SGBPs may reflect ongoing evolutionary experiments to enhance glycan uptake efficiency. Whether organisms that express longer SGBPs, extending further above the cell surface toward the extracellular environment, are better equipped to compete for available carbohydrates is presently unknown. However, the natural diversity of these proteins represents a rich source for the discovery of unique carbohydrate-binding motifs to both inform gut microbiology and generate new, specific carbohydrate analytical reagents.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>44381</offset><text>In conclusion, the present study further illuminates the essential role that surface-glycan binding proteins play in facilitating the catabolism of complex dietary carbohydrates by Bacteroidetes. The ability of our resident gut bacteria to recognize polysaccharides is the first committed step of glycan consumption by these organisms, a critical process that influences the community structure and thus the metabolic output (i.e., short-chain fatty acid and metabolite profile) of these organisms. A molecular understanding of glycan uptake by human gut bacteria is therefore central to the development of strategies to improve human health through manipulation of the microbiota.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>45063</offset><text>MATERIALS AND METHODS</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>45085</offset><text>Protein production and purification.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>45122</offset><text>The gene fragments corresponding to Bacova_02650 (encoding SGBP-B residues 34 to 489) and Bacova_02651 (encoding SGBP-A residues 28 to 546) were amplified from Bacteroidetes ovatus ATCC 8483 genomic DNA by PCR using forward primers, including NdeI restriction sites, and reverse primers, including XhoI. The gene products were ligated into a modified version of pET-28a (EMD Biosciences) containing a recombinant tobacco etch virus (rTEV) protease recognition site (pET-28aTEV) preceding an N-terminal 6-His tag for affinity purification. The expression vector (pET-28TEV) containing SGBP-B was used for subsequent cloning of the domains A (residues 34 to 133), B (residues 134 to 229), and CD (residues 230 to 489). The pET-28TEV vector expressing residues 28 to 546 of SGBP-A was utilized for carbohydrate-binding experiments and crystallization of the apo structure. To obtain crystals of SGBP-A with XyGO2, the DNA sequence coding for residues 36 to 546 was PCR amplified from genomic DNA for ligation-independent cloning into the pETite N-His vector (Lucigen, Madison, WI) according to the manufacturer’s instructions. The N-terminal primer for pETite N-His insertion contained a TEV cleavage site immediately downstream of the complementary 18-bp overlap (encoding the His tag) to create a TEV-cleavable His-tagged protein. The site-directed mutants of SGBP-A and SGBP-B in pET-28TEV were created using the QuikChange II site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions. The sequences of all primers to generate these constructs are displayed in Table S1 in the supplemental material.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>46758</offset><text>The plasmids containing the SGBP-A and SGBP-B genes were transformed into Escherichia coli BL21(DE3) or Rosetta(DE3) cells. For native protein expression, cells were cultured in Terrific Broth containing kanamycin (50 µg/ml) and chloramphenicol (20 µg/ml) at 37°C to the mid-exponential phase (A600 of ≈0.6), induced by the addition of 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), and then incubated for 2 days at 16°C or 1 day at 20°C. Cells were harvested by centrifugation and frozen at −80°C prior to protein purification. For selenomethionine-substituted SGBP-B, the pET-28TEV-SGBP-B plasmid was transformed into E. coli Rosetta(DE3)/pLysS and plated onto LB supplemented with kanamycin (50 µg/ml) and chloramphenicol (20 µg/ml). After 16 h of growth at 37°C, colonies were harvested from the plates, used to inoculate 100 ml of M9 minimal medium supplemented with kanamycin (30 µg/ml) and chloramphenicol (20 µg/ml), and then grown at 37°C for 16 h. This overnight culture was used to inoculate a 2-liter baffled flask containing 1 liter of Molecular Dimensions SelenoMet premade medium supplemented with 50 ml of the recommended sterile nutrient mix, chloramphenicol, and kanamycin. Cultures were grown at 37°C to an A600 of ≈0.45 before adjusting the temperature to 20°C and supplementing each flask with 100 mg each of l-lysine, l-threonine, and l-phenylalanine and 50 mg each of l-leucine, l-isoleucine, l-valine, and l-selenomethionine. After 20 additional minutes of growth, the cells were induced with 0.5 mM IPTG, and cultures were grown for an additional 48 h.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>48382</offset><text>For the purification of native and selenomethionine-substituted protein, cells were thawed and lysed via sonication in His buffer (25 mM NaH2PO4, 500 mM NaCl, 20 mM imidazole, pH 7.5) and purified via immobilized nickel affinity chromatography (His-Trap; GE Healthcare) using a gradient of 20 to 300 mM imidazole, according to the manufacturer’s instructions. The His tag was removed by incubation with TEV protease (1:100 molar ratio relative to protein) at room temperature for 2 h and then overnight at 4°C while being dialyzed against His buffer. The cleaved protein was then repurified via nickel affinity chromatography to remove undigested target protein, the cleaved His tag, and His-tagged TEV protease. Purified proteins were dialyzed against 20 mM HEPES–100 mM NaCl (pH 7.0) prior to crystallization and concentrated using Vivaspin 15 (10,000-molecular-weight-cutoff [MWCO]) centrifugal concentrators (Vivaproducts, Inc.).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>49330</offset><text>Glycans.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>49339</offset><text>Xyloglucan from tamarind seed, β-glucan from barley, and konjac glucomannan were purchased from Megazyme. Starch, guar, and mucin were purchased from Sigma. Hydroxyethyl cellulose was purchased from AMRESCO. Carboxymethyl cellulose was purchased from Acros Organics. Xylogluco-oligosaccharides XyGO1 and XyGO2 for biophysical analyses were prepared from tamarind seed XyG according to the method of Martinez-Fleites et al. with minor modifications. XyGO2 for cocrystallization was purchased from Megazyme (O-XGHDP).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>49858</offset><text>Affinity gel electrophoresis.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>49888</offset><text>Affinity PAGE was performed as described previously, with minor modification. Various polysaccharides were used at a concentration of 0.05 to 0.1% (wt/vol), and electrophoresis was carried out for 90 min at room temperature in native 10% (wt/vol) polyacrylamide gels. BSA was used as noninteracting negative-control protein.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>50214</offset><text>ITC.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>50219</offset><text>Isothermal titration calorimetry (ITC) of glycan binding by the SGPB-A mutants was performed using the TA Nano isothermal titration calorimeter calibrated to 25°C. Proteins were dialyzed against 20 mM HEPES–100 mM NaCl (pH 7.0), and sugars were prepared using the dialysis buffer. The protein (45 to 50 µM) was placed in the sample cell, and the syringe was loaded with 2.5 to 4 mg/ml XyG polysaccharide. Following an initial injection of 0.5 µl, 26 subsequent injections of 2 µl were performed with stirring at 350 rpm, and the resulting heat of reaction was recorded. Data were analyzed using the Nano Analyze software. All other ITC experiments were performed using a MicroCal VP-ITC titration calorimeter calibrated to 25°C. Proteins were dialyzed into 20 mM HEPES–100 mM NaCl (pH 7.0), and polysaccharides were prepared using the dialysis buffer. Proteins (micromolar concentrations) were placed in the sample cell, and a first injection of 2 µl was performed followed by 24 subsequent injections of 10 µl of 2 to 20 mM oligosaccharide (cellotetraose, cellohexaose, XyGO1, or XyGO2) or 1 to 2.5 mg/ml XyG polysaccharide. The solution was stirred at 280 rpm, and the resulting heat of reaction was recorded. Data were analyzed using the Origin software program.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>51512</offset><text>DSC.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>51517</offset><text>Structural integrity of the SGBP-B mutants was verified by differential scanning calorimetry (DSC). DSC studies were performed on a MicroCal VP-DSC (Malvern Instruments). Experiments were carried out in 50 mM HEPES (pH 7.0) at a scan rate of 60°C/h. All samples (40 µM protein) were degassed for 7 min with gentle stirring under vacuum prior to being loaded into the calorimeter. Background excess thermal power scans were obtained with buffer in both the sample and reference cells and subtracted from the scans for each sample solution to generate excess heat capacity versus temperature thermograms.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>52126</offset><text>The melting temperature decreased from 57.8 ± 0.9°C for the wild-type SGBP-B protein to 54.6 ± 0.1°C for the Y363A mutant, 54.2 ± 0.1°C for the W364A mutant, and 52 ± 1°C for the F414A mutant. All proteins were therefore in their stable folded state for the ITC measurements (see Fig. S9 in the supplemental material).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>52454</offset><text>Bacteroides ovatus mutagenesis.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>52486</offset><text>Gene deletions and complementations were performed via allelic exchange in a Bacteroides ovatus thymidine kinase gene (Bacova_03071) deletion (Δtdk) derivative strain of ATCC 8483 using the vector pExchange-tdk, as previously described. Primers for the construction of B. ovatus mutants are listed in Table S1. The B. ovatus Δtdk strain and the B. ovatus ΔXyGUL mutant were a generous gift from Eric Martens, University of Michigan Medical School.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>52946</offset><text>Bacteroides growth experiments.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>52978</offset><text>All Bacteroides ovatus culturing was performed in a Coy anaerobic chamber (85% N2, 10% H2, 5% CO2) at 37°C. Prior to growth on minimal medium plus the carbohydrates indicated (Fig. 6; see Fig. S8 in the supplemental material), each strain was grown for 16 h from a freezer stock in tryptone-yeast extract-glucose (TYG) medium and then back diluted 1:100 into Bacteroides minimal medium supplemented with 5 mg/ml glucose, as previously described. After growth for 24 h, cultures were back-diluted 1:100 into Bacteroides minimal medium supplemented with 5 mg/ml of glucose, xylose, XyG, XyGO1, or XyGO2. Growth experiments were performed in replicates of 12 (glucose, xylose, and xyloglucan) or 5 (XyGO1 and XyGO2) as 200-µl cultures in 96-well plates. Plates were loaded into a Biostack automated plate handling device coupled to a Powerwave HT absorbance reader (both devices from Biotek Instruments, Winooski, VT). Absorbance at 600 nm (A600; i.e., optical density at 600 nm [OD600]) was measured for each well at 20-min intervals. Data were processed using Gen5 software (BioTek) and Microsoft Excel. Growth was quantified in each assay by first identifying a minimum time point (Amin) at which A600 had increased by 15% over a baseline reading taken during the first 500 min of incubation. Next, we identified the time point at which A600 reached its maximum (Amax) immediately after exponential growth. The growth rate for each well was defined by (Amax − Amin)/(Tmax − Tmin), where Tmax and Tmin are the corresponding time values for each absorbance. To account for variations in inoculum density, for each strain, the lag time (Tmin) on glucose was subtracted from the lag time for the substrate of interest; in all cases, cultures had shorter lag times on glucose than other glycans.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>54783</offset><text>Immunofluorescence.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>54803</offset><text>Custom rabbit antibodies to recombinant SGBP-A and SGBP-B were generated by Cocalico Biologicals, Inc. (Reamstown, PA). The B. ovatus ATCC 8483 Δtdk and ΔSGBP-B strains were grown in 1 ml minimal Bacteroides medium supplemented with 5 mg/ml tamarind xyloglucan to an A600 of ≈0.6 and then harvested via centrifugation (7,000 × g for 3 min) and washed twice with phosphate-buffered saline (PBS). Cells were resuspended in 0.25 ml PBS, and 0.75 ml of 6% formalin in PBS was added. Cells were incubated with rocking at 20°C for 1.5 h and then washed twice with PBS. Cells were resuspended in 0.5 to 1 ml blocking solution (2% goat serum, 0.02% NaN3 in PBS) and incubated for 16 h at 4°C. Cells were centrifuged and resuspended in 0.5 ml of a 1/100 dilution of custom rabbit antibody sera in blocking solution and incubated by rocking for 2 h at 20°C. Cells were washed with PBS and then resuspended in 0.4 ml of a 1/500 dilution of Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies) in blocking solution and incubated with rocking for 1 h at 20°C. Cells were washed three times with an excess of PBS and then resuspended in 20 µl of PBS plus 1 µl of ProLong Gold antifade (Life Technologies). Cells were spotted on 0.8% agarose pads and imaged at the Center for Live Cell Imaging at the University of Michigan Medical School, using an Olympus IX70 inverted confocal microscope. Images were processed with Metamorph Software.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>56256</offset><text>Crystallization and data collection.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>56293</offset><text>All X-ray diffraction data for both native and selenomethionine-substituted protein crystals were collected at the Life Science Consortium (LS-CAT) at the Advance Photon Source at Argonne National Laboratory in Argonne, IL. The native protein SGBP-B (residues 34 to 489) was concentrated to an A280 of 12.25 prior to crystallization and mixed with 10 mM XyGO2 (Megazyme, O-XGHDP). Hanging drop vapor diffusion was performed against mother liquor consisting of 1.1 to 1.5 M ammonium sulfate and 30 to 70 mM sodium cacodylate (pH 6.5). To decrease crystal nucleation, 0.3 ml of paraffin oil was overlaid on top of 0.5 ml of mother liquor yielding diffraction-quality crystals within 2 weeks. Selenomethionine-substituted crystals of SGBP-B were generated using the same conditions as the native crystals. Crystals of the truncated SGBP-B (domains CD, residues 230 to 489) were obtained by mixing concentrated protein (A600 of 20.6) with 10 mM XyGO2 for hanging drop vapor diffusion against a solution containing 2 M sodium formate and 0.1 M sodium acetate (pH 4.6). All SGBP-B crystals were flash-frozen prior to data collection by briefly soaking in a solution of 80% mother liquor–20% glycerol plus 10 mM xylogluco-oligosaccharide. Data were processed and scaled using HKL2000 and Scalepack. SAD phasing from a selenomethionine-substituted protein crystals was used to determine the structure of SGBP-B. The AutoSol and Autobuild algorithms within the Phenix suite of programs were used to locate and refine the selenium positions and automatically build an initial model of the protein structure, respectively. Successive rounds of manual model building and refinement in Coot and Phenix, respectively, were utilized to build a 2.7-Å model of the selenomethionine-substituted protein, which then was placed in the unit cell of the native data set. Additional rounds of manual model building and refinement were performed to complete the 2.37-Å structure of SGBP-B with XyGO2. The structure of the truncated protein (CD domains, residues 230 to 489) was solved via molecular replacement with Phaser using the CD domains of the full-length protein as a model.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>58465</offset><text>The native protein SGBP-A (residues 28 to 546) was concentrated to an A280 of 28.6 and crystallized via hanging drop vapor diffusion from the Morpheus crystal screen (Molecular Dimensions). Crystals formed in well A1 (30 mM MgCl2, 30 mM CaCl2, 20% polyethylene glycol [PEG 500], 10% PEG 20K, 0.1 M imidazole-MES [morpholinethanesulfonic acid], pH 6.5), and were flash-frozen in liquid nitrogen without additional cryoprotectant. The truncated SGBP-A (residues 36 to 546) concentrated to an A280 of 38.2 yielded crystals with 10 mM XyGO2 via hanging drop vapor diffusion against 1.2 to 1.8 M sodium citrate (pH 6.15 to 6.25), and were flash-frozen in a cryoprotectant solution of 80% mother liquor–20% ethylene glycol with the glycan. Data were processed and scaled using HKL2000 and Scalepack. The structure of the apo protein was solved via molecular replacement with BALBES using the homologous structure PDB 3JYS, followed by successive rounds of automatic and manual model building with Autobuild and Coot. The structure of SGBP-A with XyGO2 was solved via molecular replacement with Phaser and refined with Phenix. X-data collection and refinement statistics are presented in Table 2.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>59664</offset><text>SUPPLEMENTAL MATERIAL</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>59686</offset><text>Citation Tauzin AS, Kwiatkowski KJ, Orlovsky NI, Smith CJ, Creagh AL, Haynes CA, Wawrzak Z, Brumer H, Koropatkin NM. 2016. Molecular dissection of xyloglucan recognition in a prominent human gut symbiont. mBio 7(2):e02134-15. doi:10.1128/mBio.02134-15.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>59939</offset><text>REFERENCES</text></passage><passage><infon key="fpage">107</infon><infon key="lpage">118</infon><infon key="name_0">surname:Mazmanian;given-names:SK</infon><infon key="name_1">surname:Liu;given-names:CH</infon><infon key="name_2">surname:Tzianabos;given-names:AO</infon><infon key="name_3">surname:Kasper;given-names:DL</infon><infon key="pub-id_doi">10.1016/j.cell.2005.05.007</infon><infon key="pub-id_pmid">16009137</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">122</infon><infon key="year">2005</infon><offset>59950</offset><text>An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system</text></passage><passage><infon key="fpage">62</infon><infon key="name_0">surname:Sommer;given-names:F</infon><infon key="name_1">surname:Nookaew;given-names:I</infon><infon key="name_2">surname:Sommer;given-names:N</infon><infon key="name_3">surname:Fogelstrand;given-names:P</infon><infon key="name_4">surname:Bäckhed;given-names:F</infon><infon key="pub-id_doi">10.1186/s13059-015-0614-4</infon><infon key="pub-id_pmid">25887251</infon><infon key="section_type">REF</infon><infon key="source">Genome Biol</infon><infon key="type">ref</infon><infon key="volume">16</infon><infon key="year">2015</infon><offset>60046</offset><text>Site-specific programming of the host epithelial transcriptome by the gut microbiota</text></passage><passage><infon key="comment">1241214</infon><infon key="name_0">surname:Ridaura;given-names:VK</infon><infon key="name_1">surname:Faith;given-names:JJ</infon><infon key="name_10">surname:Muehlbauer;given-names:MJ</infon><infon key="name_11">surname:Ilkayeva;given-names:O</infon><infon key="name_12">surname:Semenkovich;given-names:CF</infon><infon key="name_13">surname:Funai;given-names:K</infon><infon key="name_14">surname:Hayashi;given-names:DK</infon><infon key="name_15">surname:Lyle;given-names:BJ</infon><infon key="name_16">surname:Martini;given-names:MC</infon><infon key="name_17">surname:Ursell;given-names:LK</infon><infon key="name_18">surname:Clemente;given-names:JC</infon><infon key="name_19">surname:Van Treuren;given-names:W</infon><infon key="name_2">surname:Rey;given-names:FE</infon><infon key="name_3">surname:Cheng;given-names:J</infon><infon key="name_4">surname:Duncan;given-names:AE</infon><infon key="name_5">surname:Kau;given-names:AL</infon><infon key="name_6">surname:Griffin;given-names:NW</infon><infon key="name_7">surname:Lombard;given-names:V</infon><infon key="name_8">surname:Henrissat;given-names:B</infon><infon key="name_9">surname:Bain;given-names:JR</infon><infon key="pub-id_doi">10.1126/science.1241214</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">341</infon><infon key="year">2013</infon><offset>60131</offset><text>Gut microbiota from twins discordant for obesity modulate metabolism in mice</text></passage><passage><infon key="fpage">979</infon><infon key="lpage">984</infon><infon key="name_0">surname:Bäckhed;given-names:F</infon><infon key="name_1">surname:Manchester;given-names:JK</infon><infon key="name_2">surname:Semenkovich;given-names:CF</infon><infon key="name_3">surname:Gordon;given-names:JI</infon><infon key="pub-id_doi">10.1073/pnas.0605374104</infon><infon key="pub-id_pmid">17210919</infon><infon key="section_type">REF</infon><infon key="source">Proc Natl Acad Sci U S A</infon><infon key="type">ref</infon><infon key="volume">104</infon><infon key="year">2007</infon><offset>60208</offset><text>Mechanisms underlying the resistance to diet-induced obesity in germ-free mice</text></passage><passage><infon key="fpage">1635</infon><infon key="lpage">1638</infon><infon key="name_0">surname:Eckburg;given-names:PB</infon><infon key="name_1">surname:Bik;given-names:EM</infon><infon key="name_2">surname:Bernstein;given-names:CN</infon><infon key="name_3">surname:Purdom;given-names:E</infon><infon key="name_4">surname:Dethlefsen;given-names:L</infon><infon key="name_5">surname:Sargent;given-names:M</infon><infon key="name_6">surname:Gill;given-names:SR</infon><infon key="name_7">surname:Nelson;given-names:KE</infon><infon key="name_8">surname:Relman;given-names:DA</infon><infon key="pub-id_doi">10.1126/science.1110591</infon><infon key="pub-id_pmid">15831718</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">308</infon><infon key="year">2005</infon><offset>60287</offset><text>Diversity of the human intestinal microbial flora</text></passage><passage><infon key="fpage">357</infon><infon key="lpage">360</infon><infon key="name_0">surname:Ding;given-names:T</infon><infon key="name_1">surname:Schloss;given-names:PD</infon><infon key="pub-id_doi">10.1038/nature13178</infon><infon key="pub-id_pmid">24739969</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">509</infon><infon key="year">2014</infon><offset>60337</offset><text>Dynamics and associations of microbial community types across the human body</text></passage><passage><infon key="fpage">497</infon><infon key="lpage">504</infon><infon key="name_0">surname:El Kaoutari;given-names:A</infon><infon key="name_1">surname:Armougom;given-names:F</infon><infon key="name_2">surname:Gordon;given-names:JI</infon><infon key="name_3">surname:Raoult;given-names:D</infon><infon key="name_4">surname:Henrissat;given-names:B</infon><infon key="pub-id_doi">10.1038/nrmicro3050</infon><infon key="pub-id_pmid">23748339</infon><infon key="section_type">REF</infon><infon key="source">Nat Rev Microbiol</infon><infon key="type">ref</infon><infon key="volume">11</infon><infon key="year">2013</infon><offset>60414</offset><text>The abundance and variety of carbohydrate-active enzymes in the human gut microbiota</text></passage><passage><infon key="fpage">e156</infon><infon key="name_0">surname:Xu;given-names:J</infon><infon key="name_1">surname:Mahowald;given-names:MA</infon><infon key="name_10">surname:Cordum;given-names:H</infon><infon key="name_11">surname:Van Brunt;given-names:A</infon><infon key="name_12">surname:Kim;given-names:K</infon><infon key="name_13">surname:Fulton;given-names:RS</infon><infon key="name_14">surname:Fulton;given-names:LA</infon><infon key="name_15">surname:Clifton;given-names:SW</infon><infon key="name_16">surname:Wilson;given-names:RK</infon><infon key="name_17">surname:Knight;given-names:RD</infon><infon key="name_18">surname:Gordon;given-names:JI</infon><infon key="name_2">surname:Ley;given-names:RE</infon><infon key="name_3">surname:Lozupone;given-names:CA</infon><infon key="name_4">surname:Hamady;given-names:M</infon><infon key="name_5">surname:Martens;given-names:EC</infon><infon key="name_6">surname:Henrissat;given-names:B</infon><infon key="name_7">surname:Coutinho;given-names:PM</infon><infon key="name_8">surname:Minx;given-names:P</infon><infon key="name_9">surname:Latreille;given-names:P</infon><infon key="pub-id_doi">10.1371/journal.pbio.0050156</infon><infon key="pub-id_pmid">17579514</infon><infon key="section_type">REF</infon><infon key="source">PLoS Biol</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2007</infon><offset>60499</offset><text>Evolution of symbiotic bacteria in the distal human intestine</text></passage><passage><infon key="fpage">2074</infon><infon key="lpage">2076</infon><infon key="name_0">surname:Xu;given-names:J</infon><infon key="name_1">surname:Bjursell;given-names:MK</infon><infon key="name_2">surname:Himrod;given-names:J</infon><infon key="name_3">surname:Deng;given-names:S</infon><infon key="name_4">surname:Carmichael;given-names:LK</infon><infon key="name_5">surname:Chiang;given-names:HC</infon><infon key="name_6">surname:Hooper;given-names:LV</infon><infon key="name_7">surname:Gordon;given-names:JI</infon><infon key="pub-id_doi">10.1126/science.1080029</infon><infon key="pub-id_pmid">12663928</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">299</infon><infon key="year">2003</infon><offset>60561</offset><text>A genomic view of the human-Bacteroides thetaiotaomicron symbiosis</text></passage><passage><infon key="fpage">3851</infon><infon key="lpage">3865</infon><infon key="name_0">surname:Martens;given-names:EC</infon><infon key="name_1">surname:Kelly;given-names:AG</infon><infon key="name_2">surname:Tauzin;given-names:AS</infon><infon key="name_3">surname:Brumer;given-names:H</infon><infon key="pub-id_doi">10.1016/j.jmb.2014.06.022</infon><infon key="pub-id_pmid">25026064</infon><infon key="section_type">REF</infon><infon key="source">J Mol Biol</infon><infon key="type">ref</infon><infon key="volume">426</infon><infon key="year">2014</infon><offset>60628</offset><text>The devil lies in the details: how variations in polysaccharide fine-structure impact the physiology and evolution of gut microbes</text></passage><passage><infon key="fpage">e1001221</infon><infon key="name_0">surname:Martens;given-names:EC</infon><infon key="name_1">surname:Lowe;given-names:EC</infon><infon key="name_10">surname:Gordon;given-names:JI</infon><infon key="name_2">surname:Chiang;given-names:H</infon><infon key="name_3">surname:Pudlo;given-names:NA</infon><infon key="name_4">surname:Wu;given-names:M</infon><infon key="name_5">surname:McNulty;given-names:NP</infon><infon key="name_6">surname:Abbott;given-names:DW</infon><infon key="name_7">surname:Henrissat;given-names:B</infon><infon key="name_8">surname:Gilbert;given-names:HJ</infon><infon key="name_9">surname:Bolam;given-names:DN</infon><infon key="pub-id_doi">10.1371/journal.pbio.1001221</infon><infon key="pub-id_pmid">22205877</infon><infon key="section_type">REF</infon><infon key="source">PLoS Biol</infon><infon key="type">ref</infon><infon key="volume">9</infon><infon key="year">2011</infon><offset>60759</offset><text>Recognition and degradation of plant cell wall polysaccharides by two human gut symbionts</text></passage><passage><infon key="fpage">5609</infon><infon key="lpage">5616</infon><infon key="name_0">surname:Tancula;given-names:E</infon><infon key="name_1">surname:Feldhaus;given-names:MJ</infon><infon key="name_2">surname:Bedzyk;given-names:LA</infon><infon key="name_3">surname:Salyers;given-names:AA</infon><infon key="pub-id_pmid">1512196</infon><infon key="section_type">REF</infon><infon key="source">J Bacteriol</infon><infon key="type">ref</infon><infon key="volume">174</infon><infon key="year">1992</infon><offset>60849</offset><text>Location and characterization of genes involved in binding of starch to the surface of Bacteroides thetaiotaomicron</text></passage><passage><infon key="fpage">5365</infon><infon key="lpage">5372</infon><infon key="name_0">surname:Shipman;given-names:JA</infon><infon key="name_1">surname:Berleman;given-names:JE</infon><infon key="name_2">surname:Salyers;given-names:AA</infon><infon key="pub-id_doi">10.1128/JB.182.19.5365-5372.2000</infon><infon key="pub-id_pmid">10986238</infon><infon key="section_type">REF</infon><infon key="source">J Bacteriol</infon><infon key="type">ref</infon><infon key="volume">182</infon><infon key="year">2000</infon><offset>60965</offset><text>Characterization of four outer membrane proteins involved in binding starch to the cell surface of Bacteroides thetaiotaomicron</text></passage><passage><infon key="fpage">7206</infon><infon key="lpage">7211</infon><infon key="name_0">surname:Shipman;given-names:JA</infon><infon key="name_1">surname:Cho;given-names:KH</infon><infon key="name_2">surname:Siegel;given-names:HA</infon><infon key="name_3">surname:Salyers;given-names:AA</infon><infon key="pub-id_pmid">10572122</infon><infon key="section_type">REF</infon><infon key="source">J Bacteriol</infon><infon key="type">ref</infon><infon key="volume">181</infon><infon key="year">1999</infon><offset>61093</offset><text>Physiological characterization of SusG, an outer membrane protein essential for starch utilization by Bacteroides thetaiotaomicron</text></passage><passage><infon key="fpage">200</infon><infon key="lpage">215</infon><infon key="name_0">surname:Koropatkin;given-names:NM</infon><infon key="name_1">surname:Smith;given-names:TJ</infon><infon key="pub-id_doi">10.1016/j.str.2009.12.010</infon><infon key="pub-id_pmid">20159465</infon><infon key="section_type">REF</infon><infon key="source">Structure</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">2010</infon><offset>61224</offset><text>SusG: A unique cell-membrane-associated alpha-amylase from a prominent human gut symbiont targets complex starch molecules</text></passage><passage><infon key="fpage">1105</infon><infon key="lpage">1115</infon><infon key="name_0">surname:Koropatkin;given-names:NM</infon><infon key="name_1">surname:Martens;given-names:EC</infon><infon key="name_2">surname:Gordon;given-names:JI</infon><infon key="name_3">surname:Smith;given-names:TJ</infon><infon key="pub-id_doi">10.1016/j.str.2008.03.017</infon><infon key="pub-id_pmid">18611383</infon><infon key="section_type">REF</infon><infon key="source">Structure</infon><infon key="type">ref</infon><infon key="volume">16</infon><infon key="year">2008</infon><offset>61347</offset><text>Starch catabolism by a prominent human gut symbiont is directed by the recognition of amylose helices</text></passage><passage><infon key="fpage">823</infon><infon key="lpage">830</infon><infon key="name_0">surname:Reeves;given-names:AR</infon><infon key="name_1">surname:D’Elia;given-names:JN</infon><infon key="name_2">surname:Frias;given-names:J</infon><infon key="name_3">surname:Salyers;given-names:AA</infon><infon key="pub-id_pmid">8550519</infon><infon key="section_type">REF</infon><infon key="source">J Bacteriol</infon><infon key="type">ref</infon><infon key="volume">178</infon><infon key="year">1996</infon><offset>61449</offset><text>A Bacteroides thetaiotaomicron outer membrane protein that is essential for utilization of maltooligosaccharides and starch</text></passage><passage><infon key="fpage">34614</infon><infon key="lpage">34625</infon><infon key="name_0">surname:Cameron;given-names:EA</infon><infon key="name_1">surname:Maynard;given-names:MA</infon><infon key="name_2">surname:Smith;given-names:CJ</infon><infon key="name_3">surname:Smith;given-names:TJ</infon><infon key="name_4">surname:Koropatkin;given-names:NM</infon><infon key="name_5">surname:Martens;given-names:EC</infon><infon key="pub-id_doi">10.1074/jbc.M112.397380</infon><infon key="pub-id_pmid">22910908</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">287</infon><infon key="year">2012</infon><offset>61573</offset><text>Multidomain carbohydrate-binding proteins involved in Bacteroides thetaiotaomicron starch metabolism</text></passage><passage><infon key="fpage">7173</infon><infon key="lpage">7179</infon><infon key="name_0">surname:D’Elia;given-names:JN</infon><infon key="name_1">surname:Salyers;given-names:AA</infon><infon key="pub-id_pmid">8955399</infon><infon key="section_type">REF</infon><infon key="source">J Bacteriol</infon><infon key="type">ref</infon><infon key="volume">178</infon><infon key="year">1996</infon><offset>61674</offset><text>Contribution of a neopullulanase, a pullulanase, and an alpha-glucosidase to growth of Bacteroides thetaiotaomicron on starch</text></passage><passage><infon key="fpage">19786</infon><infon key="lpage">19791</infon><infon key="name_0">surname:Hehemann;given-names:JH</infon><infon key="name_1">surname:Kelly;given-names:AG</infon><infon key="name_2">surname:Pudlo;given-names:NA</infon><infon key="name_3">surname:Martens;given-names:EC</infon><infon key="name_4">surname:Boraston;given-names:AB</infon><infon key="pub-id_doi">10.1073/pnas.1211002109</infon><infon key="pub-id_pmid">23150581</infon><infon key="section_type">REF</infon><infon key="source">Proc Natl Acad Sci U S A</infon><infon key="type">ref</infon><infon key="volume">109</infon><infon key="year">2012</infon><offset>61800</offset><text>Bacteria of the human gut microbiome catabolize red seaweed glycans with carbohydrate-active enzyme updates from extrinsic microbes</text></passage><passage><infon key="fpage">165</infon><infon key="lpage">169</infon><infon key="name_0">surname:Cuskin;given-names:F</infon><infon key="name_1">surname:Lowe;given-names:EC</infon><infon key="name_10">surname:Rogowski;given-names:A</infon><infon key="name_11">surname:Hamilton;given-names:BS</infon><infon key="name_12">surname:Chen;given-names:R</infon><infon key="name_13">surname:Tolbert;given-names:TJ</infon><infon key="name_14">surname:Piens;given-names:K</infon><infon key="name_15">surname:Bracke;given-names:D</infon><infon key="name_16">surname:Vervecken;given-names:W</infon><infon key="name_17">surname:Hakki;given-names:Z</infon><infon key="name_18">surname:Speciale;given-names:G</infon><infon key="name_19">surname:Munōz-Munōz;given-names:JL</infon><infon key="name_2">surname:Temple;given-names:MJ</infon><infon key="name_20">surname:Day;given-names:A</infon><infon key="name_21">surname:Pena;given-names:MJ</infon><infon key="name_22">surname:McLean;given-names:R</infon><infon key="name_23">surname:Suits;given-names:MD</infon><infon key="name_24">surname:Boraston;given-names:AB</infon><infon key="name_25">surname:Atherly;given-names:T</infon><infon key="name_26">surname:Ziemer;given-names:CJ</infon><infon key="name_27">surname:Williams;given-names:SJ</infon><infon key="name_28">surname:Davies;given-names:GJ</infon><infon key="name_29">surname:Abbott;given-names:DW</infon><infon key="name_3">surname:Zhu;given-names:Y</infon><infon key="name_30">surname:Martens;given-names:EC</infon><infon key="name_31">surname:Gilbert;given-names:HJ</infon><infon key="name_4">surname:Cameron;given-names:EA</infon><infon key="name_5">surname:Pudlo;given-names:NA</infon><infon key="name_6">surname:Porter;given-names:NT</infon><infon key="name_7">surname:Urs;given-names:K</infon><infon key="name_8">surname:Thompson;given-names:AJ</infon><infon key="name_9">surname:Cartmell;given-names:A</infon><infon key="pub-id_doi">10.1038/nature13995</infon><infon key="pub-id_pmid">25567280</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">517</infon><infon key="year">2015</infon><offset>61932</offset><text>Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism</text></passage><passage><infon key="fpage">7481</infon><infon key="name_0">surname:Rogowski;given-names:A</infon><infon key="name_1">surname:Briggs;given-names:JA</infon><infon key="name_10">surname:Rogers;given-names:TE</infon><infon key="name_11">surname:Thompson;given-names:P</infon><infon key="name_12">surname:Hawkins;given-names:AR</infon><infon key="name_13">surname:Yadav;given-names:MP</infon><infon key="name_14">surname:Henrissat;given-names:B</infon><infon key="name_15">surname:Martens;given-names:EC</infon><infon key="name_16">surname:Dupree;given-names:P</infon><infon key="name_17">surname:Gilbert;given-names:HJ</infon><infon key="name_18">surname:Bolam;given-names:DN</infon><infon key="name_2">surname:Mortimer;given-names:JC</infon><infon key="name_3">surname:Tryfona;given-names:T</infon><infon key="name_4">surname:Terrapon;given-names:N</infon><infon key="name_5">surname:Lowe;given-names:EC</infon><infon key="name_6">surname:Basle;given-names:A</infon><infon key="name_7">surname:Morland;given-names:C</infon><infon key="name_8">surname:Day;given-names:AM</infon><infon key="name_9">surname:Zheng;given-names:H</infon><infon key="pub-id_doi">10.1038/ncomms8481</infon><infon key="pub-id_pmid">26112186</infon><infon key="section_type">REF</infon><infon key="source">Nat Commun</infon><infon key="type">ref</infon><infon key="volume">6</infon><infon key="year">2015</infon><offset>62009</offset><text>Glycan complexity dictates microbial resource allocation in the large intestine</text></passage><passage><infon key="fpage">498</infon><infon key="lpage">502</infon><infon key="name_0">surname:Larsbrink;given-names:J</infon><infon key="name_1">surname:Rogers;given-names:TE</infon><infon key="name_10">surname:Creagh;given-names:AL</infon><infon key="name_11">surname:Haynes;given-names:CA</infon><infon key="name_12">surname:Kelly;given-names:AG</infon><infon key="name_13">surname:Cederholm;given-names:SN</infon><infon key="name_14">surname:Davies;given-names:GJ</infon><infon key="name_15">surname:Martens;given-names:EC</infon><infon key="name_16">surname:Brumer;given-names:H</infon><infon key="name_2">surname:Hemsworth;given-names:GR</infon><infon key="name_3">surname:McKee;given-names:LS</infon><infon key="name_4">surname:Tauzin;given-names:AS</infon><infon key="name_5">surname:Spadiut;given-names:O</infon><infon key="name_6">surname:Klinter;given-names:S</infon><infon key="name_7">surname:Pudlo;given-names:NA</infon><infon key="name_8">surname:Urs;given-names:K</infon><infon key="name_9">surname:Koropatkin;given-names:NM</infon><infon key="pub-id_doi">10.1038/nature12907</infon><infon key="pub-id_pmid">24463512</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">506</infon><infon key="year">2014</infon><offset>62089</offset><text>A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes</text></passage><passage><infon key="fpage">133</infon><infon key="lpage">150</infon><infon key="name_0">surname:McDougall;given-names:GJ</infon><infon key="name_1">surname:Morrison;given-names:IM</infon><infon key="name_2">surname:Stewart;given-names:D</infon><infon key="name_3">surname:Hillman;given-names:JR</infon><infon key="section_type">REF</infon><infon key="source">J Sci Food Agric</infon><infon key="type">ref</infon><infon key="volume">70</infon><infon key="year">1996</infon><offset>62178</offset><text>Plant cell walls as dietary fibre: range, structure, processing and function</text></passage><passage><infon key="fpage">526</infon><infon key="lpage">542</infon><infon key="name_0">surname:Schultink;given-names:A</infon><infon key="name_1">surname:Liu;given-names:L</infon><infon key="name_2">surname:Zhu;given-names:L</infon><infon key="name_3">surname:Pauly;given-names:M</infon><infon key="pub-id_doi">10.3390/plants3040526</infon><infon key="section_type">REF</infon><infon key="source">Plants</infon><infon key="type">ref</infon><infon key="volume">3</infon><infon key="year">2014</infon><offset>62255</offset><text>Structural diversity and function of xyloglucan sidechain substituents</text></passage><passage><infon key="fpage">e01441-01414</infon><infon key="name_0">surname:Cameron;given-names:EA</infon><infon key="name_1">surname:Kwiatkowski;given-names:KJ</infon><infon key="name_2">surname:Lee;given-names:BH</infon><infon key="name_3">surname:Hamaker;given-names:BR</infon><infon key="name_4">surname:Koropatkin;given-names:NM</infon><infon key="name_5">surname:Martens;given-names:EC</infon><infon key="pub-id_doi">10.1128/mBio.01441-14</infon><infon key="pub-id_pmid">25205092</infon><infon key="section_type">REF</infon><infon key="source">mBio</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2014</infon><offset>62326</offset><text>Multifunctional nutrient-binding proteins adapt human symbiotic bacteria for glycan competition in the gut by separately promoting enhanced sensing and catalysis</text></passage><passage><infon key="fpage">647</infon><infon key="lpage">655</infon><infon key="name_0">surname:Terrapon;given-names:N</infon><infon key="name_1">surname:Lombard;given-names:V</infon><infon key="name_2">surname:Gilbert;given-names:HJ</infon><infon key="name_3">surname:Henrissat;given-names:B</infon><infon key="pub-id_doi">10.1093/bioinformatics/btu716</infon><infon key="pub-id_pmid">25355788</infon><infon key="section_type">REF</infon><infon key="source">Bioinformatics</infon><infon key="type">ref</infon><infon key="volume">31</infon><infon key="year">2015</infon><offset>62488</offset><text>Automatic prediction of polysaccharide utilization loci in Bacteroidetes species</text></passage><passage><infon key="fpage">24673</infon><infon key="lpage">24677</infon><infon key="name_0">surname:Martens;given-names:EC</infon><infon key="name_1">surname:Koropatkin;given-names:NM</infon><infon key="name_2">surname:Smith;given-names:TJ</infon><infon key="name_3">surname:Gordon;given-names:JI</infon><infon key="pub-id_doi">10.1074/jbc.R109.022848</infon><infon key="pub-id_pmid">19553672</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">284</infon><infon key="year">2009</infon><offset>62569</offset><text>Complex glycan catabolism by the human gut microbiota: the Bacteroidetes Sus-like paradigm</text></passage><passage><infon key="fpage">86</infon><infon key="lpage">90</infon><infon key="name_0">surname:Bolam;given-names:DN</infon><infon key="name_1">surname:Sonnenburg;given-names:JL</infon><infon key="pub-id_doi">10.4161/gmic.2.2.15232</infon><infon key="pub-id_pmid">21637023</infon><infon key="section_type">REF</infon><infon key="source">Gut Microbes</infon><infon key="type">ref</infon><infon key="volume">2</infon><infon key="year">2011</infon><offset>62660</offset><text>Mechanistic insight into polysaccharide use within the intestinal microbiota</text></passage><passage><infon key="fpage">e1000798</infon><infon key="name_0">surname:Ellrott;given-names:K</infon><infon key="name_1">surname:Jaroszewski;given-names:L</infon><infon key="name_2">surname:Li;given-names:W</infon><infon key="name_3">surname:Wooley;given-names:JC</infon><infon key="name_4">surname:Godzik;given-names:A</infon><infon key="pub-id_doi">10.1371/journal.pcbi.1000798</infon><infon key="pub-id_pmid">20532204</infon><infon key="section_type">REF</infon><infon key="source">PLoS Comput Biol</infon><infon key="type">ref</infon><infon key="volume">6</infon><infon key="year">2010</infon><offset>62737</offset><text>Expansion of the protein repertoire in newly explored environments: human gut microbiome specific protein families</text></passage><passage><infon key="fpage">563</infon><infon key="lpage">569</infon><infon key="name_0">surname:Bolam;given-names:DN</infon><infon key="name_1">surname:Koropatkin;given-names:NM</infon><infon key="pub-id_doi">10.1016/j.sbi.2012.06.006</infon><infon key="pub-id_pmid">22819666</infon><infon key="section_type">REF</infon><infon key="source">Curr Opin Struct Biol</infon><infon key="type">ref</infon><infon key="volume">22</infon><infon key="year">2012</infon><offset>62852</offset><text>Glycan recognition by the Bacteroidetes Sus-like systems</text></passage><passage><infon key="fpage">625</infon><infon key="lpage">641</infon><infon key="name_0">surname:Zhou;given-names:Q</infon><infon key="name_1">surname:Rutland;given-names:MW</infon><infon key="name_2">surname:Teeri;given-names:TT</infon><infon key="name_3">surname:Brumer;given-names:H</infon><infon key="pub-id_doi">10.1007/s10570-007-9109-0</infon><infon key="section_type">REF</infon><infon key="source">Cellulose</infon><infon key="type">ref</infon><infon key="volume">14</infon><infon key="year">2007</infon><offset>62909</offset><text>Xyloglucan in cellulose modification</text></passage><passage><infon key="fpage">1532</infon><infon key="lpage">1542</infon><infon key="name_0">surname:Koropatkin;given-names:N</infon><infon key="name_1">surname:Martens;given-names:EC</infon><infon key="name_2">surname:Gordon;given-names:JI</infon><infon key="name_3">surname:Smith;given-names:TJ</infon><infon key="pub-id_doi">10.1021/bi801942a</infon><infon key="pub-id_pmid">19191477</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry</infon><infon key="type">ref</infon><infon key="volume">48</infon><infon key="year">2009</infon><offset>62946</offset><text>Structure of a SusD homologue, BT1043, involved in mucin O-glycan utilization in a prominent human gut symbiont</text></passage><passage><infon key="fpage">3466</infon><infon key="lpage">3475</infon><infon key="name_0">surname:von Schantz;given-names:L</infon><infon key="name_1">surname:Håkansson;given-names:M</infon><infon key="name_2">surname:Logan;given-names:DT</infon><infon key="name_3">surname:Nordberg-Karlsson;given-names:E</infon><infon key="name_4">surname:Ohlin;given-names:M</infon><infon key="pub-id_doi">10.1002/prot.24700</infon><infon key="pub-id_pmid">25302425</infon><infon key="section_type">REF</infon><infon key="source">Proteins</infon><infon key="type">ref</infon><infon key="volume">82</infon><infon key="year">2014</infon><offset>63058</offset><text>Carbohydrate binding module recognition of xyloglucan defined by polar contacts with branching xyloses and CH-Pi interactions</text></passage><passage><infon key="fpage">4799</infon><infon key="lpage">4809</infon><infon key="name_0">surname:Luís;given-names:AS</infon><infon key="name_1">surname:Venditto;given-names:I</infon><infon key="name_10">surname:Najmudin;given-names:S</infon><infon key="name_11">surname:Gilbert;given-names:HJ</infon><infon key="name_2">surname:Temple;given-names:MJ</infon><infon key="name_3">surname:Rogowski;given-names:A</infon><infon key="name_4">surname:Baslé;given-names:A</infon><infon key="name_5">surname:Xue;given-names:J</infon><infon key="name_6">surname:Knox;given-names:JP</infon><infon key="name_7">surname:Prates;given-names:JA</infon><infon key="name_8">surname:Ferreira;given-names:LM</infon><infon key="name_9">surname:Fontes;given-names:CM</infon><infon key="pub-id_doi">10.1074/jbc.M112.432781</infon><infon key="pub-id_pmid">23229556</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">288</infon><infon key="year">2013</infon><offset>63184</offset><text>Understanding how noncatalytic carbohydrate binding modules can display specificity for xyloglucan</text></passage><passage><infon key="fpage">1205</infon><infon key="lpage">1211</infon><infon key="name_0">surname:Tsukimoto;given-names:K</infon><infon key="name_1">surname:Takada;given-names:R</infon><infon key="name_2">surname:Araki;given-names:Y</infon><infon key="name_3">surname:Suzuki;given-names:K</infon><infon key="name_4">surname:Karita;given-names:S</infon><infon key="name_5">surname:Wakagi;given-names:T</infon><infon key="name_6">surname:Shoun;given-names:H</infon><infon key="name_7">surname:Watanabe;given-names:T</infon><infon key="name_8">surname:Fushinobu;given-names:S</infon><infon key="pub-id_doi">10.1016/j.febslet.2010.02.027</infon><infon key="pub-id_pmid">20159017</infon><infon key="section_type">REF</infon><infon key="source">FEBS Lett</infon><infon key="type">ref</infon><infon key="volume">584</infon><infon key="year">2010</infon><offset>63283</offset><text>Recognition of cellooligosaccharides by a family 28 carbohydrate-binding module</text></passage><passage><infon key="fpage">7224</infon><infon key="lpage">7230</infon><infon key="name_0">surname:Cho;given-names:KH</infon><infon key="name_1">surname:Salyers;given-names:AA</infon><infon key="pub-id_doi">10.1128/JB.183.24.7224-7230.2001</infon><infon key="pub-id_pmid">11717282</infon><infon key="section_type">REF</infon><infon key="source">J Bacteriol</infon><infon key="type">ref</infon><infon key="volume">183</infon><infon key="year">2001</infon><offset>63363</offset><text>Biochemical analysis of interactions between outer membrane proteins that contribute to starch utilization by Bacteroides thetaiotaomicron</text></passage><passage><infon key="fpage">40</infon><infon key="lpage">49</infon><infon key="name_0">surname:Rakoff-Nahoum;given-names:S</infon><infon key="name_1">surname:Coyne;given-names:MJ</infon><infon key="name_2">surname:Comstock;given-names:LE</infon><infon key="pub-id_doi">10.1016/j.cub.2013.10.077</infon><infon key="pub-id_pmid">24332541</infon><infon key="section_type">REF</infon><infon key="source">Curr Biol</infon><infon key="type">ref</infon><infon key="volume">24</infon><infon key="year">2014</infon><offset>63502</offset><text>An ecological network of polysaccharide utilization among human intestinal symbionts</text></passage><passage><infon key="fpage">e00909-14</infon><infon key="name_0">surname:Elhenawy;given-names:W</infon><infon key="name_1">surname:Debelyy;given-names:MO</infon><infon key="name_2">surname:Feldman;given-names:MF</infon><infon key="pub-id_doi">10.1128/mBio.00909-14</infon><infon key="pub-id_pmid">24618254</infon><infon key="section_type">REF</infon><infon key="source">mBio</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2014</infon><offset>63587</offset><text>Preferential packing of acidic glycosidases and proteases into Bacteroides outer membrane vesicles</text></passage><passage><infon key="fpage">94</infon><infon key="lpage">108</infon><infon key="name_0">surname:Hemsworth;given-names:GR</infon><infon key="name_1">surname:Déjean;given-names:G</infon><infon key="name_2">surname:Davies;given-names:GJ</infon><infon key="name_3">surname:Brumer;given-names:H</infon><infon key="pub-id_doi">10.1042/BST20150180</infon><infon key="pub-id_pmid">26862194</infon><infon key="section_type">REF</infon><infon key="source">Biochem Soc Trans</infon><infon key="type">ref</infon><infon key="volume">44</infon><infon key="year">2016</infon><offset>63686</offset><text>Learning from microbial strategies for polysaccharide degradation</text></passage><passage><infon key="fpage">43</infon><infon key="lpage">60</infon><infon key="name_0">surname:Noinaj;given-names:N</infon><infon key="name_1">surname:Guillier;given-names:M</infon><infon key="name_2">surname:Barnard;given-names:TJ</infon><infon key="name_3">surname:Buchanan;given-names:SK</infon><infon key="pub-id_doi">10.1146/annurev.micro.112408.134247</infon><infon key="pub-id_pmid">20420522</infon><infon key="section_type">REF</infon><infon key="source">Annu Rev Microbiol</infon><infon key="type">ref</infon><infon key="volume">64</infon><infon key="year">2010</infon><offset>63752</offset><text>TonB-dependent transporters: regulation, structure, and function</text></passage><passage><infon key="fpage">e1002118</infon><infon key="name_0">surname:Renzi;given-names:F</infon><infon key="name_1">surname:Manfredi;given-names:P</infon><infon key="name_2">surname:Mally;given-names:M</infon><infon key="name_3">surname:Moes;given-names:S</infon><infon key="name_4">surname:enö;given-names:P</infon><infon key="name_5">surname:Cornelis;given-names:GR</infon><infon key="pub-id_doi">10.1371/journal.ppat.1002118</infon><infon key="pub-id_pmid">21738475</infon><infon key="section_type">REF</infon><infon key="source">PLoS Pathog</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2011</infon><offset>63817</offset><text>The N-glycan glycoprotein deglycosylation complex (Gpd) from Capnocytophaga canimorsus deglycosylates human IgG</text></passage><passage><infon key="fpage">499</infon><infon key="lpage">511</infon><infon key="name_0">surname:Phansopa;given-names:C</infon><infon key="name_1">surname:Roy;given-names:S</infon><infon key="name_2">surname:Rafferty;given-names:JB</infon><infon key="name_3">surname:Douglas;given-names:CW</infon><infon key="name_4">surname:Pandhal;given-names:J</infon><infon key="name_5">surname:Wright;given-names:PC</infon><infon key="name_6">surname:Kelly;given-names:DJ</infon><infon key="name_7">surname:Stafford;given-names:GP</infon><infon key="pub-id_doi">10.1042/BJ20131415</infon><infon key="pub-id_pmid">24351045</infon><infon key="section_type">REF</infon><infon key="source">Biochem J</infon><infon key="type">ref</infon><infon key="volume">458</infon><infon key="year">2014</infon><offset>63929</offset><text>Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species</text></passage><passage><infon key="fpage">1241</infon><infon key="lpage">1252</infon><infon key="name_0">surname:Sonnenburg;given-names:ED</infon><infon key="name_1">surname:Zheng;given-names:H</infon><infon key="name_2">surname:Joglekar;given-names:P</infon><infon key="name_3">surname:Higginbottom;given-names:SK</infon><infon key="name_4">surname:Firbank;given-names:SJ</infon><infon key="name_5">surname:Bolam;given-names:DN</infon><infon key="name_6">surname:Sonnenburg;given-names:JL</infon><infon key="pub-id_doi">10.1016/j.cell.2010.05.005</infon><infon key="pub-id_pmid">20603004</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">141</infon><infon key="year">2010</infon><offset>64088</offset><text>Specificity of polysaccharide use in intestinal Bacteroides species determines diet-induced microbiota alterations</text></passage><passage><infon key="fpage">e224</infon><infon key="name_0">surname:Blanvillain;given-names:S</infon><infon key="name_1">surname:Meyer;given-names:D</infon><infon key="name_2">surname:Boulanger;given-names:A</infon><infon key="name_3">surname:Lautier;given-names:M</infon><infon key="name_4">surname:Guynet;given-names:C</infon><infon key="name_5">surname:Denancé;given-names:N</infon><infon key="name_6">surname:Vasse;given-names:J</infon><infon key="name_7">surname:Lauber;given-names:E</infon><infon key="name_8">surname:Arlat;given-names:M</infon><infon key="pub-id_doi">10.1371/journal.pone.0000224</infon><infon key="pub-id_pmid">17311090</infon><infon key="section_type">REF</infon><infon key="source">PLoS One</infon><infon key="type">ref</infon><infon key="volume">2</infon><infon key="year">2007</infon><offset>64203</offset><text>Plant carbohydrate scavenging through tonB-dependent receptors: a feature shared by phytopathogenic and aquatic bacteria</text></passage><passage><infon key="fpage">669</infon><infon key="lpage">677</infon><infon key="name_0">surname:Gilbert;given-names:HJ</infon><infon key="name_1">surname:Knox;given-names:JP</infon><infon key="name_2">surname:Boraston;given-names:AB</infon><infon key="pub-id_doi">10.1016/j.sbi.2013.05.005</infon><infon key="pub-id_pmid">23769966</infon><infon key="section_type">REF</infon><infon key="source">Curr Opin Struct Biol</infon><infon key="type">ref</infon><infon key="volume">23</infon><infon key="year">2013</infon><offset>64324</offset><text>Advances in understanding the molecular basis of plant cell wall polysaccharide recognition by carbohydrate-binding modules</text></passage><passage><infon key="fpage">1251</infon><infon key="lpage">1253</infon><infon key="name_0">surname:Gordon;given-names:JI</infon><infon key="pub-id_doi">10.1126/science.1224686</infon><infon key="pub-id_pmid">22674326</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">336</infon><infon key="year">2012</infon><offset>64448</offset><text>Honor thy gut symbionts redux</text></passage><passage><infon key="fpage">1</infon><infon key="lpage">7</infon><infon key="name_0">surname:Hutkins;given-names:RW</infon><infon key="name_1">surname:Krumbeck;given-names:JA</infon><infon key="name_10">surname:Vaughan;given-names:E</infon><infon key="name_11">surname:Sanders;given-names:ME</infon><infon key="name_2">surname:Bindels;given-names:LB</infon><infon key="name_3">surname:Cani;given-names:PD</infon><infon key="name_4">surname:Fahey;given-names:G;suffix:Jr</infon><infon key="name_5">surname:Goh;given-names:YJ</infon><infon key="name_6">surname:Hamaker;given-names:B</infon><infon key="name_7">surname:Martens;given-names:EC</infon><infon key="name_8">surname:Mills;given-names:DA</infon><infon key="name_9">surname:Rastal;given-names:RA</infon><infon key="pub-id_doi">10.1016/j.copbio.2015.09.001</infon><infon key="pub-id_pmid">26431716</infon><infon key="section_type">REF</infon><infon key="source">Curr Opin Biotechnol</infon><infon key="type">ref</infon><infon key="volume">37</infon><infon key="year">2016</infon><offset>64478</offset><text>Prebiotics: why definitions matter</text></passage><passage><infon key="fpage">105</infon><infon key="lpage">124</infon><infon key="name_0">surname:Van Duyne;given-names:GD</infon><infon key="name_1">surname:Standaert;given-names:RF</infon><infon key="name_2">surname:Karplus;given-names:PA</infon><infon key="name_3">surname:Schreiber;given-names:SL</infon><infon key="name_4">surname:Clardy;given-names:J</infon><infon key="pub-id_doi">10.1006/jmbi.1993.1012</infon><infon key="pub-id_pmid">7678431</infon><infon key="section_type">REF</infon><infon key="source">J Mol Biol</infon><infon key="type">ref</infon><infon key="volume">229</infon><infon key="year">1993</infon><offset>64513</offset><text>Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin</text></passage><passage><infon key="fpage">43010</infon><infon key="lpage">43017</infon><infon key="name_0">surname:Freelove;given-names:AC</infon><infon key="name_1">surname:Bolam;given-names:DN</infon><infon key="name_2">surname:White;given-names:P</infon><infon key="name_3">surname:Hazlewood;given-names:GP</infon><infon key="name_4">surname:Gilbert;given-names:HJ</infon><infon key="pub-id_doi">10.1074/jbc.M107143200</infon><infon key="pub-id_pmid">11560933</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">276</infon><infon key="year">2001</infon><offset>64600</offset><text>A novel carbohydrate-binding protein is a component of the plant cell wall-degrading complex of Piromyces equi</text></passage><passage><infon key="name_0">surname:Holdeman;given-names:LV</infon><infon key="name_1">surname:Cato;given-names:ED</infon><infon key="name_2">surname:Moore;given-names:WEC</infon><infon key="section_type">REF</infon><infon key="source">Anaerobe laboratory manual</infon><infon key="type">ref</infon><infon key="year">1977</infon><offset>64711</offset></passage><passage><infon key="fpage">447</infon><infon key="lpage">457</infon><infon key="name_0">surname:Martens;given-names:EC</infon><infon key="name_1">surname:Chiang;given-names:HC</infon><infon key="name_2">surname:Gordon;given-names:JI</infon><infon key="pub-id_doi">10.1016/j.chom.2008.09.007</infon><infon key="pub-id_pmid">18996345</infon><infon key="section_type">REF</infon><infon key="source">Cell Host Microbe</infon><infon key="type">ref</infon><infon key="volume">4</infon><infon key="year">2008</infon><offset>64712</offset><text>Mucosal glycan foraging enhances fitness and transmission of a saccharolytic human gut bacterial symbiont</text></passage><passage><infon key="fpage">307</infon><infon key="lpage">326</infon><infon key="name_0">surname:Otwinowski;given-names:Z</infon><infon key="name_1">surname:Minor;given-names:W</infon><infon key="section_type">REF</infon><infon key="source">Methods Enzymol</infon><infon key="type">ref</infon><infon key="volume">276</infon><infon key="year">1997</infon><offset>64818</offset><text>Processing of X-ray diffraction data collected in oscillation mode</text></passage><passage><infon key="fpage">582</infon><infon key="lpage">601</infon><infon key="name_0">surname:Terwilliger;given-names:TC</infon><infon key="name_1">surname:Adams;given-names:PD</infon><infon key="name_2">surname:Read;given-names:RJ</infon><infon key="name_3">surname:McCoy;given-names:AJ</infon><infon key="name_4">surname:Moriarty;given-names:NW</infon><infon key="name_5">surname:Grosse-Kunstleve;given-names:RW</infon><infon key="name_6">surname:Afonine;given-names:PV</infon><infon key="name_7">surname:Zwart;given-names:PH</infon><infon key="name_8">surname:Hung;given-names:LW</infon><infon key="pub-id_doi">10.1107/S0907444909012098</infon><infon key="pub-id_pmid">19465773</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">65</infon><infon key="year">2009</infon><offset>64885</offset><text>Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard</text></passage><passage><infon key="fpage">61</infon><infon key="lpage">69</infon><infon key="name_0">surname:Terwilliger;given-names:TC</infon><infon key="name_1">surname:Grosse-Kunstleve;given-names:RW</infon><infon key="name_2">surname:Afonine;given-names:PV</infon><infon key="name_3">surname:Moriarty;given-names:NW</infon><infon key="name_4">surname:Zwart;given-names:PH</infon><infon key="name_5">surname:Hung;given-names:LW</infon><infon key="name_6">surname:Read;given-names:RJ</infon><infon key="name_7">surname:Adams;given-names:PD</infon><infon key="pub-id_doi">10.1107/S090744490705024X</infon><infon key="pub-id_pmid">18094468</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">64</infon><infon key="year">2008</infon><offset>64990</offset><text>Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard</text></passage><passage><infon key="fpage">1948</infon><infon key="lpage">1954</infon><infon key="name_0">surname:Adams;given-names:PD</infon><infon key="name_1">surname:Grosse-Kunstleve;given-names:RW</infon><infon key="name_2">surname:Hung;given-names:LW</infon><infon key="name_3">surname:Ioerger;given-names:TR</infon><infon key="name_4">surname:McCoy;given-names:AJ</infon><infon key="name_5">surname:Moriarty;given-names:NW</infon><infon key="name_6">surname:Read;given-names:RJ</infon><infon key="name_7">surname:Sacchettini;given-names:JC</infon><infon key="name_8">surname:Sauter;given-names:NK</infon><infon key="name_9">surname:Terwilliger;given-names:TC</infon><infon key="pub-id_doi">10.1107/S0907444902016657</infon><infon key="pub-id_pmid">12393927</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">58</infon><infon key="year">2002</infon><offset>65095</offset><text>PHENIX: building new software for automated crystallographic structure determination</text></passage><passage><infon key="fpage">2126</infon><infon key="lpage">2132</infon><infon key="name_0">surname:Emsley;given-names:P</infon><infon key="name_1">surname:Cowtan;given-names:K</infon><infon key="pub-id_doi">10.1107/S0907444904019158</infon><infon key="pub-id_pmid">15572765</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">60</infon><infon key="year">2004</infon><offset>65180</offset><text>Coot: model-building tools for molecular graphics</text></passage><passage><infon key="fpage">658</infon><infon key="lpage">674</infon><infon key="name_0">surname:McCoy;given-names:AJ</infon><infon key="name_1">surname:Grosse-Kunstleve;given-names:RW</infon><infon key="name_2">surname:Adams;given-names:PD</infon><infon key="name_3">surname:Winn;given-names:MD</infon><infon key="name_4">surname:Storoni;given-names:LC</infon><infon key="name_5">surname:Read;given-names:RJ</infon><infon key="pub-id_doi">10.1107/S0021889807021206</infon><infon key="pub-id_pmid">19461840</infon><infon key="section_type">REF</infon><infon key="source">J Appl Crystallogr</infon><infon key="type">ref</infon><infon key="volume">40</infon><infon key="year">2007</infon><offset>65230</offset><text>Phaser crystallographic software</text></passage><passage><infon key="fpage">125</infon><infon key="lpage">132</infon><infon key="name_0">surname:Long;given-names:F</infon><infon key="name_1">surname:Vagin;given-names:AA</infon><infon key="name_2">surname:Young;given-names:P</infon><infon key="name_3">surname:Murshudov;given-names:GN</infon><infon key="pub-id_doi">10.1107/S0907444907050172</infon><infon key="pub-id_pmid">18094476</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">64</infon><infon key="year">2008</infon><offset>65263</offset><text>BALBES: a molecular-replacement pipeline</text></passage><passage><infon key="fpage">56</infon><infon key="lpage">66</infon><infon key="name_0">surname:Tuomivaara;given-names:ST</infon><infon key="name_1">surname:Yaoi;given-names:K</infon><infon key="name_2">surname:O’Neill;given-names:MA</infon><infon key="name_3">surname:York;given-names:WS</infon><infon key="pub-id_doi">10.1016/j.carres.2014.06.031</infon><infon key="pub-id_pmid">25497333</infon><infon key="section_type">REF</infon><infon key="source">Carbohydr Res</infon><infon key="type">ref</infon><infon key="volume">402</infon><infon key="year">2015</infon><offset>65304</offset><text>Generation and structural validation of a library of diverse xyloglucan-derived oligosaccharides, including an update on xyloglucan nomenclature</text></passage><passage><infon key="fpage">24922</infon><infon key="lpage">24933</infon><infon key="name_0">surname:Martinez-Fleites;given-names:C</infon><infon key="name_1">surname:Guerreiro;given-names:CI</infon><infon key="name_2">surname:Baumann;given-names:MJ</infon><infon key="name_3">surname:Taylor;given-names:EJ</infon><infon key="name_4">surname:Prates;given-names:JA</infon><infon key="name_5">surname:Ferreira;given-names:LM</infon><infon key="name_6">surname:Fontes;given-names:CM</infon><infon key="name_7">surname:Brumer;given-names:H</infon><infon key="name_8">surname:Davies;given-names:GJ</infon><infon key="pub-id_doi">10.1074/jbc.M603583200</infon><infon key="pub-id_pmid">16772298</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">281</infon><infon key="year">2006</infon><offset>65449</offset><text>Crystal structures of Clostridium thermocellum xyloglucanase, XGH74A, reveal the structural basis for xyloglucan recognition and degradation</text></passage></document></collection>
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+<collection><source>PMC</source><date>20201216</date><key>pmc.key</key><document><id>4850288</id><infon key="license">CC BY</infon><passage><infon key="alt-title">P. merdae C11 Cysteine Peptidase</infon><infon key="article-id_doi">10.1074/jbc.M115.706143</infon><infon key="article-id_pmc">4850288</infon><infon key="article-id_pmid">26940874</infon><infon key="article-id_publisher-id">M115.706143</infon><infon key="fpage">9482</infon><infon key="issue">18</infon><infon key="kwd">C-terminal domain (carboxyl tail domain, CTD) crystal structure cysteine protease enzyme proteolysis active site domain kinteoplast</infon><infon key="license">Author's Choice—Final version free via Creative Commons CC-BY license.</infon><infon key="lpage">9491</infon><infon key="name_0">surname:McLuskey;given-names:Karen</infon><infon key="name_1">surname:Grewal;given-names:Jaspreet S.</infon><infon key="name_2">surname:Das;given-names:Debanu</infon><infon key="name_3">surname:Godzik;given-names:Adam</infon><infon key="name_4">surname:Lesley;given-names:Scott A.</infon><infon key="name_5">surname:Deacon;given-names:Ashley M.</infon><infon key="name_6">surname:Coombs;given-names:Graham H.</infon><infon key="name_7">surname:Elsliger;given-names:Marc-André</infon><infon key="name_8">surname:Wilson;given-names:Ian A.</infon><infon key="name_9">surname:Mottram;given-names:Jeremy C.</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">291</infon><infon key="year">2016</infon><offset>0</offset><text>Crystal Structure and Activity Studies of the C11 Cysteine Peptidase from Parabacteroides merdae in the Human Gut Microbiome*</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>126</offset><text>Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. However, for the clostripain family (denoted C11), little is currently known. Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/β/α sandwich and an unusual C-terminal domain. It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other families in the clan. These studies also revealed a well ordered break in the polypeptide chain at Lys147, resulting in a large conformational rearrangement close to the active site. Biochemical and kinetic analysis revealed Lys147 to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca2+ for activity. Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1565</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1578</offset><text>Cysteine peptidases play crucial roles in the virulence of bacterial and other eukaryotic pathogens. In the MEROPS peptidase database, clan CD contains groups (or families) of cysteine peptidases that share some highly conserved structural elements. Clan CD families are typically described using the name of their archetypal, or founding, member and also given an identification number preceded by a “C,” to denote cysteine peptidase. Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80). No structural information is available for clostripain (C11), separase (C50), or PrtH-peptidase (C85).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2445</offset><text>Clan CD enzymes have a highly conserved His/Cys catalytic dyad and exhibit strict specificity for the P1 residue of their substrates. However, despite these similarities, clan CD forms a functionally diverse group of enzymes: the overall structural diversity between (and at times within) the various families provides these peptidases with a wide variety of substrate specificities and activation mechanisms. Several members are initially expressed as proenzymes, demonstrating self-inhibition prior to full activation.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2966</offset><text>The archetypal and arguably most notable family in the clan is that of the mammalian caspases (C14a), although clan CD members are distributed throughout the entire phylogenetic kingdom and are often required in fundamental biological processes. Interestingly, little is known about the structure or function of the C11 proteins, despite their widespread distribution and its archetypal member, clostripain from Clostridium histolyticum, first reported in the literature in 1938. Clostripain has been described as an arginine-specific peptidase with a requirement for Ca2+ and loss of an internal nonapeptide for full activation; lack of structural information on the family appears to have prohibited further investigation.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3691</offset><text>As part of an ongoing project to characterize commensal bacteria in the microbiome that inhabit the human gut, the structure of C11 peptidase, PmC11, from Parabacteroides merdae was determined using the Joint Center for Structural Genomics (JCSG)4 HTP structural biology pipeline. The structure was analyzed, and the enzyme was biochemically characterized to provide the first structure/function correlation for a C11 peptidase.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>4120</offset><text>Experimental Procedures</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>4144</offset><text>Cloning, expression, purification, crystallization, and structure determination of PmC11 were carried out using standard JCSG protocols as follows.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>4292</offset><text>Cloning</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>4300</offset><text>Clones were generated using the polymerase incomplete primer extension (PIPE) cloning method. The gene encoding PmC11 (SP5111E) was amplified by polymerase chain reaction (PCR) from P. merdae genomic DNA using PfuTurbo DNA polymerase (Stratagene), using I-PIPE primers that included sequences for the predicted 5′ and 3′ ends (shown below). The expression vector, pSpeedET, which encodes an amino-terminal tobacco etch virus protease-cleavable expression and purification tag (MGSDKIHHHHHHENLYFQ/G), was PCR amplified with V-PIPE (Vector) primers. V-PIPE and I-PIPE PCR products were mixed to anneal the amplified DNA fragments together. Escherichia coli GeneHogs (Invitrogen) competent cells were transformed with the I-PIPE/V-PIPE mixture and dispensed on selective LB-agar plates. The cloning junctions were confirmed by DNA sequencing. The plasmid encoding the full-length protein was deposited in the PSI:Biology Materials Repository at the DNASU plasmid repository (PmCD00547516). For structure determination, to obtain soluble protein using the PIPE, method the gene segment encoding residues Met1-Asn22 was deleted because these residues were predicted to correspond to a signal peptide using SignalP.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>5514</offset><text>Protein Expression and Selenomethionine Incorporation</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>5568</offset><text>The expression plasmid for the truncated PmC11 construct was transformed into E. coli GeneHogs competent cells and grown in minimal media supplemented with selenomethionine and 30 μg ml−1 of kanamycin at 37 °C using a GNF fermentor. A methionine auxotrophic strain was not required as selenomethionine is incorporated via the inhibition of methionine biosynthesis. Protein expression was induced using 0.1% (w/v) l-arabinose and the cells were left to grow for a further 3 h at 37 °C. At the end of the cell culture, lysozyme was added to all samples to a final concentration of 250 μg ml−1 and the cells were harvested and stored at −20 °C, until required. Primers used in this section are as follows: I-PIPE (forward): CTGTACTTCCAGGGCGAGACTCCGGAACCCCGGACAACCCGC; I-PIPE (reverse): AATTAAGTCGCGTTATTCATAAACTGCCTTATACCAGCCGAC; V-PIPE (forward): TAACGCGACTTAATTAACTCGTTTAAACGGTCTCCAGC; and V-PIPE (reverse): GCCCTGGAAGTACAGGTTTTCGTGATGATGATGATGAT.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>6526</offset><text>Protein Purification for Crystallization</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>6567</offset><text>Cells were resuspended, homogenized, and lysed by sonication in 40 mm Tris (pH 8.0), 300 mm NaCl, 10 mm imidazole, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Lysis Buffer 1) containing 0.4 mm MgSO4 and 1 μl of 250 unit/μl−1 of benzonase (Sigma). The cell lysate was then clarified by centrifugation (32,500 × g for 25 min at 4 °C) before being passed over Ni2+-chelating resin equilibrated in Lysis Buffer 1 and washed in the same buffer supplemented with 40 mm imidazole and 10% (v/v) glycerol. The protein was subsequently eluted in 20 mm Tris (pH 8.0), 150 mm NaCl, 10% (v/v) glycerol, 1 mm TCEP, and 300 mm imidazole, and the fractions containing the protein were pooled.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>7267</offset><text>To remove the His tag, PmC11 was exchanged into 20 mm Tris (pH 8.0), 150 mm NaCl, 30 mm imidazole, and 1 mm TCEP using a PD-10 column (GE Healthcare), followed by incubation with 1 mg of His-tagged tobacco etch virus protease per 15 mg of protein for 2 h at room temperature and subsequent overnight incubation at 4 °C. The sample was centrifuged to remove any precipitated material (13,000 × g for 10 min at 4 °C) and the supernatant loaded onto Ni2+-chelating resin equilibrated with 20 mm Tris (pH 8.0), 150 mm NaCl, 30 mm imidazole, and 1 mm TCEP and washed with the same buffer. The flow-through and wash fractions were collected and concentrated to 13.3 mg ml−1 using Amicon Ultra-15 5K centrifugal concentrators (Millipore).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>8004</offset><text>Crystallization and Data Collection</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>8040</offset><text>PmC11 was crystallized using the nanodroplet vapor diffusion method using standard JCSG crystallization protocols. Drops were comprised of 200 nl of protein solution mixed with 200 nl of crystallization solution in 96-well sitting-drop plates, equilibrated against a 50-μl reservoir. Crystals of PmC11 were grown at 4 °C in mother liquor consisting of 0.2 m NH4H2PO4, 20% PEG-3350 (JCSG Core Suite I). Crystals were flash cooled in liquid nitrogen using 10% ethylene glycol as a cryoprotectant prior to data collection and initial screening for diffraction was carried out using the Stanford Automated Mounting system at the Stanford Synchrotron Radiation Lightsource (SSRL, Menlo Park, CA). Single wavelength anomalous dispersion data were collected using a wavelength of 0.9793 Å, at the Advanced Light Source (ALS, beamline 8.2.2, Berkeley, CA) on an ADSC Quantum 315 CCD detector. The data were indexed and integrated with XDS and scaled using XSCALE. The diffraction data were indexed in space group P21 with a = 39.11, b = 108.68, c = 77.97 Å, and β = 94.32°. The unit cell contained two molecules in the asymmetric unit resulting in a solvent content of 39% (Matthews' coefficient (Vm) of 2.4 Å3 Da−1).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>9262</offset><text>Structure Determination</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>9286</offset><text>The PmC11 structure was determined by the single wavelength anomalous dispersion method using an x-ray wavelength corresponding to the peak of the selenium K edge. Initial phases were derived using the autoSHARP interface, which included density modification with SOLOMON. Good quality electron density was obtained at 1.7-Å resolution, allowing an initial model to be obtained by automated model building with ARP/wARP. Model completion and refinement were iteratively performed with COOT and REFMAC to produce a final model with an Rcryst and Rfree of 14.3 and 17.5%, respectively. The refinement included experimental phase restraints in the form of Hendrickson-Lattman coefficients, TLS refinement with one TLS group per molecule in the asymmetric unit, and NCS restraints. The refined structure contains residues 24–375 and 28–375 for the two molecules in the crystallographic asymmetric unit. Structural validation was carried using the JCSG Quality Control Server that analyzes both the coordinates and data using a variety of structural validation tools to confirm the stereochemical quality of the model (ADIT, MOLPROBITY, and WHATIF 5.0) and agreement between model and data (SGCHECK and RESOLVE). All of the main-chain torsion angles were in the allowed regions of the Ramachandran plot and the MolProbity overall clash score for the structure was 2.09 (within the 99th percentile for its resolution). The atomic coordinates and structure factors for PmC11 have been deposited in the Protein Data Bank (PDB) with the accession code 3UWS. Data collection, model, and refinement statistics are reported in Table 1.</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>10916</offset><text>Crystallographic statistics for PDB code 3UWS</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>10962</offset><text>Values in parentheses are for the highest resolution shell.</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;tbody valign=&quot;top&quot;&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;Data collection&lt;/bold&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Wavelength (Å)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.9793&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Space group&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;P2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Unit cell dimensions &lt;italic&gt;a&lt;/italic&gt;, &lt;italic&gt;b&lt;/italic&gt;, &lt;italic&gt;c&lt;/italic&gt; (Å); β&lt;sup&gt;°&lt;/sup&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;39.11, 108.68, 77.97; β = 94.32°&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Resolution range (Å)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;28.73–1.70 (1.79–1.70)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Unique reflections&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;70,913&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    &lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;merge&lt;/sub&gt;&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;TF1-1&quot;&gt;&lt;italic&gt;&lt;sup&gt;a&lt;/sup&gt;&lt;/italic&gt;&lt;/xref&gt; on &lt;italic&gt;I&lt;/italic&gt; (%)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;10.2 (49.0)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    &lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;meas&lt;/sub&gt;&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;TF1-2&quot;&gt;&lt;italic&gt;&lt;sup&gt;b&lt;/sup&gt;&lt;/italic&gt;&lt;/xref&gt; on &lt;italic&gt;I&lt;/italic&gt; (%)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;11.0 (52.7)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    &lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;pim&lt;/sub&gt;&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;TF1-3&quot;&gt;&lt;italic&gt;&lt;sup&gt;c&lt;/sup&gt;&lt;/italic&gt;&lt;/xref&gt; on &lt;italic&gt;I&lt;/italic&gt; (%)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4.1 (19.2)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    &lt;italic&gt;I&lt;/italic&gt;/σ&lt;italic&gt;&lt;sub&gt;I&lt;/sub&gt;&lt;/italic&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;15.6 (4.6)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Wilson B (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;15.9&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Completeness (%)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.6 (99.8)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Multiplicity&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;7.3 (7.5)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;2&quot; rowspan=&quot;1&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;Model and refinement&lt;/bold&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Reflections (total/test)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;70,883/3,577&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    &lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;cryst&lt;/sub&gt;/&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt;&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;TF1-4&quot;&gt;&lt;italic&gt;&lt;sup&gt;d&lt;/sup&gt;&lt;/italic&gt;&lt;/xref&gt; (%)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;14.3/17.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    No. protein residues/atoms&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;700/5612&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    No. of water/EDO molecules&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;690/7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    ESU&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;TF1-5&quot;&gt;&lt;italic&gt;&lt;sup&gt;e&lt;/sup&gt;&lt;/italic&gt;&lt;/xref&gt; based on &lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt; (Å)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.095&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    B-values (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;        Average isotropic B (overall)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;20.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;        Protein overall&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;18.8&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;        All main/side chains&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;16.7/20.8&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;        Solvent/EDO&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;29.4/35.6&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    RMSD&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;TF1-7&quot;&gt;&lt;italic&gt;&lt;sup&gt;g&lt;/sup&gt;&lt;/italic&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;        Bond lengths (Å)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.01&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;        Bond angles (°)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.6&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Ramachandran analysis (%)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;        Favored regions&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;97.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;        Allowed regions&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;        Outliers&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.0&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>11022</offset><text>Data collection		 	    Wavelength (Å)	0.9793	 	    Space group	P21	 	    Unit cell dimensions a, b, c (Å); β°	39.11, 108.68, 77.97; β = 94.32°	 	    Resolution range (Å)	28.73–1.70 (1.79–1.70)	 	    Unique reflections	70,913	 	    Rmergea on I (%)	10.2 (49.0)	 	    Rmeasb on I (%)	11.0 (52.7)	 	    Rpimc on I (%)	4.1 (19.2)	 	    I/σI	15.6 (4.6)	 	    Wilson B (Å2)	15.9	 	    Completeness (%)	99.6 (99.8)	 	    Multiplicity	7.3 (7.5)	 		 	Model and refinement		 	    Reflections (total/test)	70,883/3,577	 	    Rcryst/Rfreed (%)	14.3/17.5	 	    No. protein residues/atoms	700/5612	 	    No. of water/EDO molecules	690/7	 	    ESUe based on Rfree (Å)	0.095	 	    B-values (Å2)		 	        Average isotropic B (overall)	20.0	 	        Protein overall	18.8	 	        All main/side chains	16.7/20.8	 	        Solvent/EDO	29.4/35.6	 	    RMSDg		 	        Bond lengths (Å)	0.01	 	        Bond angles (°)	1.6	 	    Ramachandran analysis (%)		 	        Favored regions	97.0	 	        Allowed regions	3.0	 	        Outliers	0.0	 	</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>12367</offset><text>a Rmerge = ΣhklΣi|Ii(hkl) − 〈I(hkl)〉|/Σhkl Σi(hkl).</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>12435</offset><text>b Rmeas = Σhkl[N/(N-1)]1/2Σi|Ii(hkl) − 〈I(hkl)〉|/ΣhklΣiIi(hkl).</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>12515</offset><text>c Rpim (precision-indicating Rmerge) = Σhkl[(1/(N-1)]1/2 Σi|Ii (hkl) − 〈I(hkl)〉|/ΣhklΣi Ii(hkl), where n is the multiplicity of reflection hkl, and Ii(hkl) and 〈I(hkl)〉 are the intensity of the ith measurement and the average intensity of reflection hkl, respectively.</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>12800</offset><text>d Rcryst and Rfree = Σ‖Fobs| − |Fcalc‖/Σ|Fobs| for reflections in the working and test sets, respectively, where Fobs and Fcalc are the observed and calculated structure-factor amplitudes, respectively. Rfree is the same as Rcryst but for 5% of the total reflections chosen at random and omitted from structural refinement.</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>13134</offset><text>e ESU is the estimated standard uncertainties of atoms.</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>13190</offset><text>f The average isotropic B includes TLS and residual B components.</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>13256</offset><text>g RMSD, root-mean-square deviation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>13292</offset><text>Structural Analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>13312</offset><text>The primary sequence alignment with assigned secondary structure was prepared using CLUSTAL OMEGA and ALINE. The topology diagram was produced with TOPDRAW and all three-dimensional structural figures were prepared with PyMol with the electrostatic surface potential calculated with APBS and contoured at ±5 kT/e. Architectural comparisons with known structures revealed that PmC11 was most structurally similar to caspase-7, gingipain-K, and legumain (PBD codes 4hq0, 4tkx, and 4aw9, respectively). The statistical significance of the structural alignment between PmC11 and both caspase-7 and gingipain-K is equivalent (Z-score of 9.2) with legumain giving a very similar result (Z-score of 9.1). Of note, the β-strand topology of the CDP domains of Clostridium difficile toxin B (family C80; TcdB; PDB code 3pee) is identical to that observed in the PmC11 β-sheet, but the Z-score from DaliLite was notably less at 7.6. It is possible that the PmC11 structure is more closely related to the C80 family than other families in clan CD, and appear to reside on the same branch of the phylogenetic tree based on structure.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>14440</offset><text>Protein Production for Biochemical Assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>14482</offset><text>The PmCD00547516 plasmid described above was obtained from the PSI:Biology Materials Repository and used to generate a cleavage site mutant PmC11K147A and an active-site mutant PmC11C179A using the QuikChange Site-directed Mutagenesis kit (Stratagene) as per the manufacturer's instructions using the following primers: K147A mutant (forward): CAGAATAAGCTGGCAGCGTTCGGACAGGACG, and K147A mutant (reverse): CGTCCTGTCCGAACGCTGCCAGCTTATTCTG; C179A mutant (forward): CCTGTTCGATGCCGCCTACATGGCAAGC, and C179A mutant (reverse): GCTTGCCATGTAGGCGGCATCGAACAGG. The expression plasmids containing PmC11 were transformed into E. coli BL21 Star (DE3) and grown in Luria-Bertani media containing 30 μg ml−1 of kanamycin at 37 °C until an optical density (600 nm) of ∼0.6 was reached. l-Arabinose was added to a final concentration of 0.2% (w/v) and the cells incubated overnight at 25 °C.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>15363</offset><text>Compared with the protein production for crystallography, a slightly modified purification protocol was employed for biochemical assays. Initially, the cells were resuspended in 20 mm sodium phosphate (pH 7.5), 150 mm NaCl (Lysis Buffer 2) containing an EDTA-free protease inhibitor mixture (cOmplete, Roche Applied Science). Cells were disrupted by three passages (15 KPSI) through a One-Shot cell disruptor (Constant Systems) followed by centrifugation at 20,000 × g for 20 min at 4 °C. The supernatant was collected and sterile-filtered (0.2 μm) before being applied to a 5-ml HisTrap HP column (GE Healthcare) equilibrated in Lysis Buffer 2 containing 25 mm imidazole, and the protein was eluted in the same buffer containing 250 mm imidazole. The peak fractions were pooled and buffer exchanged into the assay buffer (20 mm Tris, 150 mm NaCl, pH 8.0) using a PD-10 column. When required, purified PmC11 was concentrated using Vivaspin 2 30-K centrifugal concentrators (Sartorius). Protein concentration was routinely measured using Bradford's reagent (Bio-Rad) with a BSA standard.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>16453</offset><text>Fluorogenic Substrate Activity Assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>16491</offset><text>The release of the fluorescent group AMC (7-amino-4-methylcoumarin) from potential peptide substrates was used to assess the activity of PmC11. Peptidase activity was tested using 20 μg of PmC11 and 100 μm substrate (unless otherwise stated) in assay buffer to a final reaction volume of 200 μl and all samples were incubated (without substrate) at 37 °C for 16 h prior to carrying out the assay. The substrate and plate reader were brought to 37 °C for 20 min prior to the addition of the PmC11 and samples prepared without PmC11 were used as blanks (negative controls). The curves were plotted using the blank-corrected fluorescence units against the time of acquisition (in min). The assays were carried out in black 96-well flat-bottomed plates (Greiner). AMC fluorescence was measured using a PHERAstar FS plate reader (BMG Labtech) with excitation and emission wavelengths of 355 and 460 nm, respectively.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>17408</offset><text>To investigate the substrate specificity of PmC11, substrates Z-GGR-AMC, Bz-R-AMC, Z-GP-AMC, Z-HGP-AMC, Ac-DEVD-AMC (all Bachem), BOC-VLK-AMC, and BOC-K-AMC (both PeptaNova) were prepared at 100 mm in 100% dimethyl sulfoxide. The amount of AMC (micromoles) released was calculated by generating an AMC standard curve (as described in Ref.) and the specific activity of PmC11 was calculated as picomoles of AMC released per min per mg of the protein preparation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>17870</offset><text>The reaction rates (Vmax) and Km values were determined for mutants PmC11K147A and PmC11C179A by carrying out the activity assay at varying concentrations of Bz-R-AMC between 0 and 600 μm. The blank-corrected relative fluorescence units were plotted against time (min) with ΔFU/T giving the reaction rate. The Km and Vmax of PmC11 and PmC11K147A against an R-AMC substrate were determined from the Lineweaver-Burk plot as described, calculated using GraphPad Prism6 software. All experiments were carried out in triplicate.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>18399</offset><text>Effect of VRPR-FMK on PmC11</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>18427</offset><text>To test the effect of the inhibitor on the activity of PmC11, 25 μm Z-VRPR-FMK (100 mm stock in 100% dimethyl sulfoxide, Enzo Life Sciences), 20 μg of PmC11, 100 μm R-AMC substrate, 1 mm EGTA were prepared in the assay buffer and the activity assay carried out as described above. A gel-shift assay, to observe Z-VRPR-FMK binding to PmC11, was also set up using 20 μg of PmC11, 25 μm inhibitor, 1 mm EGTA in assay buffer. The reactions were incubated at 37 °C for 20 min before being stopped by the addition SDS-PAGE sample buffer. Samples were analyzed by loading the reaction mixture on a 10% NuPAGE BisTris gel using MES buffer.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>19065</offset><text>Effect of Cations on PmC11</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>19092</offset><text>The enzyme activity of PmC11 was tested in the presence of various divalent cations: Mg2+, Ca2+, Mn2+, Co2+, Fe2+, Zn2+, and Cu2+. The final concentration of the salts (MgSO4, CaCl2, MnCl2, CoCl2, FeSO4, ZnCl2, and CuSO4) was 1 mm and the control was set up without divalent ions but with addition of 1 mm EGTA. The assay was set up using 20 mg of PmC11, 1 mm salts, 100 μm R-AMC substrate, and the assay buffer, and incubated at 37 °C for 16 h. The activity assay was carried out as described above.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>19595</offset><text>Size Exclusion Chromatography</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>19625</offset><text>Affinity-purified PmC11 was loaded onto a HiLoad 16/60 Superdex 200 gel filtration column (GE Healthcare) equilibrated in the assay buffer. The apparent molecular weight of PmC11 was determined from calibration curves based on protein standards of known molecular weights.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>19898</offset><text>Autoprocessing Profile of PmC11</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>19930</offset><text>Autoprocessing of PmC11 was evaluated by incubating the enzyme at 37 °C and removing samples at 1-h intervals from 0 to 16 h and placing into SDS-PAGE loading buffer to stop the processing. Samples were then analyzed on a 4–12% NuPAGE (Thermo Fisher) Novex BisTris gel run in MES buffer.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>20221</offset><text>Autoprocessing Cleavage Site Analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20259</offset><text>To investigate whether processing is a result of intra- or inter-molecular cleavage, the PmC11C179A mutant was incubated with increasing concentrations of activated PmC11 (0, 0.1, 0.2, 0.5, 1, 2, and 5 μg). The final assay volume was 40 μl and the proteins were incubated at 37 °C for 16 h in the PmC11 assay buffer. To stop the reaction, NuPAGE sample buffer was added to the protein samples and 20 μl was analyzed on 10% NuPAGE Novex BisTris gel using MES buffer. These studies revealed no apparent cleavage of PmC11C179A by the active enzyme at low concentrations of PmC11 and that only limited cleavage was observed when the ratio of active enzyme (PmC11: PmC11C179A) was increased to ∼1:10 and 1:4.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>20969</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>20977</offset><text>Structure of PmC11</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20996</offset><text>The crystal structure of the catalytically active form of PmC11 revealed an extended caspase-like α/β/α sandwich architecture comprised of a central nine-stranded β-sheet, with an unusual C-terminal domain (CTD), starting at Lys250. A single cleavage was observed in the polypeptide chain at Lys147 (Fig. 1, A and B), where both ends of the cleavage site are fully visible and well ordered in the electron density. The central nine-stranded β-sheet (β1–β9) of PmC11 consists of six parallel and three anti-parallel β-strands with 4↑3↓2↑1↑5↑6↑7↓8↓9↑ topology (Fig. 1A) and the overall structure includes 14 α-helices with six (α1–α2 and α4–α7) closely surrounding the β-sheet in an approximately parallel orientation. Helices α1, α7, and α6 are located on one side of the β-sheet with α2, α4, and α5 on the opposite side (Fig. 1A). Helix α3 sits at the end of the loop following β5 (L5), just preceding the Lys147 cleavage site, with both L5 and α3 pointing away from the central β-sheet and toward the CTD, which starts with α8. The structure also includes two short β-hairpins (βA–βB and βD–βE) and a small β-sheet (βC–βF), which is formed from two distinct regions of the sequence (βC precedes α11, α12 and β9, whereas βF follows the βD-βE hairpin) in the middle of the CTD (Fig. 1B).</text></passage><passage><infon key="file">zbc0191642560001.jpg</infon><infon key="id">F1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>22424</offset><text>Crystal structure of a C11 peptidase from P. merdae.
+A, primary sequence alignment of PmC11 (Uniprot ID A7A9N3) and clostripain (Uniprot ID P09870) from C. histolyticum with identical residues highlighted in gray shading. The secondary structure of PmC11 from the crystal structure is mapped onto its sequence with the position of the PmC11 catalytic dyad, autocatalytic cleavage site (Lys147), and S1 binding pocket Asp (Asp177) highlighted by a red star, a red downturned triangle, and a red upturned triangle, respectively. Connecting loops are colored gray, the main β-sheet is in orange, with other strands in olive, α-helices are in blue, and the nonapeptide linker of clostripain that is excised upon autocleavage is underlined in red. Sequences around the catalytic site of clostripain and PmC11 align well. B, topology diagram of PmC11 colored as in A except that additional (non-core) β-strands are in yellow. Helices found on either side of the central β-sheet are shown above and below the sheet, respectively. The position of the catalytic dyad (H, C) and the processing site (Lys147) are highlighted. Helices (1–14) and β-strands (1–9 and A-F) are numbered from the N terminus. The core caspase-fold is highlighted in a box. C, tertiary structure of PmC11. The N and C termini (N and C) of PmC11 along with the central β-sheet (1–9), helix α5, and helices α8, α11, and α13 from the C-terminal domain, are all labeled. Loops are colored gray, the main β-sheet is in orange, with other β-strands in yellow, and α-helices are in blue.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24015</offset><text>The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. Of the interacting secondary structure elements, α5 is perhaps the most interesting. This helix makes a total of eight hydrogen bonds with the CTD, including one salt bridge (Arg191-Asp255) and is surrounded by the CTD on one side and the main core of the enzyme on the other, acting like a linchpin holding both components together (Fig. 1C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>24647</offset><text>Structural Comparisons</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24670</offset><text>PmC11 is, as expected, most structurally similar to other members of clan CD with the top hits in a search of known structures being caspase-7, gingipain-K, and legumain (PBD codes 4hq0, 4tkx, and 4aw9, respectively) (Table 2). The C-terminal domain is unique to PmC11 within clan CD and structure comparisons for this domain alone does not produce any hits in the PDB (DaliLite, PDBeFold), suggesting a completely novel fold. As the archetypal and arguably most well studied member of clan CD, the caspases were used as the basis to investigate the structure/function relationships in PmC11, with caspase-7 as the representative member. Six of the central β-strands in PmC11 (β1–β2 and β5–β8) share the same topology as the six-stranded β-sheet found in caspases, with strands β3, β4, and β9 located on the outside of this core structure (Fig. 1B, box). His133 and Cys179 were found at locations structurally homologous to the caspase catalytic dyad, and other clan CD structures, at the C termini of strands β5 and β6, respectively (Figs. 1, A and B, and 2A). A multiple sequence alignment of C11 proteins revealed that these residues are highly conserved (data not shown).</text></passage><passage><infon key="file">T2.xml</infon><infon key="id">T2</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>25879</offset><text>Summary of PDBeFOLD superposition of structures found to be most similar to PmC11 in the PBD based on DaliLite </text></passage><passage><infon key="file">T2.xml</infon><infon key="id">T2</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>25991</offset><text>The results are ordered in terms of structural homology (QH), where %SSEPC-X is the percentage of the SSEs in the PmC11 that can be identified in the target X (where X = caspase-7, legumain, gingipain, and TcdB-CPD; % SSEX-PC is the percentage of SSEs in X (as above) that can be identified in PmC11 (as above); % sequence ID is the percentage sequence identity after structural alignment; Nalign is the number of matched residues; and r.m.s. deviation the root mean squared deviation on the Cα positions of the matched residues.</text></passage><passage><infon key="file">T2.xml</infon><infon key="id">T2</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table xmlns:xlink=&quot;http://www.w3.org/1999/xlink&quot; frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;thead valign=&quot;bottom&quot;&gt;&lt;tr&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Enzyme&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Family&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PDB code&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Q&lt;sup&gt;H&lt;/sup&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Z-score&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;%SSE&lt;sup&gt;PC-X&lt;/sup&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;%SSE&lt;sup&gt;X-PC&lt;/sup&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;% Seq. ID&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;N&lt;/italic&gt;&lt;sub&gt;align&lt;/sub&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;RMSD (Å)&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;N&lt;/italic&gt;&lt;sub&gt;Strands&lt;/sub&gt;&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign=&quot;top&quot;&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PmC11&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;C11&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;ext-link ext-link-type=&quot;pdb&quot; xlink:href=&quot;3UWS&quot;&gt;3UWS&lt;/ext-link&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.00&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;33.4&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;352&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.00&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;9&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Caspase-7&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;C14A&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;ext-link ext-link-type=&quot;pdb&quot; xlink:href=&quot;4HQ0&quot;&gt;4HQ0&lt;/ext-link&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.16&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4.3&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;38&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;79&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;14&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;162&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.27&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Legumain&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;C13&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;ext-link ext-link-type=&quot;pdb&quot; xlink:href=&quot;4AW9&quot;&gt;4AW9&lt;/ext-link&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.13&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5.5&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;31&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;53&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;13&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;161&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.05&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;TcdB-CPD&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;C80&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;ext-link ext-link-type=&quot;pdb&quot; xlink:href=&quot;3PEE&quot;&gt;3PEE&lt;/ext-link&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.10&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4.9&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;28&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;50&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;12&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;138&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.18&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;9&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Gingipain&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;C25&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;ext-link ext-link-type=&quot;pdb&quot; xlink:href=&quot;4TKX&quot;&gt;4TKX&lt;/ext-link&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.07&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5.4&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;28&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;27&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;12&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;153&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.97&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;10&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>26525</offset><text>Enzyme	Family	PDB code	QH	Z-score	%SSEPC-X	%SSEX-PC	% Seq. ID	Nalign	RMSD (Å)	NStrands	 	PmC11	C11	3UWS	1.00	33.4	100	100	100	352	0.00	9	 	Caspase-7	C14A	4HQ0	0.16	4.3	38	79	14	162	3.27	6	 	Legumain	C13	4AW9	0.13	5.5	31	53	13	161	2.05	6	 	TcdB-CPD	C80	3PEE	0.10	4.9	28	50	12	138	3.18	9	 	Gingipain	C25	4TKX	0.07	5.4	28	27	12	153	2.97	10	 	</text></passage><passage><infon key="file">zbc0191642560002.jpg</infon><infon key="id">F2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>26866</offset><text>Biochemical and structural characterization of PmC11.
+A, ribbon representation of the overall structure of PmC11 illustrating the catalytic site, cleavage site displacement, and potential S1 binding site. The overall structure of PmC11 is shown in gray, looking down into the catalytic site with the catalytic dyad in red. The two ends of the autolytic cleavage site (Lys147 and Ala148, green) are displaced by 19.5 Å (thin black line) from one another and residues in the potential substrate binding pocket are highlighted in blue. B, size exclusion chromatography of PmC11. PmC11 migrates as a monomer with a molecular mass around 41 kDa calculated from protein standards of known molecular weights. Elution fractions across the major peak (1–6) were analyzed by SDS-PAGE on a 4–12% gel in MES buffer. C, the active form of PmC11 and two mutants, PmC11C179A (C) and PmC11K147A (K), were examined by SDS-PAGE (lane 1) and Western blot analysis using an anti-His antibody (lane 2) show that PmC11 autoprocesses, whereas mutants, PmC11C179A and PmC11K147A, do not show autoprocessing in vitro. D, cysteine peptidase activity of PmC11. Km and Vmax of PmC11 and K147A mutant were determined by monitoring change in the fluorescence corresponding to AMC release from Bz-R-AMC. Reactions were performed in triplicate and representative values ± S.D. are shown. E, intermolecular processing of PmC11C179A by PmC11. PmC11C179A (20 μg) was incubated overnight at 37 °C with increasing amounts of processed PmC11 and analyzed on a 10% SDS-PAGE gel. Inactive PmC11C179A was not processed to a major extent by active PmC11 until around a ratio of 1:4 (5 μg of active PmC11). A single lane of 20 μg of active PmC11 (labeled 20) is shown for comparison. F, activity of PmC11 against basic substrates. Specific activity is shown ± S.D. from three independent experiments. G, electrostatic surface potential of PmC11 shown in a similar orientation, where blue and red denote positively and negatively charged surface potential, respectively, contoured at ±5 kT/e. The position of the catalytic dyad, one potential key substrate binding residue Asp177, and the ends of the cleavage site Lys147 and Ala148 are indicated.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>29083</offset><text>Five of the α-helices surrounding the β-sheet of PmC11 (α1, α2, α4, α6, and α7) are found in similar positions to the five structurally conserved helices in caspases and other members of clan CD, apart from family C80. Other than its more extended β-sheet, PmC11 differs most significantly from other clan CD members at its C terminus, where the CTD contains a further seven α-helices and four β-strands after β8.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>29537</offset><text>Autoprocessing of PmC11</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>29561</offset><text>Purification of recombinant PmC11 (molecular mass = 42.6 kDa) revealed partial processing into two cleavage products of 26.4 and 16.2 kDa, related to the observed cleavage at Lys147 in the crystal structure (Fig. 2A). Incubation of PmC11 at 37 °C for 16 h, resulted in a fully processed enzyme that remained as an intact monomer when applied to a size-exclusion column (Fig. 2B). The single cleavage site of PmC11 at Lys147 is found immediately after α3, in loop L5 within the central β-sheet (Figs. 1, A and B, and 2A). The two ends of the cleavage site are remarkably well ordered in the crystal structure and displaced from one another by 19.5 Å (Fig. 2A). Moreover, the C-terminal side of the cleavage site resides near the catalytic dyad with Ala148 being 4.5 and 5.7 Å from His133 and Cys179, respectively. Consequently, it appears feasible that the helix attached to Lys147 (α3) could be responsible for steric autoinhibition of PmC11 when Lys147 is covalently bonded to Ala148. Thus, the cleavage would be required for full activation of PmC11. To investigate this possibility, two mutant forms of the enzyme were created: PmC11C179A (a catalytically inactive mutant) and PmC11K147A (a cleavage-site mutant). Initial SDS-PAGE and Western blot analysis of both mutants revealed no discernible processing occurred as compared with active PmC11 (Fig. 2C). The PmC11K147A mutant enzyme had a markedly different reaction rate (Vmax) compared with WT, where the reaction velocity of PmC11 was 10 times greater than that of PmC11K147A (Fig. 2D). Taken together, these data reveal that PmC11 requires processing at Lys147 for optimum activity.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>31216</offset><text>To investigate whether processing is a result of intra- or intermolecular cleavage, the PmC11C179A mutant was incubated with increasing concentrations of processed and activated PmC11. These studies revealed that there was no apparent cleavage of PmC11C179A by the active enzyme at low concentrations of PmC11 and that only limited cleavage was observed when the ratio of active enzyme (PmC11:PmC11C179A) was increased to ∼1:10 and 1:4, with complete cleavage observed at a ratio of 1:1 (Fig. 2E). This suggests that cleavage of PmC11C179A was most likely an effect of the increasing concentration of PmC11 and intermolecular cleavage. Collectively, these data suggest that the pro-form of PmC11 is autoinhibited by a section of L5 blocking access to the active site, prior to intramolecular cleavage at Lys147. This cleavage subsequently allows movement of the region containing Lys147 and the active site to open up for substrate access.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>32158</offset><text>Substrate Specificity of PmC11</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>32189</offset><text>The autocatalytic cleavage of PmC11 at Lys147 (sequence KLK∧A) demonstrates that the enzyme accepts substrates with Lys in the P1 position. The substrate specificity of the enzyme was further tested using a variety of fluorogenic substrates. As expected, PmC11 showed no activity against substrates with Pro or Asp in P1 but was active toward substrates with a basic residue in P1 such as Bz-R-AMC, Z-GGR-AMC, and BOC-VLK-AMC. The rate of cleavage was ∼3-fold greater toward the single Arg substrate Bz-R-AMC than for the other two (Fig. 2F) and, unexpectedly, PmC11 showed no activity toward BOC-K-AMC. These results confirm that PmC11 accepts substrates containing Arg or Lys in P1 with a possible preference for Arg.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>32913</offset><text>The catalytic dyad of PmC11 sits near the bottom of an open pocket on the surface of the enzyme at a conserved location in the clan CD family. The PmC11 structure reveals that the catalytic dyad forms part of a large acidic pocket (Fig. 2G), consistent with a binding site for a basic substrate. This pocket is lined with the potential functional side chains of Asn50, Asp177, and Thr204 with Gly134, Asp207, and Met205 also contributing to the pocket (Fig. 2A). Interestingly, these residues are in regions that are structurally similar to those involved in the S1 binding pockets of other clan CD members (shown in Ref.).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>33537</offset><text>Because PmC11 recognizes basic substrates, the tetrapeptide inhibitor Z-VRPR-FMK was tested as an enzyme inhibitor and was found to inhibit both the autoprocessing and activity of PmC11 (Fig. 3A). Z-VRPR-FMK was also shown to bind to the enzyme: a size-shift was observed, by SDS-PAGE analysis, in the larger processed product of PmC11 suggesting that the inhibitor bound to the active site (Fig. 3B). A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). Asp177 is located near the catalytic cysteine and is conserved throughout the C11 family, suggesting it is the primary S1 binding site residue. In the structure of PmC11, Asp207 resides on a flexible loop pointing away from the S1 binding pocket (Fig. 3C). However, this loop has been shown to be important for substrate binding in clan CD and this residue could easily rotate and be involved in substrate binding in PmC11. Thus, Asn50, Asp177, and Asp207 are most likely responsible for the substrate specificity of PmC11. Asp177 is highly conserved throughout the clan CD C11 peptidases and is thought to be primarily responsible for substrate specificity of the clan CD enzymes, as also illustrated from the proximity of these residues relative to the inhibitor Z-VRPR-FMK when PmC11 is overlaid on the MALT1-P structure (Fig. 3C).</text></passage><passage><infon key="file">zbc0191642560003.jpg</infon><infon key="id">F3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>35240</offset><text>PmC11 binds and is inhibited by Z-VRPR-FMK and does not require Ca2+ for activity.
+A, PmC11 activity is inhibited by Z-VRPR-FMK. Cleavage of Bz-R-AMC by PmC11 was measured in a fluorometric activity assay with (+, purple) and without (−, red) Z-VRPR-FMK. The relative fluorescence units of AMC released are plotted against time (min) (n = 3; ±S.D.). B, gel-shift assay reveals that Z-VRPR-FMK binds to PmC11. PmC11 was incubated with (+) or without (−) Z-VRPR-FMK and the samples analyzed on a 10% SDS-PAGE gel. A size shift can be observed in the larger processed product of PmC11 (26.1 kDa). C, PmC11 with the Z-VRPR-FMK from the MALT1-paracacaspase (MALT1-P) superimposed. A three-dimensional structural overlay of Z-VRPR-FMK from the MALT1-P complex onto PmC11. The position and orientation of Z-VRPR-FMK was taken from superposition of the PmC11 and MALTI_P structures and indicates the presumed active site of PmC11. Residues surrounding the inhibitor are labeled and represent potentially important binding site residues, labeled in black and shown in an atomic representation. Carbon atoms are shown in gray, nitrogen in blue, and oxygen in red. C, divalent cations do not increase the activity of PmC11. The cleavage of Bz-R-AMC by PmC11 was measured in the presence of the cations Ca2+, Mn2+, Zn2+, Co2+, Cu2+, Mg2+, and Fe3+ with EGTA as a negative control, and relative fluorescence measured against time (min). The addition of cations produced no improvement in activity of PmC11 when compared in the presence of EGTA, suggesting that PmC11 does not require metal ions for proteolytic activity. Furthermore, Cu2+, Fe2+, and Zn2+ appear to inhibit PmC11.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>36912</offset><text>Comparison with Clostripain</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>36940</offset><text>Clostripain from C. histolyticum is the founding member of the C11 family of peptidases and contains an additional 149 residues compared with PmC11. A multiple sequence alignment revealed that most of the secondary structural elements are conserved between the two enzymes, although they are only ∼23% identical (Fig. 1A). Nevertheless, PmC11 may be a good model for the core structure of clostripain.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>37344</offset><text>The primary structural alignment also shows that the catalytic dyad in PmC11 is structurally conserved in clostripain (Fig. 1A). Unlike PmC11, clostripain has two cleavage sites (Arg181 and Arg190), which results in the removal of a nonapeptide, and is required for full activation of the enzyme (highlighted in Fig. 1A). Interestingly, Arg190 was found to align with Lys147 in PmC11. In addition, the predicted primary S1-binding residue in PmC11 Asp177 also overlays with the residue predicted to be the P1 specificity determining residue in clostripain (Asp229, Fig. 1A).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>37919</offset><text>As studies on clostripain revealed addition of Ca2+ ions are required for full activation, the Ca2+ dependence of PmC11 was examined. Surprisingly, Ca2+ did not enhance PmC11 activity and, furthermore, other divalent cations, Mg2+, Mn2+, Co2+, Fe2+, Zn2+, and Cu2+, were not necessary for PmC11 activity (Fig. 3D). In support of these findings, EGTA did not inhibit PmC11 suggesting that, unlike clostripain, PmC11 does not require Ca2+ or other divalent cations, for activity.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>38397</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>38408</offset><text>The crystal structure of PmC11 now provides three-dimensional information for a member of the clostripain C11 family of cysteine peptidases. The enzyme exhibits all of the key structural elements of clan CD members, but is unusual in that it has a nine-stranded central β-sheet with a novel C-terminal domain. The structural similarity of PmC11 with its nearest structural neighbors in the PDB is decidedly low, overlaying better with six-stranded caspase-7 than any of the other larger members of the clan (Table 2). The substrate specificity of PmC11 is Arg/Lys and the crystal structure revealed an acidic pocket for specific binding of such basic substrates. In addition, the structure suggested a mechanism of self-inhibition in both PmC11 and clostripain and an activation mechanism that requires autoprocessing. PmC11 differs from clostripain in that is does not appear to require divalent cations for activation.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>39332</offset><text>Several other members of clan CD require processing for full activation including legumain, gingipain-R, MARTX-CPD, and the effector caspases, e.g. caspase-7. To date, the effector caspases are the only group of enzymes that require cleavage of a loop within the central β-sheet. This is also the case in PmC11, although the cleavage loop is structurally different to that found in the caspases and follows the catalytic His (Fig. 1A), as opposed to the Cys in the caspases.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>39810</offset><text>All other clan CD members requiring cleavage for full activation do so at sites external to their central sheets. The caspases and gingipain-R both undergo intermolecular (trans) cleavage and legumain and MARTX-CPD are reported to perform intramolecular (cis) cleavage. In addition, several members of clan CD exhibit self-inhibition, whereby regions of the enzyme block access to the active site. Like PmC11, these structures show preformed catalytic machinery and, for a substrate to gain access, movement and/or cleavage of the blocking region is required.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>40370</offset><text>The structure of PmC11 gives the first insight into this class of relatively unexplored family of proteins and should allow important catalytic and substrate binding residues to be identified in a variety of orthologues. Indeed, insights gained from an analysis of the PmC11 structure revealed the identity of the Trypanosoma brucei PNT1 protein as a C11 cysteine peptidase with an essential role in organelle replication. The PmC11 structure should provide a good basis for structural modeling and, given the importance of other clan CD enzymes, this work should also advance the exploration of these peptidases and potentially identify new biologically important substrates.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">title_1</infon><offset>41047</offset><text>Author Contributions</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">paragraph</infon><offset>41068</offset><text>K. M., J. S. G., D. D., I. A. W., and J. C. M. designed the research; K. M., J. S. G., and D. D. performed the research; K. M., J. S. G., D. D., G. H. C., A. S., M. A. E., and J. C. M. analyzed the data; A. G., S. A. L., A. M. D., M. A. E., and I. A. W. supervised various components of the JCSG structural genomics pipeline; M. K. G., A. G., S. A. L., A. M. D., and M. A. E. contributed reagents, materials, and analysis tools; and K. M., J. S. G., G. H. C., M. A. E., I. A. W., and J. C. M. wrote the paper.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>41578</offset><text>This work was supported by the Medical Research Council Grant MR/K019384, Wellcome Trust Grants 091790 and 104111, and National Institutes of Health Grant U54 GM094586 (JCSG). The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and National Institutes of Health (NIH), National Center for Research Resources, Biomedical Technology Program Grant P41RR001209, and the NIMGS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIGMS or NIH. The authors declare that they have no conflicts of interest with the contents of this article.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42243</offset><text>The atomic coordinates and structure factors (code 3UWS) have been deposited in the Protein Data Bank (http://wwpdb.org/).</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42366</offset><text>JCSG</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42371</offset><text>Joint Center for Structural Genomics</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42408</offset><text>PIPE</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42413</offset><text>polymerase incomplete primer extension</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42452</offset><text>TCEP</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42457</offset><text>Tris(2-carboxyethyl)phosphine</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42487</offset><text>AMC</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42491</offset><text>7-amino-4-methylcoumarin</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42516</offset><text>PDB</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42520</offset><text>Protein Data Bank</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42538</offset><text>BisTris</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42546</offset><text>2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42609</offset><text>Z</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42611</offset><text>benzyloxycarbonyl</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42629</offset><text>FMK</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42633</offset><text>fluoromethyl ketone</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42653</offset><text>CTD</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42657</offset><text>C-terminal domain</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42675</offset><text>Bz-R-AMC</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42684</offset><text>benzoyl-l-Arg-4-methylcoumaryl-7-amide</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42723</offset><text>Z-GGR-AMC</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42733</offset><text>benzyloxycarbonyl-Gly-Gly-Arg-AMC</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42767</offset><text>BOC-VLK-AMC</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42779</offset><text>t-butyloxycarbonyl-Val-Leu-Lys.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>42811</offset><text>The abbreviations used are: </text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>42840</offset><text>References</text></passage><passage><infon key="fpage">D343</infon><infon key="lpage">350</infon><infon key="name_0">surname:Rawlings;given-names:N. D.</infon><infon key="name_1">surname:Barrett;given-names:A. J.</infon><infon key="name_2">surname:Bateman;given-names:A.</infon><infon key="pub-id_pmid">22086950</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">40</infon><infon key="year">2012</infon><offset>42851</offset><text>MEROPS: the database of proteolytic enzymes, their substrates and inhibitors</text></passage><passage><infon key="fpage">219</infon><infon key="lpage">232</infon><infon key="name_0">surname:McLuskey;given-names:K.</infon><infon key="name_1">surname:Mottram;given-names:J. C.</infon><infon key="pub-id_pmid">25697094</infon><infon key="section_type">REF</infon><infon key="source">Biochem. J.</infon><infon key="type">ref</infon><infon key="volume">466</infon><infon key="year">2015</infon><offset>42928</offset><text>Comparative structural analysis of the caspase family with other clan CD cysteine peptidases</text></passage><passage><infon key="fpage">10940</infon><infon key="lpage">10945</infon><infon key="name_0">surname:Dall;given-names:E.</infon><infon key="name_1">surname:Brandstetter;given-names:H.</infon><infon key="pub-id_pmid">23776206</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl. Acad. Sci. U.S.A.</infon><infon key="type">ref</infon><infon key="volume">110</infon><infon key="year">2013</infon><offset>43021</offset><text>Mechanistic and structural studies on legumain explain its zymogenicity, distinct activation pathways, and regulation</text></passage><passage><infon key="fpage">343</infon><infon key="lpage">352</infon><infon key="name_0">surname:Walker;given-names:N. P.</infon><infon key="name_1">surname:Talanian;given-names:R. V.</infon><infon key="name_2">surname:Brady;given-names:K. D.</infon><infon key="name_3">surname:Dang;given-names:L. C.</infon><infon key="name_4">surname:Bump;given-names:N. J.</infon><infon key="name_5">surname:Ferenz;given-names:C. R.</infon><infon key="name_6">surname:Franklin;given-names:S.</infon><infon key="name_7">surname:Ghayur;given-names:T.</infon><infon key="name_8">surname:Hackett;given-names:M. C.</infon><infon key="name_9">surname:Hammill;given-names:L. D.</infon><infon key="pub-id_pmid">8044845</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">78</infon><infon key="year">1994</infon><offset>43139</offset><text>Crystal structure of the cysteine protease interleukin-1β-converting enzyme: a (p20/p10)2 homodimer</text></passage><passage><infon key="fpage">21004</infon><infon key="lpage">21009</infon><infon key="name_0">surname:Yu;given-names:J. W.</infon><infon key="name_1">surname:Jeffrey;given-names:P. D.</infon><infon key="name_2">surname:Ha;given-names:J. Y.</infon><infon key="name_3">surname:Yang;given-names:X.</infon><infon key="name_4">surname:Shi;given-names:Y.</infon><infon key="pub-id_pmid">22158899</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl. Acad. Sci. U.S.A.</infon><infon key="type">ref</infon><infon key="volume">108</infon><infon key="year">2011</infon><offset>43242</offset><text>Crystal structure of the mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) paracaspase region</text></passage><passage><infon key="fpage">7469</infon><infon key="lpage">7474</infon><infon key="name_0">surname:McLuskey;given-names:K.</infon><infon key="name_1">surname:Rudolf;given-names:J.</infon><infon key="name_2">surname:Proto;given-names:W. R.</infon><infon key="name_3">surname:Isaacs;given-names:N. W.</infon><infon key="name_4">surname:Coombs;given-names:G. H.</infon><infon key="name_5">surname:Moss;given-names:C. X.</infon><infon key="name_6">surname:Mottram;given-names:J. C.</infon><infon key="pub-id_pmid">22529389</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl. Acad. Sci. U.S.A.</infon><infon key="type">ref</infon><infon key="volume">109</infon><infon key="year">2012</infon><offset>43353</offset><text>Crystal structure of a Trypanosoma brucei metacaspase</text></passage><passage><infon key="fpage">5453</infon><infon key="lpage">5462</infon><infon key="name_0">surname:Eichinger;given-names:A.</infon><infon key="name_1">surname:Beisel;given-names:H. G.</infon><infon key="name_2">surname:Jacob;given-names:U.</infon><infon key="name_3">surname:Huber;given-names:R.</infon><infon key="name_4">surname:Medrano;given-names:F. J.</infon><infon key="name_5">surname:Banbula;given-names:A.</infon><infon key="name_6">surname:Potempa;given-names:J.</infon><infon key="name_7">surname:Travis;given-names:J.</infon><infon key="name_8">surname:Bode;given-names:W.</infon><infon key="pub-id_pmid">10523290</infon><infon key="section_type">REF</infon><infon key="source">EMBO J.</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">1999</infon><offset>43407</offset><text>Crystal structure of gingipain R: an Arg-specific bacterial cysteine proteinase with a caspase-like fold</text></passage><passage><infon key="fpage">265</infon><infon key="lpage">268</infon><infon key="name_0">surname:Lupardus;given-names:P. J.</infon><infon key="name_1">surname:Shen;given-names:A.</infon><infon key="name_2">surname:Bogyo;given-names:M.</infon><infon key="name_3">surname:Garcia;given-names:K. C.</infon><infon key="pub-id_pmid">18845756</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">322</infon><infon key="year">2008</infon><offset>43512</offset><text>Small molecule-induced allosteric activation of the Vibrio cholerae RTX cysteine protease domain</text></passage><passage><infon key="fpage">1685</infon><infon key="lpage">1690</infon><infon key="name_0">surname:Kocholaty;given-names:W.</infon><infon key="name_1">surname:Weil;given-names:L.</infon><infon key="name_2">surname:Smith;given-names:L.</infon><infon key="pub-id_pmid">16746798</infon><infon key="section_type">REF</infon><infon key="source">Biochem. J.</infon><infon key="type">ref</infon><infon key="volume">32</infon><infon key="year">1938</infon><offset>43609</offset><text>Proteinase secretion and growth of Clostridium histolyticum</text></passage><passage><infon key="fpage">277</infon><infon key="lpage">280</infon><infon key="name_0">surname:Kembhavi;given-names:A. A.</infon><infon key="name_1">surname:Buttle;given-names:D. J.</infon><infon key="name_2">surname:Rauber;given-names:P.</infon><infon key="name_3">surname:Barrett;given-names:A. J.</infon><infon key="pub-id_pmid">2044766</infon><infon key="section_type">REF</infon><infon key="source">FEBS Lett.</infon><infon key="type">ref</infon><infon key="volume">283</infon><infon key="year">1991</infon><offset>43669</offset><text>Clostripain: characterization of the active site</text></passage><passage><infon key="fpage">1137</infon><infon key="lpage">1142</infon><infon key="name_0">surname:Elsliger;given-names:M. A.</infon><infon key="name_1">surname:Deacon;given-names:A. M.</infon><infon key="name_2">surname:Godzik;given-names:A.</infon><infon key="name_3">surname:Lesley;given-names:S. A.</infon><infon key="name_4">surname:Wooley;given-names:J.</infon><infon key="name_5">surname:Wüthrich;given-names:K.</infon><infon key="name_6">surname:Wilson;given-names:I. A.</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>43718</offset><text>The JCSG high-throughput structural biology pipeline</text></passage><passage><infon key="fpage">91</infon><infon key="lpage">103</infon><infon key="name_0">surname:Klock;given-names:H. E.</infon><infon key="name_1">surname:Lesley;given-names:S. A.</infon><infon key="pub-id_pmid">18988020</infon><infon key="section_type">REF</infon><infon key="source">Methods Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">498</infon><infon key="year">2009</infon><offset>43771</offset><text>The Polymerase Incomplete Primer Extension (PIPE) method applied to high-throughput cloning and site-directed mutagenesis</text></passage><passage><infon key="fpage">785</infon><infon key="lpage">786</infon><infon key="name_0">surname:Petersen;given-names:T. N.</infon><infon key="name_1">surname:Brunak;given-names:S.</infon><infon key="name_2">surname:von Heijne;given-names:G.</infon><infon key="name_3">surname:Nielsen;given-names:H.</infon><infon key="pub-id_pmid">21959131</infon><infon key="section_type">REF</infon><infon key="source">Nat. Methods</infon><infon key="type">ref</infon><infon key="volume">8</infon><infon key="year">2011</infon><offset>43893</offset><text>SignalP 4.0: discriminating signal peptides from transmembrane regions</text></passage><passage><infon key="fpage">11664</infon><infon key="lpage">11669</infon><infon key="name_0">surname:Lesley;given-names:S. A.</infon><infon key="name_1">surname:Kuhn;given-names:P.</infon><infon key="name_10">surname:Vincent;given-names:J.</infon><infon key="name_11">surname:Robb;given-names:A.</infon><infon key="name_12">surname:Brinen;given-names:L. S.</infon><infon key="name_13">surname:Miller;given-names:M. D.</infon><infon key="name_14">surname:McPhillips;given-names:T. M.</infon><infon key="name_15">surname:Miller;given-names:M. A.</infon><infon key="name_16">surname:Scheibe;given-names:D.</infon><infon key="name_17">surname:Canaves;given-names:J. M.</infon><infon key="name_18">surname:Guda;given-names:C.</infon><infon key="name_19">surname:Jaroszewski;given-names:L.</infon><infon key="name_2">surname:Godzik;given-names:A.</infon><infon key="name_20">surname:Selby;given-names:T. L.</infon><infon key="name_21">surname:Elsliger;given-names:M. A.</infon><infon key="name_22">surname:Wooley;given-names:J.</infon><infon key="name_23">surname:Taylor;given-names:S. S.</infon><infon key="name_24">surname:Hodgson;given-names:K. O.</infon><infon key="name_25">surname:Wilson;given-names:I. A.</infon><infon key="name_26">surname:Schultz;given-names:P. G.</infon><infon key="name_27">surname:Stevens;given-names:R. C.</infon><infon key="name_3">surname:Deacon;given-names:A. M.</infon><infon key="name_4">surname:Mathews;given-names:I.</infon><infon key="name_5">surname:Kreusch;given-names:A.</infon><infon key="name_6">surname:Spraggon;given-names:G.</infon><infon key="name_7">surname:Klock;given-names:H. E.</infon><infon key="name_8">surname:McMullan;given-names:D.</infon><infon key="name_9">surname:Shin;given-names:T.</infon><infon key="pub-id_pmid">12193646</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl. Acad. Sci. U.S.A.</infon><infon key="type">ref</infon><infon key="volume">99</infon><infon key="year">2002</infon><offset>43964</offset><text>Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline</text></passage><passage><infon key="fpage">91</infon><infon key="lpage">108</infon><infon key="name_0">surname:Doublié;given-names:S.</infon><infon key="pub-id_pmid">17272838</infon><infon key="section_type">REF</infon><infon key="source">Methods Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">363</infon><infon key="year">2007</infon><offset>44086</offset><text>Production of selenomethionyl proteins in prokaryotic and eukaryotic expression systems</text></passage><passage><infon key="fpage">105</infon><infon key="lpage">124</infon><infon key="name_0">surname:Van Duyne;given-names:G. D.</infon><infon key="name_1">surname:Standaert;given-names:R. F.</infon><infon key="name_2">surname:Karplus;given-names:P. A.</infon><infon key="name_3">surname:Schreiber;given-names:S. L.</infon><infon key="name_4">surname:Clardy;given-names:J.</infon><infon key="pub-id_pmid">7678431</infon><infon key="section_type">REF</infon><infon key="source">J. Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">229</infon><infon key="year">1993</infon><offset>44174</offset><text>Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin</text></passage><passage><infon key="fpage">720</infon><infon key="lpage">726</infon><infon key="name_0">surname:Cohen;given-names:A. E.</infon><infon key="name_1">surname:Ellis;given-names:P. J.</infon><infon key="name_2">surname:Miller;given-names:M. D.</infon><infon key="name_3">surname:Deacon;given-names:A. M.</infon><infon key="name_4">surname:Phizackerley;given-names:R. P.</infon><infon key="pub-id_pmid">24899734</infon><infon key="section_type">REF</infon><infon key="source">J. Appl. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">35</infon><infon key="year">2002</infon><offset>44261</offset><text>An automated system to mount cryo-cooled protein crystals on a synchrotron beam line, using compact sample cassettes and a small-scale robot</text></passage><passage><infon key="fpage">125</infon><infon key="lpage">132</infon><infon key="name_0">surname:Kabsch;given-names:W.</infon><infon key="pub-id_pmid">20124692</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>44402</offset><text>XDS</text></passage><passage><infon key="fpage">215</infon><infon key="lpage">230</infon><infon key="name_0">surname:Vonrhein;given-names:C.</infon><infon key="name_1">surname:Blanc;given-names:E.</infon><infon key="name_2">surname:Roversi;given-names:P.</infon><infon key="name_3">surname:Bricogne;given-names:G.</infon><infon key="pub-id_pmid">17172768</infon><infon key="section_type">REF</infon><infon key="source">Methods Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">364</infon><infon key="year">2007</infon><offset>44406</offset><text>Automated structure solution with autoSHARP</text></passage><passage><infon key="fpage">30</infon><infon key="lpage">42</infon><infon key="name_0">surname:Abrahams;given-names:J. P.</infon><infon key="name_1">surname:Leslie;given-names:A. G.</infon><infon key="pub-id_pmid">15299723</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">52</infon><infon key="year">1996</infon><offset>44450</offset><text>Methods used in the structure determination of bovine mitochondrial F1 ATPase</text></passage><passage><infon key="fpage">1171</infon><infon key="lpage">1179</infon><infon key="name_0">surname:Langer;given-names:G.</infon><infon key="name_1">surname:Cohen;given-names:S. X.</infon><infon key="name_2">surname:Lamzin;given-names:V. S.</infon><infon key="name_3">surname:Perrakis;given-names:A.</infon><infon key="pub-id_pmid">18600222</infon><infon key="section_type">REF</infon><infon key="source">Nat. Protoc.</infon><infon key="type">ref</infon><infon key="volume">3</infon><infon key="year">2008</infon><offset>44528</offset><text>Automated macromolecular model building for x-ray crystallography using ARP/wARP version 7</text></passage><passage><infon key="fpage">486</infon><infon key="lpage">501</infon><infon key="name_0">surname:Emsley;given-names:P.</infon><infon key="name_1">surname:Lohkamp;given-names:B.</infon><infon key="name_2">surname:Scott;given-names:W. G.</infon><infon key="name_3">surname:Cowtan;given-names:K.</infon><infon key="pub-id_pmid">20383002</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>44619</offset><text>Features and development of Coot</text></passage><passage><infon key="fpage">235</infon><infon key="lpage">242</infon><infon key="name_0">surname:Winn;given-names:M. D.</infon><infon key="name_1">surname:Ballard;given-names:C. C.</infon><infon key="name_10">surname:McNicholas;given-names:S. J.</infon><infon key="name_11">surname:Murshudov;given-names:G. N.</infon><infon key="name_12">surname:Pannu;given-names:N. S.</infon><infon key="name_13">surname:Potterton;given-names:E. A.</infon><infon key="name_14">surname:Powell;given-names:H. R.</infon><infon key="name_2">surname:Cowtan;given-names:K. D.</infon><infon key="name_3">surname:Dodson;given-names:E. J.</infon><infon key="name_4">surname:Emsley;given-names:P.</infon><infon key="name_5">surname:Evans;given-names:P. R.</infon><infon key="name_6">surname:Keegan;given-names:R. M.</infon><infon key="name_7">surname:Krissinel;given-names:E. B.</infon><infon key="name_8">surname:Leslie;given-names:A. G.</infon><infon key="name_9">surname:McCoy;given-names:A.</infon><infon key="pub-id_pmid">21460441</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">67</infon><infon key="year">2011</infon><offset>44652</offset><text>Overview of the CCP4 suite and current developments</text></passage><passage><infon key="fpage">355</infon><infon key="lpage">367</infon><infon key="name_0">surname:Murshudov;given-names:G. N.</infon><infon key="name_1">surname:Skubák;given-names:P.</infon><infon key="name_2">surname:Lebedev;given-names:A. A.</infon><infon key="name_3">surname:Pannu;given-names:N. S.</infon><infon key="name_4">surname:Steiner;given-names:R. A.</infon><infon key="name_5">surname:Nicholls;given-names:R. A.</infon><infon key="name_6">surname:Winn;given-names:M. D.</infon><infon key="name_7">surname:Long;given-names:F.</infon><infon key="name_8">surname:Vagin;given-names:A. A.</infon><infon key="pub-id_pmid">21460454</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">67</infon><infon key="year">2011</infon><offset>44704</offset><text>REFMAC5 for the refinement of macromolecular crystal structures</text></passage><passage><infon key="fpage">1833</infon><infon key="lpage">1839</infon><infon key="name_0">surname:Yang;given-names:H.</infon><infon key="name_1">surname:Guranovic;given-names:V.</infon><infon key="name_2">surname:Dutta;given-names:S.</infon><infon key="name_3">surname:Feng;given-names:Z.</infon><infon key="name_4">surname:Berman;given-names:H. M.</infon><infon key="name_5">surname:Westbrook;given-names:J. D.</infon><infon key="pub-id_pmid">15388930</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">60</infon><infon key="year">2004</infon><offset>44768</offset><text>Automated and accurate deposition of structures solved by X-ray diffraction to the Protein Data Bank</text></passage><passage><infon key="fpage">12</infon><infon key="lpage">21</infon><infon key="name_0">surname:Chen;given-names:V. B.</infon><infon key="name_1">surname:Arendall;given-names:W. B.;suffix:3rd</infon><infon key="name_2">surname:Headd;given-names:J. J.</infon><infon key="name_3">surname:Keedy;given-names:D. A.</infon><infon key="name_4">surname:Immormino;given-names:R. M.</infon><infon key="name_5">surname:Kapral;given-names:G. J.</infon><infon key="name_6">surname:Murray;given-names:L. W.</infon><infon key="name_7">surname:Richardson;given-names:J. S.</infon><infon key="name_8">surname:Richardson;given-names:D. C.</infon><infon key="pub-id_pmid">20057044</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>44869</offset><text>MolProbity: all-atom structure validation for macromolecular crystallography</text></passage><passage><infon key="fpage">52</infon><infon key="lpage">56</infon><infon key="name_0">surname:Vriend;given-names:G.</infon><infon key="pub-id_pmid">2268628</infon><infon key="section_type">REF</infon><infon key="source">J. Mol. Graph.</infon><infon key="type">ref</infon><infon key="volume">8</infon><infon key="year">1990</infon><offset>44946</offset><text>WHAT IF: a molecular modeling and drug design program</text></passage><passage><infon key="fpage">191</infon><infon key="lpage">205</infon><infon key="name_0">surname:Vaguine;given-names:A. A.</infon><infon key="name_1">surname:Richelle;given-names:J.</infon><infon key="name_2">surname:Wodak;given-names:S. J.</infon><infon key="pub-id_pmid">10089410</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">55</infon><infon key="year">1999</infon><offset>45000</offset><text>SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model</text></passage><passage><infon key="fpage">49</infon><infon key="lpage">52</infon><infon key="name_0">surname:Terwilliger;given-names:T.</infon><infon key="pub-id_pmid">14646132</infon><infon key="section_type">REF</infon><infon key="source">J. Synchrotron Radiat.</infon><infon key="type">ref</infon><infon key="volume">11</infon><infon key="year">2004</infon><offset>45146</offset><text>SOLVE and RESOLVE: automated structure solution, density modification and model building</text></passage><passage><infon key="fpage">539</infon><infon key="name_0">surname:Sievers;given-names:F.</infon><infon key="name_1">surname:Wilm;given-names:A.</infon><infon key="name_10">surname:Thompson;given-names:J. D.</infon><infon key="name_11">surname:Higgins;given-names:D. G.</infon><infon key="name_2">surname:Dineen;given-names:D.</infon><infon key="name_3">surname:Gibson;given-names:T. J.</infon><infon key="name_4">surname:Karplus;given-names:K.</infon><infon key="name_5">surname:Li;given-names:W.</infon><infon key="name_6">surname:Lopez;given-names:R.</infon><infon key="name_7">surname:McWilliam;given-names:H.</infon><infon key="name_8">surname:Remmert;given-names:M.</infon><infon key="name_9">surname:Söding;given-names:J.</infon><infon key="pub-id_pmid">21988835</infon><infon key="section_type">REF</infon><infon key="source">Mol. Syst. Biol.</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2011</infon><offset>45235</offset><text>Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega</text></passage><passage><infon key="fpage">510</infon><infon key="lpage">512</infon><infon key="name_0">surname:Bond;given-names:C. S.</infon><infon key="name_1">surname:Schüttelkopf;given-names:A. W.</infon><infon key="pub-id_pmid">19390156</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">65</infon><infon key="year">2009</infon><offset>45334</offset><text>ALINE: a WYSIWYG protein-sequence alignment editor for publication-quality alignments</text></passage><passage><infon key="fpage">311</infon><infon key="lpage">312</infon><infon key="name_0">surname:Bond;given-names:C. S.</infon><infon key="pub-id_pmid">12538265</infon><infon key="section_type">REF</infon><infon key="source">Bioinformatics</infon><infon key="type">ref</infon><infon key="volume">19</infon><infon key="year">2003</infon><offset>45420</offset><text>TopDraw: a sketchpad for protein structure topology cartoons</text></passage><passage><infon key="comment">version 1.2r3pre</infon><infon key="name_0">surname:DeLano;given-names:W.</infon><infon key="section_type">REF</infon><infon key="source">The PyMOL Molecular Graphic System</infon><infon key="type">ref</infon><infon key="year">2002</infon><offset>45481</offset></passage><passage><infon key="fpage">203</infon><infon key="lpage">221</infon><infon key="name_0">surname:McLuskey;given-names:K.</infon><infon key="name_1">surname:Moss;given-names:C. X.</infon><infon key="name_2">surname:Mottram;given-names:J. C.</infon><infon key="pub-id_pmid">24567104</infon><infon key="section_type">REF</infon><infon key="source">Methods Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">1133</infon><infon key="year">2014</infon><offset>45482</offset><text>Purification, characterization, and crystallization of Trypanosoma metacaspases</text></passage><passage><infon key="fpage">4</infon><infon key="lpage">21</infon><infon key="name_0">surname:Wiesmann;given-names:C.</infon><infon key="name_1">surname:Leder;given-names:L.</infon><infon key="name_10">surname:Freuler;given-names:F.</infon><infon key="name_11">surname:Nikolay;given-names:R.</infon><infon key="name_12">surname:Alves;given-names:J.</infon><infon key="name_13">surname:Bornancin;given-names:F.</infon><infon key="name_14">surname:Renatus;given-names:M.</infon><infon key="name_2">surname:Blank;given-names:J.</infon><infon key="name_3">surname:Bernardi;given-names:A.</infon><infon key="name_4">surname:Melkko;given-names:S.</infon><infon key="name_5">surname:Decock;given-names:A.</infon><infon key="name_6">surname:D'Arcy;given-names:A.</infon><infon key="name_7">surname:Villard;given-names:F.</infon><infon key="name_8">surname:Erbel;given-names:P.</infon><infon key="name_9">surname:Hughes;given-names:N.</infon><infon key="pub-id_pmid">22366302</infon><infon key="section_type">REF</infon><infon key="source">J. Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">419</infon><infon key="year">2012</infon><offset>45562</offset><text>Structural determinants of MALT1 protease activity</text></passage><passage><infon key="fpage">865</infon><infon key="lpage">872</infon><infon key="name_0">surname:Ullmann;given-names:D.</infon><infon key="name_1">surname:Jakubke;given-names:H. D.</infon><infon key="pub-id_pmid">8055964</infon><infon key="section_type">REF</infon><infon key="source">Eur. J. Biochem.</infon><infon key="type">ref</infon><infon key="volume">223</infon><infon key="year">1994</infon><offset>45613</offset><text>The specificity of clostripain from Clostridium histolyticum: mapping the S′ subsites via acyl transfer to amino acid amides and peptides</text></passage><passage><infon key="fpage">281</infon><infon key="lpage">286</infon><infon key="name_0">surname:Witte;given-names:V.</infon><infon key="name_1">surname:Wolf;given-names:N.</infon><infon key="name_2">surname:Dargatz;given-names:H.</infon><infon key="pub-id_pmid">8875906</infon><infon key="section_type">REF</infon><infon key="source">Curr. Microbiol.</infon><infon key="type">ref</infon><infon key="volume">33</infon><infon key="year">1996</infon><offset>45753</offset><text>Clostripain linker deletion variants yield active enzyme in Escherichia coli: a possible function of the linker peptide as intramolecular inhibitor of clostripain automaturation</text></passage><passage><infon key="fpage">983</infon><infon key="lpage">992</infon><infon key="name_0">surname:Labrou;given-names:N. E.</infon><infon key="name_1">surname:Rigden;given-names:D. J.</infon><infon key="pub-id_pmid">15009210</infon><infon key="section_type">REF</infon><infon key="source">Eur. J. Biochem.</infon><infon key="type">ref</infon><infon key="volume">271</infon><infon key="year">2004</infon><offset>45931</offset><text>The structure-function relationship in the clostripain family of peptidases</text></passage><passage><infon key="fpage">38980</infon><infon key="lpage">38990</infon><infon key="name_0">surname:Li;given-names:D. N.</infon><infon key="name_1">surname:Matthews;given-names:S. P.</infon><infon key="name_2">surname:Antoniou;given-names:A. N.</infon><infon key="name_3">surname:Mazzeo;given-names:D.</infon><infon key="name_4">surname:Watts;given-names:C.</infon><infon key="pub-id_pmid">12860980</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">278</infon><infon key="year">2003</infon><offset>46007</offset><text>Multistep autoactivation of asparaginyl endopeptidase in vitro and in vivo</text></passage><passage><infon key="fpage">10458</infon><infon key="lpage">10464</infon><infon key="name_0">surname:Mikolajczyk;given-names:J.</infon><infon key="name_1">surname:Boatright;given-names:K. M.</infon><infon key="name_2">surname:Stennicke;given-names:H. R.</infon><infon key="name_3">surname:Nazif;given-names:T.</infon><infon key="name_4">surname:Potempa;given-names:J.</infon><infon key="name_5">surname:Bogyo;given-names:M.</infon><infon key="name_6">surname:Salvesen;given-names:G. S.</infon><infon key="pub-id_pmid">12533545</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">278</infon><infon key="year">2003</infon><offset>46082</offset><text>Sequential autolytic processing activates the zymogen of Arg-gingipain</text></passage><passage><infon key="fpage">34042</infon><infon key="lpage">34050</infon><infon key="name_0">surname:Denault;given-names:J. B.</infon><infon key="name_1">surname:Salvesen;given-names:G. S.</infon><infon key="pub-id_pmid">12824163</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">278</infon><infon key="year">2003</infon><offset>46153</offset><text>Human caspase-7 activity and regulation by its N-terminal peptide</text></passage><passage><infon key="fpage">9492</infon><infon key="lpage">9500</infon><infon key="name_0">surname:Grewal;given-names:J. S.</infon><infon key="name_1">surname:McLuskey;given-names:K.</infon><infon key="name_10">surname:Schnaufer;given-names:A.</infon><infon key="name_11">surname:Mottram;given-names:J. C.</infon><infon key="name_2">surname:Das;given-names:D.</infon><infon key="name_3">surname:Myburgh;given-names:E.</infon><infon key="name_4">surname:Wilkes;given-names:J.</infon><infon key="name_5">surname:Brown;given-names:E.</infon><infon key="name_6">surname:Lemgruber;given-names:L.</infon><infon key="name_7">surname:Gould;given-names:M. K.</infon><infon key="name_8">surname:Burchmore;given-names:R. J.</infon><infon key="name_9">surname:Coombs;given-names:G. H.</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">291</infon><infon key="year">2016</infon><offset>46219</offset><text>PNT1 is a C11 cysteine peptidase essential for replication of the Trypanosome kinetoplast</text></passage><passage><infon key="fpage">291</infon><infon key="lpage">296</infon><infon key="name_0">surname:Weiss;given-names:M. S.</infon><infon key="name_1">surname:Metzner;given-names:H. J.</infon><infon key="name_2">surname:Hilgenfeld;given-names:R.</infon><infon key="pub-id_pmid">9515726</infon><infon key="section_type">REF</infon><infon key="source">FEBS Lett.</infon><infon key="type">ref</infon><infon key="volume">423</infon><infon key="year">1998</infon><offset>46309</offset><text>Two non-proline cis peptide bonds may be important for factor XIII function</text></passage><passage><infon key="fpage">269</infon><infon key="lpage">275</infon><infon key="name_0">surname:Diederichs;given-names:K.</infon><infon key="name_1">surname:Karplus;given-names:P. A.</infon><infon key="pub-id_pmid">9095194</infon><infon key="section_type">REF</infon><infon key="source">Nat. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">4</infon><infon key="year">1997</infon><offset>46385</offset><text>Improved R-factors for diffraction data analysis in macromolecular crystallography</text></passage><passage><infon key="fpage">2256</infon><infon key="lpage">2268</infon><infon key="name_0">surname:Krissinel;given-names:E.</infon><infon key="name_1">surname:Henrick;given-names:K.</infon><infon key="pub-id_pmid">15572779</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">60</infon><infon key="year">2004</infon><offset>46468</offset><text>Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions</text></passage><passage><infon key="fpage">566</infon><infon key="lpage">567</infon><infon key="name_0">surname:Holm;given-names:L.</infon><infon key="name_1">surname:Park;given-names:J.</infon><infon key="pub-id_pmid">10980157</infon><infon key="section_type">REF</infon><infon key="source">Bioinformatics</infon><infon key="type">ref</infon><infon key="volume">16</infon><infon key="year">2000</infon><offset>46572</offset><text>DaliLite workbench for protein structure comparison</text></passage><passage><infon key="fpage">1514</infon><infon key="lpage">1521</infon><infon key="name_0">surname:Kang;given-names:H. J.</infon><infon key="name_1">surname:Lee;given-names:Y. M.</infon><infon key="name_2">surname:Bae;given-names:K. H.</infon><infon key="name_3">surname:Kim;given-names:S. J.</infon><infon key="name_4">surname:Chung;given-names:S. J.</infon><infon key="pub-id_pmid">23897474</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol. Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">69</infon><infon key="year">2013</infon><offset>46624</offset><text>Structural asymmetry of procaspase-7 bound to a specific inhibitor</text></passage><passage><infon key="fpage">162</infon><infon key="lpage">166</infon><infon key="name_0">surname:Gorman;given-names:M. A.</infon><infon key="name_1">surname:Seers;given-names:C. A.</infon><infon key="name_2">surname:Michell;given-names:B. J.</infon><infon key="name_3">surname:Feil;given-names:S. C.</infon><infon key="name_4">surname:Huq;given-names:N. L.</infon><infon key="name_5">surname:Cross;given-names:K. J.</infon><infon key="name_6">surname:Reynolds;given-names:E. C.</infon><infon key="name_7">surname:Parker;given-names:M. W.</infon><infon key="pub-id_pmid">25327141</infon><infon key="section_type">REF</infon><infon key="source">Protein Sci.</infon><infon key="type">ref</infon><infon key="volume">24</infon><infon key="year">2015</infon><offset>46691</offset><text>Structure of the lysine specific protease Kgp from Porphyromonas gingivalis, a target for improved oral health</text></passage><passage><infon key="fpage">364</infon><infon key="lpage">371</infon><infon key="name_0">surname:Shen;given-names:A.</infon><infon key="name_1">surname:Lupardus;given-names:P. J.</infon><infon key="name_2">surname:Gersch;given-names:M. M.</infon><infon key="name_3">surname:Puri;given-names:A. W.</infon><infon key="name_4">surname:Albrow;given-names:V. E.</infon><infon key="name_5">surname:Garcia;given-names:K. C.</infon><infon key="name_6">surname:Bogyo;given-names:M.</infon><infon key="pub-id_pmid">21317893</infon><infon key="section_type">REF</infon><infon key="source">Nat. Struct. Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">2011</infon><offset>46802</offset><text>Defining an allosteric circuit in the cysteine protease domain of Clostridium difficile toxins</text></passage></document></collection>
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+<collection><source>PMC</source><date>20201218</date><key>pmc.key</key><document><id>4852598</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/ncomms11337</infon><infon key="article-id_pii">ncomms11337</infon><infon key="article-id_pmc">4852598</infon><infon key="article-id_pmid">27088325</infon><infon key="elocation-id">11337</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0
+International License. The images or other third party material in this article are
+included in the article's Creative Commons license, unless indicated otherwise
+in the credit line; if the material is not included under the Creative Commons
+license, users will need to obtain permission from the license holder to reproduce
+the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:van den Berg;given-names:Bert</infon><infon key="name_1">surname:Chembath;given-names:Anupama</infon><infon key="name_2">surname:Jefferies;given-names:Damien</infon><infon key="name_3">surname:Basle;given-names:Arnaud</infon><infon key="name_4">surname:Khalid;given-names:Syma</infon><infon key="name_5">surname:Rutherford;given-names:Julian C.</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">7</infon><infon key="year">2016</infon><offset>0</offset><text>Structural basis for Mep2 ammonium transceptor activation by phosphorylation</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>77</offset><text>Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1224</offset><text> Mep2 proteins are tightly regulated fungal ammonium transporters. Here, the authors report the crystal structures of closed states of Mep2 proteins and propose a model for their regulation by comparing them with the open ammonium transporters of bacteria.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1481</offset><text>Transceptors are membrane proteins that function not only as transporters but also as receptors/sensors during nutrient sensing to activate downstream signalling pathways. A common feature of transceptors is that they are induced when cells are starved for their substrate. While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). One of the most important unresolved questions in the field is how the transceptors couple to downstream signalling pathways. One hypothesis is that downstream signalling is dependent on a specific conformation of the transporter.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2271</offset><text>Mep2 (methylammonium (MA) permease) proteins are ammonium transceptors that are ubiquitous in fungi. They belong to the Amt/Mep/Rh family of transporters that are present in all kingdoms of life and they take up ammonium from the extracellular environment. Fungi typically have more than one Mep paralogue, for example, Mep1-3 in S. cerevisiae. Of these, only Mep2 proteins function as ammonium receptors/sensors in fungal development. Under conditions of nitrogen limitation, Mep2 initiates a signalling cascade that results in a switch from the yeast form to filamentous (pseudohyphal) growth that may be required for fungal pathogenicity. As is the case for other transceptors, it is not clear how Mep2 interacts with downstream signalling partners, but the protein kinase A and mitogen-activated protein kinase pathways have been proposed as downstream effectors of Mep2 (refs). Compared with Mep1 and Mep3, Mep2 is highly expressed and functions as a low-capacity, high-affinity transporter in the uptake of MA. In addition, Mep2 is also important for uptake of ammonium produced by growth on other nitrogen sources.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3393</offset><text>With the exception of the human RhCG structure, no structural information is available for eukaryotic ammonium transporters. By contrast, several bacterial Amt orthologues have been characterized in detail via high-resolution crystal structures and a number of molecular dynamics (MD) studies. All the solved structures including that of RhCG are very similar, establishing the basic architecture of ammonium transporters. The proteins form stable trimers, with each monomer having 11 transmembrane (TM) helices and a central channel for the transport of ammonium. All structures show the transporters in open conformations. Intriguingly, fundamental questions such as the nature of the transported substrate and the transport mechanism are still controversial. Where earlier studies favoured the transport of ammonia gas, recent data and theoretical considerations suggest that Amt/Mep proteins are instead active, electrogenic transporters of either NH4+ (uniport) or NH3/H+ (symport). A highly conserved pair of channel-lining histidine residues dubbed the twin-His motif may serve as a proton relay system while NH3 moves through the channel during NH3/H+ symport.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4562</offset><text>Ammonium transport is tightly regulated. In animals, this is due to toxicity of elevated intracellular ammonium levels, whereas for microorganisms ammonium is a preferred nitrogen source. In bacteria, amt genes are present in an operon with glnK, encoding a PII-like signal transduction class protein. By binding tightly to Amt proteins without inducing a conformational change in the transporter, GlnK sterically blocks ammonium conductance when nitrogen levels are sufficient. Under conditions of nitrogen limitation, GlnK becomes uridylated, blocking its ability to bind and inhibit Amt proteins. Importantly, eukaryotes do not have GlnK orthologues and have a different mechanism for regulation of ammonium transport activity. In plants, transporter phosphorylation and dephosphorylation are known to regulate activity. In S. cerevisiae, phosphorylation of Ser457 within the C-terminal region (CTR) in the cytoplasm was recently proposed to cause Mep2 opening, possibly via inducing a conformational change.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5574</offset><text>To elucidate the mechanism of Mep2 transport regulation, we present here X-ray crystal structures of the Mep2 transceptors from S. cerevisiae and C. albicans. The structures are similar to each other but show considerable differences to all other ammonium transporter structures. The most striking difference is the fact that the Mep2 proteins have closed conformations. The putative phosphorylation site is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The channels of phosphorylation-mimicking mutants of C. albicans Mep2 are still closed but show large conformational changes within a conserved part of the CTR. Together with a structure of a C-terminal Mep2 variant lacking the segment containing the phosphorylation site, the results allow us to propose a structural model for phosphorylation-based regulation of eukaryotic ammonium transport.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>6478</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>6486</offset><text>General architecture of Mep2 ammonium transceptors</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6537</offset><text>The Mep2 protein of S. cerevisiae (ScMep2) was overexpressed in S. cerevisiae in high yields, enabling structure determination by X-ray crystallography using data to 3.2 Å resolution by molecular replacement (MR) with the archaebacterial Amt-1 structure (see Methods section). Given that the modest resolution of the structure and the limited detergent stability of ScMep2 would likely complicate structure–function studies, several other fungal Mep2 orthologues were subsequently overexpressed and screened for diffraction-quality crystals. Of these, Mep2 from C. albicans (CaMep2) showed superior stability in relatively harsh detergents such as nonyl-glucoside, allowing structure determination in two different crystal forms to high resolution (up to 1.5 Å). Despite different crystal packing (Supplementary Table 1), the two CaMep2 structures are identical to each other and very similar to ScMep2 (Cα r.m.s.d. (root mean square deviation)=0.7 Å for 434 residues), with the main differences confined to the N terminus and the CTR (Fig. 1). Electron density is visible for the entire polypeptide chains, with the exception of the C-terminal 43 (ScMep2) and 25 residues (CaMep2), which are poorly conserved and presumably disordered. Both Mep2 proteins show the archetypal trimeric assemblies in which each monomer consists of 11 TM helices surrounding a central pore. Important functional features such as the extracellular ammonium binding site, the Phe gate and the twin-His motif within the hydrophobic channel are all very similar to those present in the bacterial transporters and RhCG. In the remainder of the manuscript, we will specifically discuss CaMep2 due to the superior resolution of the structure. Unless specifically stated, the drawn conclusions also apply to ScMep2.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8338</offset><text>While the overall architecture of Mep2 is similar to that of the prokaryotic transporters (Cα r.m.s.d. with Amt-1=1.4 Å for 361 residues), there are large differences within the N terminus, intracellular loops (ICLs) ICL1 and ICL3, and the CTR. The N termini of the Mep2 proteins are ∼20–25 residues longer compared with their bacterial counterparts (Figs 1 and 2), substantially increasing the size of the extracellular domain. Moreover, the N terminus of one monomer interacts with the extended extracellular loop ECL5 of a neighbouring monomer. Together with additional, smaller differences in other extracellular loops, these changes generate a distinct vestibule leading to the ammonium binding site that is much more pronounced than in the bacterial proteins. The N-terminal vestibule and the resulting inter-monomer interactions likely increase the stability of the Mep2 trimer, in support of data for plant AMT proteins. However, given that an N-terminal deletion mutant (2-27Δ) grows as well as wild-type (WT) Mep2 on minimal ammonium medium (Fig. 3 and Supplementary Fig. 1), the importance of the N terminus for Mep2 activity is not clear.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>9498</offset><text>Mep2 channels are closed by a two-tier channel block</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9551</offset><text>The largest differences between the Mep2 structures and the other known ammonium transporter structures are located on the intracellular side of the membrane. In the vicinity of the Mep2 channel exit, the cytoplasmic end of TM2 has unwound, generating a longer ICL1 even though there are no insertions in this region compared to the bacterial proteins (Figs 2 and 4). ICL1 has also moved inwards relative to its position in the bacterial Amts. The largest backbone movements of equivalent residues within ICL1 are ∼10 Å, markedly affecting the conserved basic RxK motif (Fig. 4). The head group of Arg54 has moved ∼11 Å relative to that in Amt-1, whereas the shift of the head group of the variable Lys55 residue is almost 20 Å. The side chain of Lys56 in the basic motif points in an opposite direction in the Mep2 structures compared with that of, for example, Amt-1 (Fig. 4). In addition to changing the RxK motif, the movement of ICL1 has another, crucial functional consequence. At the C-terminal end of TM1, the side-chain hydroxyl group of the relatively conserved Tyr49 (Tyr53 in ScMep2) makes a strong hydrogen bond with the ɛ2 nitrogen atom of the absolutely conserved His342 of the twin-His motif (His348 in ScMep2), closing the channel (Figs 4 and 5). In bacterial Amt proteins, this Tyr side chain is rotated ∼4 Å away as a result of the different conformation of TM1, leaving the channel open and the histidine available for its putative role in substrate transport (Supplementary Fig. 2).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11074</offset><text>Compared with ICL1, the backbone conformational changes observed for the neighbouring ICL2 are smaller, but large shifts are nevertheless observed for the conserved residues Glu140 and Arg141 (Fig. 4). Finally, the important ICL3 linking the pseudo-symmetrical halves (TM1-5 and TM6-10) of the transporter is also shifted up to ∼10 Å and forms an additional barrier that closes the channel on the cytoplasmic side (Fig. 5). This two-tier channel block likely ensures that very little ammonium transport will take place under nitrogen-sufficient conditions. The closed state of the channel might also explain why no density, which could correspond to ammonium (or water), is observed in the hydrophobic part of the Mep2 channel close to the twin-His motif. Significantly, this is also true for ScMep2, which was crystallized in the presence of 0.2 M ammonium ions (see Methods section).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11967</offset><text>The final region in Mep2 that shows large differences compared with the bacterial transporters is the CTR. In Mep2, the CTR has moved away and makes relatively few contacts with the main body of the transporter, generating a more elongated protein (Figs 1 and 4). By contrast, in the structures of bacterial proteins, the CTR is docked tightly onto the N-terminal half of the transporters (corresponding to TM1-5), resulting in a more compact structure. This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2). These residues include those of the ‘ExxGxD' motif, which when mutated generate inactive transporters. In Amt-1 and other bacterial ammonium transporters, these CTR residues interact with residues within the N-terminal half of the protein. On one side, the Tyr390 hydroxyl in Amt-1 is hydrogen bonded with the side chain of the conserved His185 at the C-terminal end of loop ICL3. At the other end of ICL3, the backbone carbonyl groups of Gly172 and Lys173 are hydrogen bonded to the side chain of Arg370. Similar interactions were also modelled in the active, non-phosphorylated plant AtAmt-1;1 structure (for example, Y467-H239 and D458-K71). The result of these interactions is that the CTR ‘hugs' the N-terminal half of the transporters (Fig. 4). Also noteworthy is Asp381, the side chain of which interacts strongly with the positive dipole on the N-terminal end of TM2. This interaction in the centre of the protein may be particularly important to stabilize the open conformations of ammonium transporters. In the Mep2 structures, none of the interactions mentioned above are present.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13717</offset><text>Phosphorylation target site is at the periphery of Mep2</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13773</offset><text>Recently Boeckstaens et al. provided evidence that Ser457 in ScMep2 (corresponding to Ser453 in CaMep2) is phosphorylated by the TORC1 effector kinase Npr1 under nitrogen-limiting conditions. In the absence of Npr1, plasmid-encoded WT Mep2 in a S. cerevisiae mep1-3Δ strain (triple mepΔ) does not allow growth on low concentrations of ammonium, suggesting that the transporter is inactive (Fig. 3 and Supplementary Fig. 1). Conversely, the phosphorylation-mimicking S457D variant is active both in the triple mepΔ background and in a triple mepΔ npr1Δ strain (Fig. 3). Mutation of other potential phosphorylation sites in the CTR did not support growth in the npr1Δ background. Collectively, these data suggest that phosphorylation of Ser457 opens the Mep2 channel to allow ammonium uptake. Ser457 is located in a part of the CTR that is conserved in a subgroup of Mep2 proteins, but which is not present in bacterial proteins (Fig. 2). This segment (residues 450–457 in ScMep2 and 446–453 in CaMep2) was dubbed an autoinhibitory (AI) region based on the fact that its removal generates an active transporter in the absence of Npr1 (Fig. 3).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14939</offset><text>Where is the AI region and the Npr1 phosphorylation site located? Our structures reveal that surprisingly, the AI region is folded back onto the CTR and is not located near the centre of the trimer as expected from the bacterial structures (Fig. 4). The AI region packs against the cytoplasmic ends of TM2 and TM4, physically linking the main body of the transporter with the CTR via main chain interactions and side-chain interactions of Val447, Asp449, Pro450 and Arg452 (Fig. 6). The AI regions have very similar conformations in CaMep2 and ScMep2, despite considerable differences in the rest of the CTR (Fig. 6). Strikingly, the Npr1 target serine residue is located at the periphery of the trimer, far away (∼30 Å) from any channel exit (Fig. 6). Despite its location at the periphery of the trimer, the electron density for the serine is well defined in both Mep2 structures and corresponds to the non-phosphorylated state (Fig. 6). This makes sense since the proteins were expressed in rich medium and confirms the recent suggestion by Boeckstaens et al. that the non-phosphorylated form of Mep2 corresponds to the inactive state. For ScMep2, Ser457 is the most C-terminal residue for which electron density is visible, indicating that the region beyond Ser457 is disordered. In CaMep2, the visible part of the sequence extends for two residues beyond Ser453 (Fig. 6). The peripheral location and disorder of the CTR beyond the kinase target site should facilitate the phosphorylation by Npr1. The disordered part of the CTR is not conserved in ammonium transporters (Fig. 2), suggesting that it is not important for transport. Interestingly, a ScMep2 457Δ truncation mutant in which a His-tag directly follows Ser457 is highly expressed but has low activity (Fig. 3 and Supplementary Fig. 1b), suggesting that the His-tag interferes with phosphorylation by Npr1. The same mutant lacking the His-tag has WT properties (Supplementary Fig. 1b), confirming that the region following the phosphorylation site is dispensable for function.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>16987</offset><text>Mep2 lacking the AI region is conformationally heterogeneous</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17048</offset><text>Given that Ser457/453 is far from any channel exit (Fig. 6), the crucial question is how phosphorylation opens the Mep2 channel to generate an active transporter. Boeckstaens et al. proposed that phosphorylation does not affect channel activity directly, but instead relieves inhibition by the AI region. The data behind this hypothesis is the observation that a ScMep2 449-485Δ deletion mutant lacking the AI region is highly active in MA uptake both in the triple mepΔ and triple mepΔ npr1Δ backgrounds, implying that this Mep2 variant has a constitutively open channel. We obtained a similar result for ammonium uptake by the 446Δ mutant (Fig. 3), supporting the data from Marini et al. We then constructed and purified the analogous CaMep2 442Δ truncation mutant and determined the crystal structure using data to 3.4 Å resolution. The structure shows that removal of the AI region markedly increases the dynamics of the cytoplasmic parts of the transporter. This is not unexpected given the fact that the AI region bridges the CTR and the main body of Mep2 (Fig. 6). Density for ICL3 and the CTR beyond residue Arg415 is missing in the 442Δ mutant, and the density for the other ICLs including ICL1 is generally poor with visible parts of the structure having high B-factors (Fig. 7). Interestingly, however, the Tyr49-His342 hydrogen bond that closes the channel in the WT protein is still present (Fig. 7 and Supplementary Fig. 2). Why then does this mutant appear to be constitutively active? We propose two possibilities. The first one is that the open state is disfavoured by crystallization because of lower stability or due to crystal packing constraints. The second possibility is that the Tyr–His hydrogen bond has to be disrupted by the incoming substrate to open the channel. The latter model would fit well with the NH3/H+ symport model in which the proton is relayed by the twin-His motif. The importance of the Tyr–His hydrogen bond is underscored by the fact that its removal in the ScMep2 Y53A mutant results in a constitutively active transporter (Fig. 3).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>19155</offset><text>Phosphorylation causes a conformational change in the CTR</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19213</offset><text>Do the Mep2 structures provide any clues regarding the potential effect of phosphorylation? The side-chain hydroxyl of Ser457/453 is located in a well-defined electronegative pocket that is solvent accessible (Fig. 6). The closest atoms to the serine hydroxyl group are the backbone carbonyl atoms of Asp419, Glu420 and Glu421, which are 3–4 Å away. We therefore predict that phosphorylation of Ser453 will result in steric clashes as well as electrostatic repulsion, which in turn might cause substantial conformational changes within the CTR. To test this hypothesis, we determined the structure of the phosphorylation-mimicking R452D/S453D protein (hereafter termed ‘DD mutant'), using data to a resolution of 2.4 Å. The additional mutation of the arginine preceding the phosphorylation site was introduced (i) to increase the negative charge density and make it more comparable to a phosphate at neutral pH, and (ii) to further destabilize the interactions of the AI region with the main body of the transporter (Fig. 6). The ammonium uptake activity of the S. cerevisiae version of the DD mutant is the same as that of WT Mep2 and the S453D mutant, indicating that the mutations do not affect transporter functionality in the triple mepΔ background (Fig. 3). Unexpectedly, the AI segment containing the mutated residues has only undergone a slight shift compared with the WT protein (Fig. 8 and Supplementary Fig. 3). By contrast, the conserved part of the CTR has undergone a large conformational change involving formation of a 12-residue-long α-helix from Leu427 to Asp438. In addition, residues Glu420-Leu423 including Glu421 of the ExxGxD motif are now disordered (Fig. 8 and Supplementary Fig. 3). Overall, ∼20 residues are affected by the introduced mutations. This is the first time a large conformational change has been observed in an ammonium transporter as a result of a mutation, and confirms previous hypotheses that phosphorylation causes structural changes in the CTR. To exclude the possibility that the additional R452D mutation is responsible for the observed changes, we also determined the structure of the ‘single D' S453D mutant. As shown in Supplementary Fig. 4, the consequence of the single D mutation is very similar to that of the DD substitution, with conformational changes and increased dynamics confined to the conserved part of the CTR (Supplementary Fig. 4). To supplement the crystal structures, we also performed modelling and MD studies of WT CaMep2, the DD mutant and phosphorylated protein (S453J). In the WT structure, the acidic residues Asp419, Glu420 and Glu421 are within hydrogen bonding distance of Ser453. After 200 ns of MD simulation, the interactions between these residues and Ser453 remain intact. The protein backbone has an average r.m.s.d. of only ∼3 Å during the 200-ns simulation, indicating that the protein is stable. There is flexibility in the side chains of the acidic residues so that they are able to form stable hydrogen bonds with Ser453. In particular, persistent hydrogen bonds are observed between the Ser453 hydroxyl group and the acidic group of Glu420, and also between the amine group of Ser453 and the backbone carbonyl of Glu420 (Supplementary Fig. 5). The DD mutant is also stable during the simulations, but the average backbone r.m.s.d of ∼3.6 Å suggests slightly more conformational flexibility than WT. As the simulation proceeds, the side chains of the acidic residues move away from Asp452 and Asp453, presumably to avoid electrostatic repulsion. For example, the distance between the Asp453 acidic oxygens and the Glu420 acidic oxygens increases from ∼7 to &gt;22 Å after 200 ns simulations, and thus these residues are not interacting. The protein is structurally stable throughout the simulation with little deviation in the other parts of the protein. Finally, the S453J mutant is also stable throughout the 200-ns simulation and has an average backbone deviation of ∼3.8 Å, which is similar to the DD mutant. The movement of the acidic residues away from Arg452 and Sep453 is more pronounced in this simulation in comparison with the movement away from Asp452 and Asp453 in the DD mutant. The distance between the phosphate of Sep453 and the acidic oxygen atoms of Glu420 is initially ∼11 Å, but increases to &gt;30 Å after 200 ns. The short helix formed by residues Leu427 to Asp438 unravels during the simulations to a disordered state. The remainder of the protein is not affected (Supplementary Fig. 5). Thus, the MD simulations support the notion from the crystal structures that phosphorylation generates conformational changes in the conserved part of the CTR. However, the conformational changes for the phosphomimetic mutants in the crystals are confined to the CTR (Fig. 8), and the channels are still closed (Supplementary Fig. 2). One possible explanation is that the mutants do not accurately mimic a phosphoserine, but the observation that the S453D and DD mutants are fully active in the absence of Npr1 suggests that the mutations do mimic the effect of phosphorylation (Fig. 3). The fact that the S453D structure was obtained in the presence of 10 mM ammonium ions suggests that the crystallization process favours closed states of the Mep2 channels.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>24519</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>24530</offset><text>Knowledge about ammonium transporter structure has been obtained from experimental and theoretical studies on bacterial family members. In addition, a number of biochemical and genetic studies are available for bacterial, fungal and plant proteins. These efforts have advanced our knowledge considerably but have not yet yielded atomic-level answers to several important mechanistic questions, including how ammonium transport is regulated in eukaryotes and the mechanism of ammonium signalling. In Arabidopsis thaliana Amt-1;1, phosphorylation of the CTR residue T460 under conditions of high ammonium inhibits transport activity, that is, the default (non-phosphorylated) state of the plant transporter is open. Interestingly, phosphomimetic mutations introduced into one monomer inactivate the entire trimer, indicating that (i) heterotrimerization occurs and (ii) the CTR mediates allosteric regulation of ammonium transport activity via phosphorylation. Owing to the lack of structural information for plant AMTs, the details of channel closure and inter-monomer crosstalk are not yet clear. Contrasting with the plant transporters, the inactive states of Mep2 proteins under conditions of high ammonium are non-phosphorylated, with channels that are closed on the cytoplasmic side. The reason why similar transporters such as A. thaliana Amt-1;1 and Mep2 are regulated in opposite ways by phosphorylation (inactivation in plants and activation in fungi) is not known. In fungi, preventing ammonium entry via channel closure in ammonium transporters would be one way to alleviate ammonium toxicity, in addition to ammonium excretion via Ato transporters and amino-acid secretion.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>26215</offset><text>By determining the first structures of closed ammonium transporters and comparing those structures with the permanently open bacterial proteins, we demonstrate that Mep2 channel closure is likely due to movements of the CTR and ICL1 and ICL3. More specifically, the close interactions between the CTR and ICL1/ICL3 present in open transporters are disrupted, causing ICL3 to move outwards and block the channel (Figs 4 and 9a). In addition, ICL1 has shifted inwards to contribute to the channel closure by engaging His2 from the twin-His motif via hydrogen bonding with a highly conserved tyrosine hydroxyl group. Upon phosphorylation by the Npr1 kinase in response to nitrogen limitation, the region around the conserved ExxGxD motif undergoes a conformational change that opens the channel (Fig. 9). Importantly, the structural similarities in the TM parts of Mep2 and AfAmt-1 (Fig. 5a) suggest that channel opening/closure does not require substantial changes in the residues lining the channel. How exactly the channel opens and whether opening is intra-monomeric are still open questions; it is possible that the change in the CTR may disrupt its interactions with ICL3 of the neighbouring monomer (Fig. 9b), which could result in opening of the neighbouring channel via inward movement of its ICL3. Owing to the crosstalk between monomers, a single phosphorylation event might lead to opening of the entire trimer, although this has not yet been tested (Fig. 9b). Whether or not Mep2 channel opening requires, in addition to phosphorylation, disruption of the Tyr–His2 interaction by the ammonium substrate is not yet clear.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>27848</offset><text>Is our model for opening and closing of Mep2 channels valid for other eukaryotic ammonium transporters? Our structural data support previous studies and clarify the central role of the CTR and cytoplasmic loops in the transition between closed and open states. However, even the otherwise highly similar Mep2 proteins of S. cerevisiae and C. albicans have different structures for their CTRs (Fig. 1 and Supplementary Fig. 6). In addition, the AI region of the CTR containing the Npr1 kinase site is conserved in only a subset of fungal transporters, suggesting that the details of the structural changes underpinning regulation vary. Nevertheless, given the central role of absolutely conserved residues within the ICL1-ICL3-CTR interaction network (Fig. 4), we propose that the structural basics of fungal ammonium transporter activation are conserved. The fact that Mep2 orthologues of distantly related fungi are fully functional in ammonium transport and signalling in S. cerevisiae supports this notion. It should also be noted that the tyrosine residue interacting with His2 is highly conserved in fungal Mep2 orthologues, suggesting that the Tyr–His2 hydrogen bond might be a general way to close Mep2 proteins.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>29070</offset><text>With regards to plant AMTs, it has been proposed that phosphorylation at T460 generates conformational changes that would close the neighbouring pore via the C terminus. This assumption was based partly on a homology model for Amt-1;1 based on the (open) archaebacterial AfAmt-1 structure, which suggested that the C terminus of Amt-1;1 would extend further to the neighbouring monomer. Our Mep2 structures show that this assumption may not be correct (Fig. 4 and Supplementary Fig. 6). In addition, the considerable differences between structurally resolved CTR domains means that the exact environment of T460 in Amt-1;1 is also not known (Supplementary Fig. 6). Based on the available structural information, we consider it more likely that phosphorylation-mediated pore closure in Amt-1;1 is intra-monomeric, via disruption of the interactions between the CTR and ICL1/ICL3 (for example, Y467-H239 and D458-K71). There is generally no equivalent for CaMep2 Tyr49 in plant AMTs, indicating that a Tyr–His2 hydrogen bond as observed in Mep2 may not contribute to the closed state in plant transporters.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>30177</offset><text>We propose that intra-monomeric CTR-ICL1/ICL3 interactions lie at the basis of regulation of both fungal and plant ammonium transporters; close interactions generate open channels, whereas the lack of ‘intra-' interactions leads to inactive states. The need to regulate in opposite ways may be the reason why the phosphorylation sites are in different parts of the CTR, that is, centrally located close to the ExxGxD motif in AMTs and peripherally in Mep2. In this way, phosphorylation can either lead to channel closing (in the case of AMTs) or channel opening in the case of Mep2. Our model also provides an explanation for the observation that certain mutations within the CTR completely abolish transport activity. An example of an inactivating residue is the glycine of the ExxGxD motif of the CTR. Mutation of this residue (G393 in EcAmtB; G456 in AtAmt-1;1) inactivates transporters as diverse as Escherichia coli AmtB and A. thaliana Amt-1;1 (refs). Such mutations likely cause structural changes in the CTR that prevent close contacts between the CTR and ICL1/ICL3, thereby stabilizing a closed state that may be similar to that observed in Mep2.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>31335</offset><text>Regulation and modulation of membrane transport by phosphorylation is known to occur in, for example, aquaporins and urea transporters, and is likely to be a common theme for eukaryotic channels and transporters. Recently, phosphorylation was also shown to modulate substrate affinity in nitrate transporters. With respect to ammonium transport, phosphorylation has thus far only been shown for A. thaliana AMTs and for S. cerevisiae Mep2 (refs). However, the absence of GlnK proteins in eukaryotes suggests that phosphorylation-based regulation of ammonium transport may be widespread. Nevertheless, as discussed above, considerable differences may exist between different species.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>32018</offset><text>With respect to Mep2-mediated signalling to induce pseudohyphal growth, two models have been put forward as to how this occurs and why it is specific to Mep2 proteins. In one model, signalling is proposed to depend on the nature of the transported substrate, which might be different in certain subfamilies of ammonium transporters (for example, Mep1/Mep3 versus Mep2). For example, NH3 uniport or symport of NH3/H+ might result in changes in local pH, but NH4+ uniport might not, and this difference might determine signalling. In the other model, signalling is thought to require a distinct conformation of the Mep2 transporter occurring during the transport cycle. While the current study does not specifically address the mechanism of signalling underlying pseudohyphal growth, our structures do show that Mep2 proteins can assume different conformations.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>32878</offset><text>It is clear that ammonium transport across biomembranes remains a fascinating and challenging field in large part due to the unique properties of the substrate. Our Mep2 structural work now provides a foundation for future studies to uncover the details of the structural changes that occur during eukaryotic ammonium transport and signaling, and to assess the possibility to utilize small molecules to shut down ammonium sensing and downstream signalling pathways in pathogenic fungi.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>33364</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>33372</offset><text>Mep2 overexpression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>33409</offset><text>Ammonium transporter genes were amplified from genomic DNA or cDNA by PCR (Phusion, New England Biolabs). In both ScMEP2 and CaMEP2, Asn4 was replaced by a glutamine to prevent glycosylation. In order to allow transformation of yeast by recombination, the following primer extensions were used: forward 5′-GAAAAAACCCCGGATTCTAGAACTAGTGGATCCTCC-3′ and reverse 5′-TGACTCGAGTTATGCACCGTGGTGGTGATGGTGATG-3′. These primers result in a construct that lacks the cleavable N- and C-terminal tags present in the original vector, and replaces these with a C-terminal hexa-histidine tag. Recombination in yeast strain W303 pep4Δ was carried out using ∼50–100 ng of SmaI-digested vector 83νΔ (ref.) and at least a fourfold molar excess of PCR product via the lithium acetate method. Transformants were selected on SCD -His plates incubated at 30 °C. Construction of mutant CaMEP2 genes was done using the Q5 site-directed mutagenesis kit (NEB) per manufacturer's instructions. Three CaMep2 mutants were made for crystallization: the first mutant is a C-terminal truncation mutant 442Δ, lacking residues 443–480 including the AI domain. The second mutant, R452D/S453D, mimics the protein phosphorylated at Ser453. Given that phosphate is predominantly charged −2 at physiological pH, we introduced the second aspartate residue for Arg452. However, we also constructed the ‘single D', S453D CaMep2 variant.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34827</offset><text>For expression, cells were grown in shaker flasks at 30 °C for ∼24 h in synthetic minimal medium lacking histidine and with 1% (w/v) glucose to a typical OD600 of 6–8. Cells were subsequently spun down for 15 min at 4,000g and resuspended in YP medium containing 1.5% (w/v) galactose, followed by another 16–20 h growth at 30 °C/160 r.p.m. and harvesting by centrifugation. Final OD600 values typically reached 18–20. Cells were lysed by bead beating (Biospec) for 5 × 1 min with 1 min intervals on ice, or by 1–2 passes through a cell disrupter operated at 35,000 p.s.i. (TS-Series 0.75 kW; Constant Systems). Membranes were collected from the suspension by centrifugation at 200,000g for 90 min (45Ti rotor; Beckmann Coulter). Membrane protein extraction was performed by homogenization in a 1:1 (w/w) mixture of dodecyl-β-D-maltoside and decyl-β-D-maltoside (DDM/DM) followed by stirring at 4 °C overnight. Typically, 1 g (1% w/v) of total detergent was used for membranes from 2 l of cells. The membrane extract was centrifuged for 35 min at 200,000g and the supernatant was loaded onto a 10-ml Nickel column (Chelating Sepharose; GE Healthcare) equilibrated in 20 mM Tris/300 mM NaCl/0.2% DDM, pH 8. The column was washed with 15 column volumes buffer containing 30 mM imidazole and eluted in 3 column volumes with 250 mM imidazole. Proteins were purified to homogeneity by gel filtration chromatography in 10 mM HEPES/100 mM NaCl/0.05% DDM, pH 7–7.5. For polishing and detergent exchange, a second gel filtration column was performed using various detergents. In the case of ScMep2, diffracting crystals were obtained only with 0.05% decyl-maltose neopentyl glycol. For the more stable CaMep2 protein, we obtained crystals in, for example, nonyl-glucoside, decyl-maltoside and octyl-glucose neopentyl glycol. Proteins were concentrated to 7–15 mg ml−1 using 100 kDa cutoff centrifugal devices (Millipore), flash-frozen and stored at −80 °C before use.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>36860</offset><text>Crystallization and structure determination</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>36904</offset><text>Crystallization screening trials by sitting drop vapour diffusion were set up at 4 and 20 °C using in-house screens and the MemGold 1 and 2 screens (Molecular Dimensions) with a Mosquito crystallization robot. Crystals were harvested directly from the initial trials or optimized by sitting or hanging drop vapour diffusion using larger drops (typically 2–3 μl total volume). Bar-shaped crystals for ScMep2 diffracting to 3.2 Å resolution were obtained from 50 mM 2-(N-morpholino)ethanesulfonic acid (MES)/0.2 M di-ammonium hydrogen phosphate/30% PEG 400, pH 6. They belong to space group P212121 and have nine molecules (three trimers) in the asymmetric unit (AU). Well-diffracting crystals for CaMep2 were obtained in space group P3 from 0.1 M MES/0.2 M lithium sulphate/20% PEG400, pH 6 (two molecules per AU). An additional crystal form in space group R3 was grown in 0.04 M Tris/0.04 M NaCl/27% PEG350 MME, pH 8 (one molecule per AU). Diffracting crystals for the phosporylation-mimicking CaMep2 DD mutant were obtained in space group P6322 from 0.1 M sodium acetate/15–20% PEG400, pH 5 (using decyl-maltoside as detergent; one molecule per AU), while S453D mutant crystals grew in 24% PEG400/0.05 M sodium acetate, pH 5.4/0.05 M magnesium acetate tetrahydrate/10 mM NH4Cl (space group R32; one molecule per AU). Finally, the 442Δ truncation mutant gave crystals under many different conditions, but most of these diffracted poorly or not at all. A reasonable low-resolution data set (3.4 Å resolution) was eventually obtained from a crystal grown in 24% PEG400/0.05 M sodium acetate/0.05 M magnesium acetate, pH 6.1 (space group R32). Diffraction data were collected at the Diamond Light Source and processed with XDS or HKL2000 (ref. ).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38689</offset><text>For MR, a search model was constructed with Sculptor within Phenix, using a sequence alignment of ScMep2 with Archaeoglobus fulgidus Amt-1 (PDB ID 2B2H; ∼40% sequence identity to ScMep2). A clear solution with nine molecules (three trimers) in the AU was obtained using Phaser. The model was subsequently completed by iterative rounds of manual building within Coot followed by refinement within Phenix. The structures for WT CaMep2 were solved using the best-defined monomer of ScMep2 (60% sequence identity with CaMep2) in MR with Phaser, followed by automated model building within Phenix. Finally, the structures of the three mutant CaMep2 proteins were solved using WT CaMep2 as the search model. The data collection and refinement statistics for all six solved structures have been summarized in Supplementary Tables 1 and 2.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>39523</offset><text>Growth assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>39537</offset><text>The S. cerevisiae haploid triple mepΔ strain (Σ1278b MATα mep1::LEU2 mep2::LEU2 mep3::G418 ura3-52) and triple mepΔ npr1Δ strain (Σ1278b MATα mep1::LEU2 mep2::LEU2 mep3::G418 npr1::NAT1 ura3-52) were generated by integrating the NAT1 resistance gene at one NPR1 locus in the diploid strain MLY131 (ref.), followed by isolation of individual haploid strains. Cells were grown in synthetic minimal medium with glucose (2%) as the carbon source and ammonium sulphate (1 mM) or glutamate (0.1%) as the nitrogen source. Yeast cells were transformed as described. All DNA sequences encoding epitope-tagged ScMep2 and its mutant derivatives were generated by PCR and homologous recombination using the vector pRS316 (ref. ). In each case, the ScMEP2 sequences included the ScMEP2 promoter (1 kb), the ScMEP2 terminator and sequences coding for a His-6 epitope at the C-terminal end of the protein. All Mep2-His fusions contain the N4Q mutation to prevent glycosylation of Mep2 (ref.). All newly generated plasmid inserts were verified by DNA sequencing. For growth assays, S. cerevisiae cells containing plasmids expressing ScMep2 or mutant derivatives were grown overnight in synthetic minimal glutamate medium, washed, spotted by robot onto solid agar plates and culture growth followed by time course photography. Images were then processed to quantify the growth of each strain over 3 days as described.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>40966</offset><text>Protein modelling</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>40984</offset><text>The MODELLER (version 9.15) software package was used to build protein structures for MD simulations. This method was required to construct two complete protein models, the double mutant R452D/S453D (with the four missing residues from the X-ray structure added) and also the construct in which the mutation at position 452 is reverted to R, and D453 is replaced with a phosphoserine. The quality of these models was assessed using normalized Discrete Optimized Protein Energy (DOPE) values and the molpdf assessment function within the MODELLER package. The model R452D/S453D mutant has a molpdf assessment score of 1854.05, and a DOPE assessment score of -60920.55. The model of the S453J mutant has a molpdf assessment score of 1857.01 and a DOPE assessment score of −61032.15.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>41767</offset><text>MD simulations</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>41782</offset><text>WT and model structures were embedded into a pre-equilibrated lipid bilayer composed of 512 dipalmitoylphosphatidylcholine lipids using the InflateGRO2 computer programme. The bilayers were then solvated with the SPC water model and counterions were added to achieve a charge neutral state. All simulations were performed with the GROMACS package (version 4.5.5), and the GROMOS96 43a1p force field. During simulation time, the temperature was maintained at 310 K using the Nosé-Hoover thermostat with a coupling constant of 0.5 ps. Pressure was maintained at 1 bar using semi-isotropic coupling with the Parrinello-Rahman barostat and a time constant of 5 ps. Electrostatic interactions were treated using the smooth particle mesh Ewald algorithm with a short-range cutoff of 0.9 nm. Van der Waals interactions were truncated at 1.4 nm with a long-range dispersion correction applied to energy and pressure. The neighbour list was updated every five steps. All bonds were constrained with the LINCS algorithm, so that a 2-fs time step could be applied throughout. The phospholipid parameters for the dipalmitoylphosphatidylcholine lipids were based on the work of Berger. The embedded proteins were simulated for 200 ns each; a repeat simulation was performed for each system with different initial velocities to ensure reproducibility. To keep the c.p.u. times within reasonable limits, all simulations were performed on Mep2 monomers. This is also consistent with previous simulations for E. coli AmtB.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>43303</offset><text>Additional information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>43326</offset><text>Accession codes: The atomic coordinates and the associated structure factors have been deposited in the Protein Data Bank (http:// www.pdbe.org) with accession codes 5AEX (ScMep2), 5AEZ(CaMep2; R3), 5AF1(CaMep2; P3), 5AID(CaMep2; 442D), 5AH3 (CaMep2; R452D/S453D) and 5FUF (CaMep2; S453D).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>43616</offset><text>How to cite this article: van den Berg, B. et al. Structural basis for Mep2 ammonium transceptor activation by phosphorylation. Nat. Commun. 7:11337 doi: 10.1038/ncomms11337 (2016).</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>43798</offset><text>Supplementary Material</text></passage><passage><infon key="fpage">556</infon><infon key="lpage">564</infon><infon key="name_0">surname:Holsbeeks;given-names:I.</infon><infon key="name_1">surname:Lagatie;given-names:O.</infon><infon key="name_2">surname:Van
+Nuland;given-names:A.</infon><infon key="name_3">surname:Van de Velde;given-names:S.</infon><infon key="name_4">surname:Thevelein;given-names:J.
+M.</infon><infon key="pub-id_pmid">15450611</infon><infon key="section_type">REF</infon><infon key="source">Trends Biochem. Sci.</infon><infon key="type">ref</infon><infon key="volume">29</infon><infon key="year">2004</infon><offset>43821</offset><text>The eukaryotic plasma membrane as a nutrient-sensing device</text></passage><passage><infon key="fpage">254</infon><infon key="lpage">299</infon><infon key="name_0">surname:Conrad;given-names:M.</infon><infon key="pub-id_pmid">24483210</infon><infon key="section_type">REF</infon><infon key="source">FEMS Microbiol. Rev.</infon><infon key="type">ref</infon><infon key="volume">38</infon><infon key="year">2014</infon><offset>43881</offset><text>Nutrient sensing and signaling in the yeast Saccharomyces cerevisiae</text></passage><passage><infon key="fpage">D251</infon><infon key="lpage">D258</infon><infon key="name_0">surname:Saier;given-names:M. H.</infon><infon key="name_1">surname:Reddy;given-names:V. S.</infon><infon key="name_2">surname:Tamang;given-names:D.
+G.</infon><infon key="name_3">surname:Vastermark;given-names:A.</infon><infon key="pub-id_pmid">24225317</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids
+Res.</infon><infon key="type">ref</infon><infon key="volume">42</infon><infon key="year">2014</infon><offset>43950</offset><text>The transporter classification database</text></passage><passage><infon key="fpage">4282</infon><infon key="lpage">4293</infon><infon key="name_0">surname:Marini;given-names:A. M.</infon><infon key="name_1">surname:Soussi-Boudekou;given-names:S.</infon><infon key="name_2">surname:Vissers;given-names:S.</infon><infon key="name_3">surname:Andre;given-names:B.</infon><infon key="pub-id_pmid">9234685</infon><infon key="section_type">REF</infon><infon key="source">Mol. Cell Biol.</infon><infon key="type">ref</infon><infon key="volume">17</infon><infon key="year">1997</infon><offset>43990</offset><text>A family of ammonium transporters in Saccharomyces cerevisiae</text></passage><passage><infon key="fpage">e24275</infon><infon key="name_0">surname:Van Zeebroeck;given-names:G.</infon><infon key="name_1">surname:Kimpe;given-names:M.</infon><infon key="name_2">surname:Vandormael;given-names:P.</infon><infon key="name_3">surname:Thevelein;given-names:J. M.</infon><infon key="pub-id_pmid">21912684</infon><infon key="section_type">REF</infon><infon key="source">PLoS ONE</infon><infon key="type">ref</infon><infon key="volume">6</infon><infon key="year">2011</infon><offset>44052</offset><text>A split-ubiquitin two-hybrid screen for proteins physically interacting with the yeast amino acid transceptor Gap1 and ammonium transceptor Mep2</text></passage><passage><infon key="fpage">1236</infon><infon key="lpage">1247</infon><infon key="name_0">surname:Lorenz;given-names:M. C.</infon><infon key="name_1">surname:Heitman;given-names:J.</infon><infon key="pub-id_pmid">9482721</infon><infon key="section_type">REF</infon><infon key="source">EMBO J.</infon><infon key="type">ref</infon><infon key="volume">17</infon><infon key="year">1998</infon><offset>44197</offset><text>The MEP2 ammonium permease regulates pseudohyphal differentiation in Saccharomyces cerevisiae</text></passage><passage><infon key="fpage">345</infon><infon key="lpage">355</infon><infon key="name_0">surname:Shnaiderman;given-names:C.</infon><infon key="name_1">surname:Miyara;given-names:I.</infon><infon key="name_2">surname:Kobiler;given-names:I.</infon><infon key="name_3">surname:Sherman;given-names:A.</infon><infon key="name_4">surname:Prusky;given-names:D.</infon><infon key="pub-id_pmid">23387470</infon><infon key="section_type">REF</infon><infon key="source">Mol. Plant Microbe Interact.</infon><infon key="type">ref</infon><infon key="volume">26</infon><infon key="year">2013</infon><offset>44291</offset><text>Differential activation of ammonium transporters during the accumulation of ammonia by Colletotrichum gloeosporioides and its effect on appressoria formation andpathogenicity</text></passage><passage><infon key="fpage">3028</infon><infon key="lpage">3039</infon><infon key="name_0">surname:Rutherford;given-names:J. C.</infon><infon key="name_1">surname:Chua;given-names:G.</infon><infon key="name_2">surname:Hughes;given-names:T.</infon><infon key="name_3">surname:Cardenas;given-names:M.
+E.</infon><infon key="name_4">surname:Heitman;given-names:J.</infon><infon key="pub-id_pmid">18434596</infon><infon key="section_type">REF</infon><infon key="source">Mol. Biol. Cell</infon><infon key="type">ref</infon><infon key="volume">19</infon><offset>44466</offset><text>A Mep2-dependent transcriptional profile links permease function to gene expression during pseudohyphal growth in Saccharomyces cerevisiae</text></passage><passage><infon key="fpage">649</infon><infon key="lpage">669</infon><infon key="name_0">surname:Biswas;given-names:K.</infon><infon key="name_1">surname:Morschhäuser;given-names:J.</infon><infon key="pub-id_pmid">15819622</infon><infon key="section_type">REF</infon><infon key="source">Mol. Microbiol.</infon><infon key="type">ref</infon><infon key="volume">56</infon><infon key="year">2005</infon><offset>44605</offset><text>The Mep2p ammonium permease controls nitrogen starvation-induced filamentous growth in Candida albicans</text></passage><passage><infon key="fpage">21362</infon><infon key="lpage">21370</infon><infon key="name_0">surname:Boeckstaens;given-names:M.</infon><infon key="name_1">surname:André;given-names:B.</infon><infon key="name_2">surname:Marini;given-names:A. M.</infon><infon key="pub-id_pmid">18508774</infon><infon key="section_type">REF</infon><infon key="source">J. Biol.
+Chem.</infon><infon key="type">ref</infon><infon key="volume">283</infon><infon key="year">2008</infon><offset>44709</offset><text>Distinct transport mechanisms in yeast ammonium transport/sensor proteins of the Mep/Amt/Rh family and impact on filamentation</text></passage><passage><infon key="fpage">534</infon><infon key="lpage">546</infon><infon key="name_0">surname:Boeckstaens;given-names:M.</infon><infon key="name_1">surname:André;given-names:B.</infon><infon key="name_2">surname:Marini;given-names:A. M.</infon><infon key="pub-id_pmid">17493133</infon><infon key="section_type">REF</infon><infon key="source">Mol. Microbiol.</infon><infon key="type">ref</infon><infon key="volume">64</infon><infon key="year">2007</infon><offset>44836</offset><text>The yeast ammonium transport protein Mep2 and its positive regulator, the Npr1 kinase, play an important role in normal and pseudohyphal growth on various nitrogen media through retrieval of excreted ammonium</text></passage><passage><infon key="fpage">9638</infon><infon key="lpage">9643</infon><infon key="name_0">surname:Gruswitz;given-names:F.</infon><infon key="pub-id_pmid">20457942</infon><infon key="section_type">REF</infon><infon key="source">Proc.
+Natl Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">107</infon><infon key="year">2010</infon><offset>45045</offset><text>Function of human Rh based on structure of RhCG at 2.1A</text></passage><passage><infon key="fpage">1587</infon><infon key="lpage">1594</infon><infon key="name_0">surname:Khademi;given-names:S.</infon><infon key="pub-id_pmid">15361618</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">305</infon><infon key="year">2004</infon><offset>45101</offset><text>Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35A</text></passage><passage><infon key="fpage">14994</infon><infon key="lpage">14999</infon><infon key="name_0">surname:Andrade;given-names:S. L.</infon><infon key="name_1">surname:Dickmanns;given-names:A.</infon><infon key="name_2">surname:Ficner;given-names:R.</infon><infon key="name_3">surname:Einsle;given-names:O.</infon><infon key="pub-id_pmid">16214888</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">102</infon><infon key="year">2005</infon><offset>45174</offset><text>Crystal structure of the archaeal ammonium transporter Amt-1 from Archaeoglobus fulgidus</text></passage><passage><infon key="fpage">39492</infon><infon key="lpage">39498</infon><infon key="name_0">surname:Javelle;given-names:A.</infon><infon key="pub-id_pmid">17040913</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">281</infon><infon key="year">2006</infon><offset>45263</offset><text>An unusual twin-his arrangement in the pore of ammonia channels is essential for substrate conductance</text></passage><passage><infon key="fpage">10419</infon><infon key="lpage">10427</infon><infon key="name_0">surname:Wang;given-names:S.</infon><infon key="name_1">surname:Orabi;given-names:E. A.</infon><infon key="name_2">surname:Baday;given-names:S.</infon><infon key="name_3">surname:Bernèche;given-names:S.</infon><infon key="name_4">surname:Lamoureux;given-names:G.</infon><infon key="pub-id_pmid">22631217</infon><infon key="section_type">REF</infon><infon key="source">J. Am. Chem. Soc.</infon><infon key="type">ref</infon><infon key="volume">134</infon><infon key="year">2012</infon><offset>45366</offset><text>Ammonium transporters achieve charge transfer by fragmenting their substrate</text></passage><passage><infon key="fpage">e62745</infon><infon key="name_0">surname:Wang;given-names:J.</infon><infon key="pub-id_pmid">23667517</infon><infon key="section_type">REF</infon><infon key="source">PLoS ONE</infon><infon key="type">ref</infon><infon key="volume">8</infon><infon key="year">2013</infon><offset>45443</offset><text>Ammonium transport proteins with changes in one of the conserved pore histidines have different performance in ammonia and methylamine conduction</text></passage><passage><infon key="fpage">10876</infon><infon key="lpage">10884</infon><infon key="name_0">surname:Lin;given-names:Y.</infon><infon key="name_1">surname:Cao;given-names:Z.</infon><infon key="name_2">surname:Mo;given-names:Y.</infon><infon key="pub-id_pmid">16910683</infon><infon key="section_type">REF</infon><infon key="source">J. Am.
+Chem. Soc.</infon><infon key="type">ref</infon><infon key="volume">128</infon><infon key="year">2006</infon><offset>45589</offset><text>Molecular dynamics simulations on the Escherichia coli ammonia channel protein AmtB: mechanism of ammonia/ammonium transport</text></passage><passage><infon key="fpage">3970</infon><infon key="lpage">3975</infon><infon key="name_0">surname:Akgun;given-names:U.</infon><infon key="name_1">surname:Khademi;given-names:S.</infon><infon key="pub-id_pmid">21368153</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl
+Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">108</infon><infon key="year">2011</infon><offset>45714</offset><text>Periplasmic vestibule plays an important role for solute recruitment, selectivity, and gating in the Rh/Amt/MEP superfamily</text></passage><passage><infon key="fpage">17090</infon><infon key="lpage">17095</infon><infon key="name_0">surname:Zheng;given-names:L.</infon><infon key="name_1">surname:Kostrewa;given-names:D.</infon><infon key="name_2">surname:Bernèche;given-names:S.</infon><infon key="name_3">surname:Winkler;given-names:F. K.</infon><infon key="name_4">surname:Li;given-names:X. D.</infon><infon key="pub-id_pmid">15563598</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">101</infon><infon key="year">2004</infon><offset>45838</offset><text>The mechanism of ammonia transport based on the crystal structure of AmtB of Escherichia coli</text></passage><passage><infon key="fpage">7091</infon><infon key="lpage">7098</infon><infon key="name_0">surname:Baday;given-names:S.</infon><infon key="name_1">surname:Wang;given-names:S.</infon><infon key="name_2">surname:Lamoureux;given-names:G.</infon><infon key="name_3">surname:Bernèche;given-names:S.</infon><infon key="pub-id_pmid">24021113</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry</infon><infon key="type">ref</infon><infon key="volume">52</infon><infon key="year">2013</infon><offset>45932</offset><text>Different hydration patterns in the pores of AmtB and RhCG could determine their transport mechanisms</text></passage><passage><infon key="fpage">628</infon><infon key="lpage">695</infon><infon key="name_0">surname:van Heeswijk;given-names:W. C.</infon><infon key="name_1">surname:Westerhoff;given-names:H. V.</infon><infon key="name_2">surname:Boogerd;given-names:F. C.</infon><infon key="pub-id_pmid">24296575</infon><infon key="section_type">REF</infon><infon key="source">Microbiol. Mol. Biol. Rev.</infon><infon key="type">ref</infon><infon key="volume">77</infon><infon key="year">2013</infon><offset>46034</offset><text>Nitrogen assimilation in Escherichia coli: putting molecular data into a systems perspective</text></passage><passage><infon key="fpage">9995</infon><infon key="lpage">10000</infon><infon key="name_0">surname:Wacker;given-names:T.</infon><infon key="name_1">surname:Garcia-Celma;given-names:J. J.</infon><infon key="name_2">surname:Lewe;given-names:P.</infon><infon key="name_3">surname:Andrade;given-names:S. L.</infon><infon key="pub-id_pmid">24958855</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl Acad. Sci. USA</infon><infon key="type">ref</infon><infon key="volume">111</infon><infon key="year">2014</infon><offset>46127</offset><text>Direct observation of electrogenic NH4(+) transport in ammonium transport (Amt) proteins</text></passage><passage><infon key="fpage">11650</infon><infon key="lpage">11655</infon><infon key="name_0">surname:Neuhäuser;given-names:B.</infon><infon key="name_1">surname:Ludewig;given-names:U.</infon><infon key="pub-id_pmid">24634212</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">289</infon><infon key="year">2014</infon><offset>46216</offset><text>Uncoupling of ionic currents from substrate transport in the plant ammonium transporter AtAMT1;2</text></passage><passage><infon key="fpage">95</infon><infon key="lpage">102</infon><infon key="name_0">surname:Monfort;given-names:P.</infon><infon key="name_1">surname:Kosenko;given-names:E.</infon><infon key="name_2">surname:Erceg;given-names:S.</infon><infon key="name_3">surname:Canales;given-names:J. J.</infon><infon key="name_4">surname:Felipo;given-names:V.</infon><infon key="pub-id_pmid">12020609</infon><infon key="section_type">REF</infon><infon key="source">Neurochem. Int.</infon><infon key="type">ref</infon><infon key="volume">41</infon><infon key="year">2002</infon><offset>46313</offset><text>Molecular mechanism of acute ammonia toxicity: role of NMDA receptors</text></passage><passage><infon key="fpage">11</infon><infon key="lpage">14</infon><infon key="name_0">surname:Thomas;given-names:G.</infon><infon key="name_1">surname:Coutts;given-names:G.</infon><infon key="name_2">surname:Merrick;given-names:M.</infon><infon key="pub-id_pmid">10637624</infon><infon key="section_type">REF</infon><infon key="source">Trends Genet.</infon><infon key="type">ref</infon><infon key="volume">16</infon><infon key="year">2000</infon><offset>46383</offset><text>The glnKamtB operon. A conserved gene pair in prokaryotes</text></passage><passage><infon key="fpage">42</infon><infon key="lpage">47</infon><infon key="name_0">surname:Gruswitz;given-names:F.</infon><infon key="name_1">surname:O'Connell;given-names:J.;suffix:III</infon><infon key="name_2">surname:Stroud;given-names:R.
+M.</infon><infon key="pub-id_pmid">17190799</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl Acad.
+Sci. USA</infon><infon key="type">ref</infon><infon key="volume">104</infon><infon key="year">2006</infon><offset>46441</offset><text>Inhibitory complex of the transmembrane ammonia channel, AmtB, and the cytosolic regulatory protein, GlnK, at 1.96A</text></passage><passage><infon key="fpage">29558</infon><infon key="lpage">29567</infon><infon key="name_0">surname:Durand;given-names:A.</infon><infon key="name_1">surname:Merrick;given-names:M.</infon><infon key="pub-id_pmid">16864585</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">281</infon><infon key="year">2006</infon><offset>46557</offset><text>In vitro analysis of the Escherichia coli AmtB-GlnK complex reveals a stoichiometric interaction and sensitivity to ATP and 2-oxoglutarate</text></passage><passage><infon key="fpage">736</infon><infon key="lpage">738</infon><infon key="name_0">surname:Lanquar;given-names:V.</infon><infon key="name_1">surname:Frommer;given-names:W. B.</infon><infon key="pub-id_pmid">20418663</infon><infon key="section_type">REF</infon><infon key="source">Plant Signal.
+Behav.</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2010</infon><offset>46696</offset><text>Adjusting ammonium uptake via phosphorylation</text></passage><passage><infon key="fpage">3101</infon><infon key="lpage">3110</infon><infon key="name_0">surname:Boeckstaens;given-names:M.</infon><infon key="name_1">surname:Llinares;given-names:E.</infon><infon key="name_2">surname:Van
+Vooren;given-names:P.</infon><infon key="name_3">surname:Marini;given-names:A. M.</infon><infon key="pub-id_pmid">24476960</infon><infon key="section_type">REF</infon><infon key="source">Nat. Commun.</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2014</infon><offset>46742</offset><text>The TORC1 effector kinase Npr1 fine tunes the inherent activity of the Mep2 ammonium transport protein</text></passage><passage><infon key="fpage">1361</infon><infon key="lpage">1373</infon><infon key="name_0">surname:Graff;given-names:L.</infon><infon key="pub-id_pmid">21127027</infon><infon key="section_type">REF</infon><infon key="source">J. Exp. Bot.</infon><infon key="type">ref</infon><infon key="volume">62</infon><infon key="year">2011</infon><offset>46845</offset><text>N- terminal cysteines affect oligomer stability of the allosterically regulated ammonium transporter LeAMT1;1</text></passage><passage><infon key="fpage">161</infon><infon key="lpage">171</infon><infon key="name_0">surname:Severi;given-names:E.</infon><infon key="name_1">surname:Javelle;given-names:A.</infon><infon key="name_2">surname:Merrick;given-names:M.</infon><infon key="pub-id_pmid">17453422</infon><infon key="section_type">REF</infon><infon key="source">Mol. Membr. Biol.</infon><infon key="type">ref</infon><infon key="volume">24</infon><infon key="year">2007</infon><offset>46955</offset><text>The conserved carboxy-terminal region of the ammonia channel AmtB plays a critical role in channel function</text></passage><passage><infon key="fpage">195</infon><infon key="lpage">198</infon><infon key="name_0">surname:Loqué;given-names:D.</infon><infon key="name_1">surname:Lalonde;given-names:S.</infon><infon key="name_2">surname:Looger;given-names:L. L.</infon><infon key="name_3">surname:von
+Wirén;given-names:N.</infon><infon key="name_4">surname:Frommer;given-names:W. B.</infon><infon key="pub-id_pmid">17293878</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">446</infon><infon key="year">2007</infon><offset>47063</offset><text>A cytosolic trans-activation domain essential for ammonium uptake</text></passage><passage><infon key="fpage">974</infon><infon key="lpage">984</infon><infon key="name_0">surname:Yuan;given-names:L.</infon><infon key="pub-id_pmid">23463773</infon><infon key="section_type">REF</infon><infon key="source">Plant
+Cell</infon><infon key="type">ref</infon><infon key="volume">25</infon><infon key="year">2013</infon><offset>47129</offset><text>Allosteric regulation of transport activity by heterotrimerization of Arabidopsis ammonium transporter complexes in vivo</text></passage><passage><infon key="fpage">e351</infon><infon key="name_0">surname:Hess;given-names:D. C.</infon><infon key="name_1">surname:Lu;given-names:W.</infon><infon key="name_2">surname:Rabinowitz;given-names:J.
+D.</infon><infon key="name_3">surname:Botstein;given-names:D.</infon><infon key="pub-id_pmid">17048990</infon><infon key="section_type">REF</infon><infon key="source">PLoS
+Biol.</infon><infon key="type">ref</infon><infon key="volume">4</infon><infon key="year">2006</infon><offset>47250</offset><text>Ammonium toxicity and potassium limitation in yeast</text></passage><passage><infon key="fpage">259</infon><infon key="lpage">275</infon><infon key="name_0">surname:Smith;given-names:D. G.</infon><infon key="name_1">surname:Garcia-Pedrajas;given-names:M. D.</infon><infon key="name_2">surname:Gold;given-names:S. E.</infon><infon key="name_3">surname:Perlin;given-names:M. H.</infon><infon key="pub-id_pmid">14507379</infon><infon key="section_type">REF</infon><infon key="source">Mol.
+Microbiol.</infon><infon key="type">ref</infon><infon key="volume">50</infon><infon key="year">2003</infon><offset>47302</offset><text>Isolation and characterization from pathogenic fungi of genes encoding ammonium permeases and their roles in dimorphism</text></passage><passage><infon key="fpage">187</infon><infon key="lpage">201</infon><infon key="name_0">surname:Teichert;given-names:S.</infon><infon key="name_1">surname:Rutherford;given-names:J. C.</infon><infon key="name_2">surname:Wottawa;given-names:M.</infon><infon key="name_3">surname:Heitman;given-names:J.</infon><infon key="name_4">surname:Tudzynski;given-names:B.</infon><infon key="pub-id_pmid">18083831</infon><infon key="section_type">REF</infon><infon key="source">Eukaryot. Cell</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2008</infon><offset>47422</offset><text>Impact of ammonium permeases mepA, mepB, and mepC on nitrogen-regulated secondary metabolism in Fusarium fujikuroi</text></passage><passage><infon key="fpage">411</infon><infon key="lpage">430</infon><infon key="name_0">surname:Javelle;given-names:A.</infon><infon key="pub-id_pmid">12519192</infon><infon key="section_type">REF</infon><infon key="source">Mol.
+Microbiol.</infon><infon key="type">ref</infon><infon key="volume">47</infon><infon key="year">2003</infon><offset>47537</offset><text>Molecular characterization, function and regulation of ammonium transporters (Amt) and ammonium-metabolizing enzymes (GS, NADP-GDH) in the ectomycorrhizal fungus Hebeloma cylindrosporum</text></passage><passage><infon key="fpage">2580</infon><infon key="lpage">2588</infon><infon key="name_0">surname:Törnroth-Horsefield;given-names:S.</infon><infon key="name_1">surname:Hedfalk;given-names:K.</infon><infon key="name_2">surname:Fischer;given-names:G.</infon><infon key="name_3">surname:Lindkvist-Petersson;given-names:K.</infon><infon key="name_4">surname:Neutze;given-names:R.</infon><infon key="section_type">REF</infon><infon key="source">FEBS
+Lett.</infon><infon key="type">ref</infon><infon key="volume">584</infon><infon key="year">2006</infon><offset>47723</offset><text>Structural insights into eukaryotic aquaporin regulation</text></passage><passage><infon key="fpage">79</infon><infon key="lpage">107</infon><infon key="name_0">surname:Klein;given-names:J. D.</infon><infon key="pub-id_pmid">25298340</infon><infon key="section_type">REF</infon><infon key="source">Subcell.
+Biochem.</infon><infon key="type">ref</infon><infon key="volume">73</infon><infon key="year">2014</infon><offset>47780</offset><text>Expression of urea transporters and their regulation</text></passage><passage><infon key="fpage">68</infon><infon key="lpage">72</infon><infon key="name_0">surname:Parker;given-names:J. L.</infon><infon key="name_1">surname:Newstead;given-names:S.</infon><infon key="pub-id_pmid">24572366</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">507</infon><infon key="year">2014</infon><offset>47833</offset><text>Molecular basis of nitrate uptake by the plant nitrate transporter NRT1.1</text></passage><passage><infon key="fpage">695</infon><infon key="lpage">707</infon><infon key="name_0">surname:Hays;given-names:F. A.</infon><infon key="name_1">surname:Roe-Zurz;given-names:Z.</infon><infon key="name_2">surname:Stroud;given-names:R. M.</infon><infon key="pub-id_pmid">20946832</infon><infon key="section_type">REF</infon><infon key="source">Methods Enzymol.</infon><infon key="type">ref</infon><infon key="volume">470</infon><infon key="year">2010</infon><offset>47907</offset><text>Overexpression and purification of integral membrane proteins in yeast</text></passage><passage><infon key="fpage">125</infon><infon key="lpage">132</infon><infon key="name_0">surname:Kabsch;given-names:W.</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">D66</infon><infon key="year">2010</infon><offset>47978</offset><text>XDS</text></passage><passage><infon key="fpage">307</infon><infon key="lpage">326</infon><infon key="name_0">surname:Otwinowski;given-names:Z.</infon><infon key="name_1">surname:Minor;given-names:W.</infon><infon key="section_type">REF</infon><infon key="source">Methods Enzymol.</infon><infon key="type">ref</infon><infon key="volume">276</infon><infon key="year">2003</infon><offset>47982</offset><text>Processing of X-ray diffraction data collected in oscillation mode</text></passage><passage><infon key="fpage">352</infon><infon key="lpage">367</infon><infon key="name_0">surname:Afonine;given-names:P. V.</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">D68</infon><infon key="year">2012</infon><offset>48049</offset><text>Towards automated crystallographic structure refinement with phenix.refine</text></passage><passage><infon key="fpage">458</infon><infon key="lpage">464</infon><infon key="name_0">surname:McCoy;given-names:A. J.</infon><infon key="name_1">surname:Grosse-Kunstleve;given-names:R. W.</infon><infon key="name_2">surname:Storoni;given-names:L. C.</infon><infon key="name_3">surname:Read;given-names:R. J.</infon><infon key="section_type">REF</infon><infon key="source">Acta
+Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">D61</infon><infon key="year">2005</infon><offset>48124</offset><text>Likelihood-enhanced fast translation functions</text></passage><passage><infon key="fpage">2126</infon><infon key="lpage">2132</infon><infon key="name_0">surname:Emsley;given-names:P.</infon><infon key="name_1">surname:Cowtan;given-names:K.</infon><infon key="section_type">REF</infon><infon key="source">Acta
+Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">D60</infon><infon key="year">2004</infon><offset>48171</offset><text>Coot: model-building tools for molecular graphics</text></passage><passage><infon key="fpage">339</infon><infon key="lpage">346</infon><infon key="name_0">surname:Schiestl;given-names:R. H.</infon><infon key="name_1">surname:Gietz;given-names:R. D.</infon><infon key="pub-id_pmid">2692852</infon><infon key="section_type">REF</infon><infon key="source">Curr. Genet.</infon><infon key="type">ref</infon><infon key="volume">16</infon><infon key="year">1989</infon><offset>48221</offset><text>High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier</text></passage><passage><infon key="fpage">201</infon><infon key="lpage">216</infon><infon key="name_0">surname:Ma;given-names:H.</infon><infon key="name_1">surname:Kunes;given-names:S.</infon><infon key="name_2">surname:Schatz;given-names:P.
+J.</infon><infon key="name_3">surname:Botstein;given-names:D.</infon><infon key="pub-id_pmid">2828185</infon><infon key="section_type">REF</infon><infon key="source">Gene</infon><infon key="type">ref</infon><infon key="volume">58</infon><infon key="year">1987</infon><offset>48323</offset><text>Plasmid construction by homologous recombination in yeast</text></passage><passage><infon key="fpage">552</infon><infon key="lpage">564</infon><infon key="name_0">surname:Marini;given-names:A. M.</infon><infon key="name_1">surname:André;given-names:B.</infon><infon key="pub-id_pmid">11069679</infon><infon key="section_type">REF</infon><infon key="source">Mol. Microbiol.</infon><infon key="type">ref</infon><infon key="volume">38</infon><infon key="year">2000</infon><offset>48381</offset><text>In vivo N-glycosylation of the Mep2 high-affinity ammonium transporter of Saccharomyces cerevisiae reveals an extracytosolic N-terminus</text></passage><passage><infon key="fpage">e1001362</infon><infon key="name_0">surname:Addinall;given-names:S. G.</infon><infon key="pub-id_pmid">21490951</infon><infon key="section_type">REF</infon><infon key="source">PLoS Genet.</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2011</infon><offset>48517</offset><text>Quantitative fitness analysis shows that NMD proteins and many other protein complexes suppress or enhance distinct telomere cap defects</text></passage><passage><infon key="fpage">291</infon><infon key="lpage">325</infon><infon key="name_0">surname:Marti-Renom;given-names:M. A.</infon><infon key="pub-id_pmid">10940251</infon><infon key="section_type">REF</infon><infon key="source">Annu. Rev. Biophys. Biomol. Struct.</infon><infon key="type">ref</infon><infon key="volume">29</infon><infon key="year">2000</infon><offset>48654</offset><text>Comparative protein structure modeling of genes and genomes</text></passage><passage><infon key="fpage">2657</infon><infon key="lpage">2669</infon><infon key="name_0">surname:Schmidt;given-names:T. H.</infon><infon key="name_1">surname:Kandt;given-names:C.</infon><infon key="pub-id_pmid">22989154</infon><infon key="section_type">REF</infon><infon key="source">J. Chem.
+Inf. Model.</infon><infon key="type">ref</infon><infon key="volume">52</infon><infon key="year">2012</infon><offset>48714</offset><text>LAMBADA and InflateGRO2: efficient membrane alignment and insertion of membrane proteins for molecular dynamics simulations</text></passage><passage><infon key="fpage">331</infon><infon key="lpage">342</infon><infon key="name_0">surname:Berendsen;given-names:H. J. C.</infon><infon key="name_1">surname:Postma;given-names:J. P.</infon><infon key="name_2">surname:van
+Gunsteren;given-names:W. F.</infon><infon key="name_3">surname:Hermans;given-names:J.</infon><infon key="section_type">REF</infon><infon key="source">Interaction models for water in relation to protein hydration Intermolecular Forces</infon><infon key="type">ref</infon><infon key="year">1981</infon><offset>48838</offset></passage><passage><infon key="fpage">1701</infon><infon key="lpage">1718</infon><infon key="name_0">surname:Van Der Spoel;given-names:D.</infon><infon key="pub-id_pmid">16211538</infon><infon key="section_type">REF</infon><infon key="source">J. Comput. Chem.</infon><infon key="type">ref</infon><infon key="volume">26</infon><infon key="year">2005</infon><offset>48839</offset><text>GROMACS: fast, flexible, and free</text></passage><passage><infon key="fpage">511</infon><infon key="lpage">519</infon><infon key="name_0">surname:Nosé;given-names:S.</infon><infon key="section_type">REF</infon><infon key="source">J. Chem. Phys.</infon><infon key="type">ref</infon><infon key="volume">81</infon><infon key="year">1984</infon><offset>48873</offset><text>A unified formulation of the constant temperature molecular dynamics methods</text></passage><passage><infon key="fpage">1695</infon><infon key="name_0">surname:Hoover;given-names:W. G.</infon><infon key="section_type">REF</infon><infon key="source">Phys.
+Rev. A</infon><infon key="type">ref</infon><infon key="volume">31</infon><infon key="year">1985</infon><offset>48950</offset><text>Canonical dynamics: equilibrium phase-space distributions</text></passage><passage><infon key="fpage">7182</infon><infon key="lpage">7190</infon><infon key="name_0">surname:Parrinello;given-names:M.</infon><infon key="name_1">surname:Rahman;given-names:A.</infon><infon key="section_type">REF</infon><infon key="source">J. Appl. Phys.</infon><infon key="type">ref</infon><infon key="volume">52</infon><infon key="year">1981</infon><offset>49008</offset><text>Polymorphic transitions in single crystals: a new molecular dynamics method</text></passage><passage><infon key="fpage">8577</infon><infon key="lpage">8593</infon><infon key="name_0">surname:Essmann;given-names:U.</infon><infon key="section_type">REF</infon><infon key="source">J. Chem. Phys.</infon><infon key="type">ref</infon><infon key="volume">103</infon><infon key="year">1995</infon><offset>49084</offset><text>A smooth particle mesh Ewald method</text></passage><passage><infon key="fpage">1463</infon><infon key="lpage">1472</infon><infon key="name_0">surname:Hess;given-names:B.</infon><infon key="name_1">surname:Bekker;given-names:H.</infon><infon key="name_2">surname:Berendsen;given-names:H.
+J.</infon><infon key="name_3">surname:Fraaije;given-names:J. G.</infon><infon key="section_type">REF</infon><infon key="source">J.
+Comput. Chem.</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">1997</infon><offset>49120</offset><text>LINCS: a linear constraint solver for molecular simulations</text></passage><passage><infon key="fpage">2002</infon><infon key="lpage">2013</infon><infon key="name_0">surname:Berger;given-names:O.</infon><infon key="name_1">surname:Edholm;given-names:O.</infon><infon key="name_2">surname:Jähnig;given-names:F.</infon><infon key="pub-id_pmid">9129804</infon><infon key="section_type">REF</infon><infon key="source">Biophys. J.</infon><infon key="type">ref</infon><infon key="volume">72</infon><infon key="year">1997</infon><offset>49180</offset><text>Molecular dynamics simulations of a fluid bilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>49327</offset><text> The PyMOL Molecular Graphics System. version 1.7.4 (Schrödinger, LLC).</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>49400</offset><text>Author contributions B.v.d.B. performed the experiments related to Mep2 structure determination, designed research and wrote the paper. A.C. performed ammonium growth experiments of Mep variants. D.J. and S.K. performed modelling studies and MD simulations. A.B. collected the X-ray synchrotron data and maintained the Newcastle Structural Biology Laboratory. J.C.R. designed research related to the S. cerevisiae growth assays.</text></passage><passage><infon key="file">ncomms11337-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>49829</offset><text>X-ray crystal structures of Mep2 transceptors.</text></passage><passage><infon key="file">ncomms11337-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>49876</offset><text>(a) Monomer cartoon models viewed from the side for (left) A.
+fulgidus Amt-1 (PDB ID 2B2H), S. cerevisiae Mep2 (middle) and
+C. albicans Mep2 (right). The cartoons are in rainbow
+representation. The region showing ICL1 (blue), ICL3 (green) and the CTR
+(red) is boxed for comparison. (b) CaMep2 trimer viewed from the
+intracellular side (right). One monomer is coloured as in a and one
+monomer is coloured by B-factor (blue, low; red; high). The CTR is boxed.
+(c) Overlay of ScMep2 (grey) and CaMep2 (rainbow), illustrating
+the differences in the CTRs. All structure figures were generated with
+Pymol.</text></passage><passage><infon key="file">ncomms11337-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>50476</offset><text>Sequence conservation in ammonium transporters.</text></passage><passage><infon key="file">ncomms11337-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>50524</offset><text>ClustalW alignment of CaMep2, ScMep2, A. fulgidus Amt-1, E.
+coli AmtB and A. thaliana Amt-1;1. The secondary structure
+elements observed for CaMep2 are indicated, with the numbers corresponding
+to the centre of the TM segment. Important regions are labelled. The
+conserved RxK motif in ICL1 is boxed in blue, the ER motif in ICL2 in cyan,
+the conserved ExxGxD motif of the CTR in red and the AI region in yellow.
+Coloured residues are functionally important and correspond to those of the
+Phe gate (blue), the binding site Trp residue (magenta) and the twin-His
+motif (red). The Npr1 kinase site in the AI region is highlighted pink. The
+grey sequences at the C termini of CaMep2 and ScMep2 are not visible in the
+structures and are likely disordered.</text></passage><passage><infon key="file">ncomms11337-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>51276</offset><text>Growth of ScMep2 variants on low ammonium medium.</text></passage><passage><infon key="file">ncomms11337-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>51326</offset><text>(a) The triple mepΔ strain (black) and triple
+mepΔ npr1Δ strain (grey) containing plasmids
+expressing WT and variant ScMep2 were grown on minimal medium containing
+1 mM ammonium sulphate. The quantified cell density reflects
+logarithmic growth after 24 h. Error bars are the s.d. for three
+replicates of each strain (b) The strains used in a were also
+serially diluted and spotted onto minimal agar plates containing glutamate
+(0.1%) or ammonium sulphate (1 mM), and grown for 3 days at
+30 °C.</text></passage><passage><infon key="file">ncomms11337-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>51832</offset><text>Structural differences between Mep2 and bacterial ammonium
+transporters.</text></passage><passage><infon key="file">ncomms11337-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>51905</offset><text>(a) ICL1 in AfAmt-1 (light blue) and CaMep2 (dark blue), showing
+unwinding and inward movement in the fungal protein. (b) Stereo
+diagram viewed from the cytosol of ICL1, ICL3 (green) and the CTR (red) in
+AfAmt-1 (light colours) and CaMep2 (dark colours). The side chains of
+residues in the RxK motif as well as those of Tyr49 and His342 are labelled.
+The numbering is for CaMep2. (c) Conserved residues in ICL1-3 and the
+CTR. Views from the cytosol for CaMep2 (left) and AfAmt-1, highlighting the
+large differences in conformation of the conserved residues in ICL1 (RxK
+motif; blue), ICL2 (ER motif; cyan), ICL3 (green) and the CTR (red). The
+labelled residues are analogous within both structures. In b and
+c, the centre of the trimer is at top.</text></passage><passage><infon key="file">ncomms11337-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>52652</offset><text>Channel closures in Mep2.</text></passage><passage><infon key="file">ncomms11337-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>52678</offset><text>(a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of
+the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1
+that forms a hydrogen bond with His2 in CaMep2. (b) Surface views
+from the side in rainbow colouring, showing the two-tier channel block
+(indicated by the arrows) in CaMep2.</text></passage><passage><infon key="file">ncomms11337-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>53000</offset><text>The Npr1 kinase target Ser453 is dephosphorylated and located in an
+electronegative pocket.</text></passage><passage><infon key="file">ncomms11337-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>53092</offset><text>(a) Stereoviews of CaMep2 showing 2Fo–Fc
+electron density (contoured at 1.0 σ) for CTR residues
+Asp419-Met422 and for Tyr446-Thr455 of the AI region. For clarity, the
+residues shown are coloured white, with oxygen atoms in red and nitrogen
+atoms in blue. The phosphorylation target residue Ser453 is labelled in
+bold. (b) Overlay of the CTRs of ScMep2 (grey) and CaMep2 (green),
+showing the similar electronegative environment surrounding the
+phosphorylation site (P). The AI regions are coloured magenta. (c)
+Cytoplasmic view of the Mep2 trimer indicating the large distance between
+Ser453 and the channel exits (circles; Ile52 lining the channel exit is
+shown).</text></passage><passage><infon key="file">ncomms11337-f7.jpg</infon><infon key="id">f7</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>53761</offset><text>Effect of removal of the AI region on Mep2 structure.</text></passage><passage><infon key="file">ncomms11337-f7.jpg</infon><infon key="id">f7</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>53815</offset><text>(a) Side views for WT CaMep2 (left) and the truncation mutant
+442Δ (right). The latter is shown as a putty model according to
+B-factors to illustrate the disorder in the protein on the cytoplasmic side.
+Missing regions are labelled. (b) Stereo superpositions of WT CaMep2
+and the truncation mutant. 2Fo–Fc electron
+density (contoured at 1.0 σ) for residues Tyr49 and His342 is
+shown for the truncation mutant.</text></passage><passage><infon key="file">ncomms11337-f8.jpg</infon><infon key="id">f8</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>54233</offset><text>Phosphorylation causes conformational changes in the CTR.</text></passage><passage><infon key="file">ncomms11337-f8.jpg</infon><infon key="id">f8</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>54291</offset><text>(a) Cytoplasmic view of the DD mutant trimer, with WT CaMep2
+superposed in grey for one of the monomers. The arrow indicates the
+phosphorylation site. The AI region is coloured magenta. (b) Monomer
+side-view superposition of WT CaMep2 and the DD mutant, showing the
+conformational change and disorder around the ExxGxD motif. Side chains for
+residues 452 and 453 are shown as stick models.</text></passage><passage><infon key="file">ncomms11337-f9.jpg</infon><infon key="id">f9</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>54681</offset><text>Schematic model for phosphorylation-based regulation of Mep2 ammonium
+transporters.</text></passage><passage><infon key="file">ncomms11337-f9.jpg</infon><infon key="id">f9</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>54765</offset><text>(a) In the closed, non-phosphorylated state (i), the CTR (magenta) and
+ICL3 (green) are far apart with the latter blocking the intracellular
+channel exit (indicated with a hatched circle). Upon phosphorylation and
+mimicked by the CaMep2 S453D and DD mutants (ii), the region around the
+ExxGxD motif undergoes a conformational change that results in the CTR
+interacting with the inward-moving ICL3, opening the channel (full circle)
+(iii). The arrows depict the movements of important structural elements. The
+open-channel Mep2 structure is represented by archaebacterial Amt-1 and
+shown in lighter colours consistent with Fig. 4. As
+discussed in the text, similar structural arrangements may occur in plant
+AMTs. In this case however, the open channel corresponds to the
+non-phosphorylated state; phosphorylation breaks the CTR–ICL3
+interactions leading to channel closure. (b) Model based on AMT
+transporter analogy showing how phosphorylation of a
+Mep2 monomer might allosterically open channels in the entire trimer via
+disruption of the interactions between the CTR and ICL3 of a neighbouring
+monomer (arrow).</text></passage></document></collection>
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+<collection><source>PMC</source><date>20201222</date><key>pmc.key</key><document><id>4854314</id><infon key="license">CC BY</infon><passage><infon key="alt-title">RNA protects a nucleoprotein complex against radiation damage</infon><infon key="article-id_coden">ACSDAD</infon><infon key="article-id_doi">10.1107/S2059798316003351</infon><infon key="article-id_pii">S2059798316003351</infon><infon key="article-id_pmc">4854314</infon><infon key="article-id_pmid">27139628</infon><infon key="article-id_publisher-id">rr5121</infon><infon key="fpage">648</infon><infon key="issue">Pt 5</infon><infon key="kwd">radiation damage protein–RNA complex electron difference density specific damage decarboxylation</infon><infon key="license">This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits
+unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.</infon><infon key="lpage">657</infon><infon key="name_0">surname:Bury;given-names:Charles S.</infon><infon key="name_1">surname:McGeehan;given-names:John E.</infon><infon key="name_2">surname:Antson;given-names:Alfred A.</infon><infon key="name_3">surname:Carmichael;given-names:Ian</infon><infon key="name_4">surname:Gerstel;given-names:Markus</infon><infon key="name_5">surname:Shevtsov;given-names:Mikhail B.</infon><infon key="name_6">surname:Garman;given-names:Elspeth F.</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">72</infon><infon key="year">2016</infon><offset>0</offset><text>RNA protects a nucleoprotein complex against radiation damage</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>62</offset><text>Systematic analysis of radiation damage within a protein–RNA complex over a large dose range (1.3–25 MGy) reveals significant differential susceptibility of RNA and protein. A new method of difference electron-density quantification is presented.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>315</offset><text>Radiation damage during macromolecular X-ray crystallographic data collection is still the main impediment for many macromolecular structure determinations. Even when an eventual model results from the crystallographic pipeline, the manifestations of radiation-induced structural and conformation changes, the so-called specific damage, within crystalline macromolecules can lead to false interpretations of biological mechanisms. Although this has been well characterized within protein crystals, far less is known about specific damage effects within the larger class of nucleoprotein complexes. Here, a methodology has been developed whereby per-atom density changes could be quantified with increasing dose over a wide (1.3–25.0 MGy) range and at higher resolution (1.98 Å) than the previous systematic specific damage study on a protein–DNA complex. Specific damage manifestations were determined within the large trp RNA-binding attenuation protein (TRAP) bound to a single-stranded RNA that forms a belt around the protein. Over a large dose range, the RNA was found to be far less susceptible to radiation-induced chemical changes than the protein. The availability of two TRAP molecules in the asymmetric unit, of which only one contained bound RNA, allowed a controlled investigation into the exact role of RNA binding in protein specific damage susceptibility. The 11-fold symmetry within each TRAP ring permitted statistically significant analysis of the Glu and Asp damage patterns, with RNA binding unexpectedly being observed to protect these otherwise highly sensitive residues within the 11 RNA-binding pockets distributed around the outside of the protein molecule. Additionally, the method enabled a quantification of the reduction in radiation-induced Lys and Phe disordering upon RNA binding directly from the electron density.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>2173</offset><text>Introduction   </text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2190</offset><text>With the wide use of high-flux third-generation synchrotron sources, radiation damage (RD) has once again become a dominant reason for the failure of structure determination using macromolecular crystallography (MX) in experiments conducted both at room temperature and under cryocooled conditions (100 K). Significant progress has been made in recent years in understanding the inevitable manifestations of X-ray-induced RD within protein crystals, and there is now a body of literature on possible strategies to mitigate the effects of RD (e.g. Zeldin, Brockhauser et al., 2013; Bourenkov &amp; Popov, 2010). However, there is still no general consensus within the field on how to minimize RD during MX data collection, and debates on the dependence of RD progression on incident X-ray energy (Shimizu et al., 2007; Liebschner et al., 2015) and the efficacy of radical scavengers (Allan et al., 2013) have yet to be resolved.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3116</offset><text>RD manifests in two forms. Global radiation damage is observed within reciprocal space as the overall decay of the summed intensity of reflections detected within the diffraction pattern as dose increases (Garman, 2010; Murray &amp; Garman, 2002). Dose is defined as the absorbed energy per unit mass of crystal in grays (Gy; 1 Gy = 1 J kg−1), and is the metric against which damage progression should be monitored during MX data collection, as opposed to time. At 100 K, an experimental dose limit of 30 MGy has been recommended as an upper limit beyond which the biological information derived from any macromolecular crystal may be compromised (Owen et al., 2006).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3792</offset><text> Specific radiation damage (SRD) is observed in the real-space electron density, and has been detected at much lower doses than any observable decay in the intensity of reflections. Indeed, the C—Se bond in selenomethionine, the stability of which is key for the success of experimental phasing methods, can be cleaved at a dose as low as 2 MGy for a crystal maintained at 100 K (Holton, 2007). SRD has been well characterized in a large range of proteins, and is seen to follow a reproducible order: metallo-centre reduction, disulfide-bond cleavage, acidic residue decarboxylation and methionine methylthio cleavage (Ravelli &amp; McSweeney, 2000; Burmeister, 2000; Weik et al., 2000; Yano et al., 2005). Furthermore, damage susceptibility within each residue type follows a preferential ordering influenced by a combination of local environment factors (solvent accessibility, conformational strain, proximity to active sites/high X-ray cross-section atoms; Holton, 2009). Deconvoluting the individual roles of these parameters has been surprisingly challenging, with factors such as solvent accessibility currently under active investigation (Weik et al., 2000; Fioravanti et al., 2007; Gerstel et al., 2015).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5008</offset><text>There are a number of cases where SRD manifestations have compromised the biological information extracted from MX-determined structures at much lower doses than the recommended 30 MGy limit, leading to false structural interpretations of protein mechanisms. Active-site residues appear to be particularly susceptible, particularly for photosensitive proteins and in instances where chemical strain is an intrinsic feature of the reaction mechanism. For instance, structure determination of the purple membrane protein bacterio­rhodopsin required careful corrections for radiation-induced structural changes before the correct photosensitive intermediate states could be isolated (Matsui et al., 2002). The significant chemical strain required for catalysis within the active site of phosphoserine aminotransferase has been observed to diminish during X-ray exposure (Dubnovitsky et al., 2005).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5906</offset><text>Since the majority of SRD studies to date have focused on proteins, much less is known about the effects of X-ray irradiation on the wider class of crystalline nucleoprotein complexes or how to correct for such radiation-induced structural changes. Understanding RD to such complexes is crucial, since DNA is rarely naked within a cell, instead dynamically interacting with proteins, facilitating replication, transcription, modification and DNA repair. As of early 2016, &gt;5400 nucleoprotein complex structures have been deposited within the PDB, with 91% solved by MX. It is essential to understand how these increasingly complex macromolecular structures are affected by the radiation used to solve them. Nucleoproteins also represent one of the main targets of radiotherapy, and an insight into the damage mechanisms induced by X-ray irradiation could inform innovative treatments.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6791</offset><text>When a typical macromolecular crystal is irradiated with ionizing X-rays, each photoelectron produced via interactions with both the macromolecule (direct damage) and solvent (indirect damage) can induce cascades of up to 500 secondary low-energy electrons (LEEs) that are capable of inducing further ionizations. Investigations on sub-ionization-level LEEs (0–15 eV) interacting with both dried and aqueous oligonucleotides (Alizadeh &amp; Sanche, 2014; Simons, 2006) concluded that resonant electron attachment to DNA bases and the sugar-phosphate backbone could lead to the preferential cleavage of strong (∼4 eV, 385 kJ mol−1) sugar-phosphate C—O covalent bonds within the DNA backbone and then base-sugar N1—C bonds, eventually leading to single-strand breakages (SSBs; Ptasińska &amp; Sanche, 2007). Electrons have been shown to be mobile at 77 K by electron spin resonance spectroscopy studies (Symons, 1997; Jones et al., 1987), with rapid electron quantum tunnelling and positive hole migration along the protein backbone and through stacked DNA bases indicated as a dominant mechanism by which oxidative and reductive damage localizes at distances from initial ionization sites at 100 K (O’Neill et al., 2002).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>8029</offset><text>The investigation of naturally forming nucleoprotein complexes circumvents the inherent challenges in making controlled comparisons of damage mechanisms between protein and nucleic acids crystallized separately. Recently, for a well characterized bacterial protein–DNA complex (C.Esp1396I; PDB entry 3clc; resolution 2.8 Å; McGeehan et al., 2008) it was concluded that over a wide dose range (2.1–44.6 MGy) the protein was far more susceptible to SRD than the DNA within the crystal (Bury et al., 2015). Only at doses above 20 MGy were precursors of phosphodiester-bond cleavage observed within AT-rich regions of the 35-mer DNA.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>8670</offset><text>For crystalline complexes such as C.Esp1396I, whether the protein is intrinsically more susceptible to X-ray-induced damage or whether the protein scavenges electrons to protect the DNA remains unclear in the absence of a non-nucleic acid-bound protein control obtained under exactly the same crystallization and data-collection conditions. To monitor the effects of nucleic acid binding on protein damage susceptibility, a crystal containing two protein molecules per asymmetric unit, only one of which was bound to RNA, is reported here (Fig. 1 ▸). Using newly developed methodology, we present a controlled SRD investigation at 1.98 Å resolution using a large (∼91 kDa) crystalline protein–RNA complex: trp RNA-binding attenuation protein (TRAP) bound to a 53 bp RNA sequence (GAGUU)10GAG (PDB entry 1gtf; Hopcroft et al., 2002). TRAP consists of 11 identical subunits assembled into a ring with 11-fold rotational symmetry. It binds with high affinity (K d ≃ 1.0 nM) to RNA segments containing 11 GAG/UAG triplets separated by two or three spacer nucleotides (Elliott et al., 2001) to regulate the transcription of tryptophan biosynthetic genes in Bacillus subtilis (Antson et al., 1999). In this structure, the bases of the G1-A2-G3 nucleotides form direct hydrogen bonds to the protein, unlike the U4-U5 nucleotides, which appear to be more flexible.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>10044</offset><text>Ten successive 1.98 Å resolution MX data sets were collected from the same TRAP–RNA crystal to analyse X-ray-induced structural changes over a large dose range (d 1 = 1.3 MGy to d 10 = 25.0 MGy). To avoid the previous necessity for visual inspection of electron-density maps to detect SRD sites, a computational approach was designed to quantify the electron-density change for each refined atom with increasing dose, thus providing a rapid systematic method for SRD study on such large multimeric complexes. By employing the high 11-fold structural symmetry within each TRAP macromolecule, this approach permitted a thorough statistical quantification of the RD effects of RNA binding to TRAP.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>10748</offset><text>Materials and methods   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>10774</offset><text>RNA synthesis and protein preparation   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>10816</offset><text>As previously described (Hopcroft et al., 2002), the 53-base RNA (GAGUU)10GAG was synthesized by in vitro transcription with T7 RNA polymerase and gel-purified. TRAP from B. stearothermophilus was overexpressed in Escherichia coli and purified.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>11061</offset><text>Crystallization   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>11081</offset><text>TRAP–RNA crystals were prepared using a previously established hanging-drop crystallization protocol (Antson et al., 1999). By using a 2:1 molar ratio of TRAP to RNA, crystals successfully formed from the protein–RNA complex (∼15 mg ml−1) in a solution containing 70 mM potassium phosphate pH 7.8 and 10 mM l-tryptophan. The reservoir consisted of 0.2 M potassium glutamate, 50 mM triethanol­amine pH 8.0, 10 mM MgCl2, 8–11% monomethyl ether PEG 2000. In order to accelerate crystallization, a further gradient was induced by adding 0.4 M KCl to the reservoir after 1.5 µl protein solution had been mixed with an equal volume of the reservoir solution. Wedge-shaped crystals of approximate length 70 µm (longest dimension) grew within 3 d and were vitrified and stored in liquid nitrogen immediately after growth. The cryosolution consisted of 12% monomethyl ether PEG 2000, 30 mM triethanolamine pH 8.0, 6 mM l-tryptophan, 0.1 M potassium glutamate, 35 mM potassium phosphate pH 7.8, 5 mM MgCl2 with 25% 2-methyl-2,4-pentanediol (MPD) included as a cryoprotectant.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>12192</offset><text>X-ray data collection   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>12218</offset><text>Data were collected at 100 K from a wedge-shaped TRAP–RNA crystal of approximate dimensions 70 × 20 × 40 µm (see Supplementary Fig. S2) on beamline ID14-4 at the ESRF using an incident wavelength of 0.940 Å (13.2 keV) and an ADSC Q315R mosaic CCD detector at 304.5 mm from the crystal throughout the data collection. The beam size was slitted to 0.100 mm (vertical) × 0.160 mm (horizontal), with a uniformly distributed profile, such that the crystal was completely bathed within the beam throughout data collection. Ten successive (1.98 Å resolution) 180° data sets (with Δφ = 1°) were collected over the same angular range from a TRAP–RNA crystal at 28.9% beam transmission. The TRAP–RNA macromolecule crystallized in space group C2, with unit-cell parameters a = 140.9, b = 110.9, c = 137.8 Å, α = γ = 90, β = 137.8° (the values quoted are for the first data set; see Supplementary Table S1 for subsequent values). For the first nine data sets the attenuated flux was recorded to be ∼5 × 1011 photons s−1. A beam refill took place immediately before data set 10, requiring a flux-scale factor increase of 1.42 to be applied, based on the ratio of observed relative intensity I D/I 1 at data set 10 to that extrapolated from data set 9.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>13507</offset><text>Dose calculation   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>13528</offset><text> RADDOSE-3D (Zeldin, Gerstel et al., 2013) was used to calculate the absorbed dose distribution during each data set (see input file; Supplementary Figs. S1 and S2). The crystal composition was calculated from the deposited TRAP–RNA structure (PDB entry 1gtf; Hopcroft et al., 2002). Crystal absorption coefficients were calculated in RADDOSE-3D using the concentration (mmol l−1) of solvent heavy elements from the crystallization conditions. The beam-intensity profile was modelled as a uniform (‘top-hat’) distribution. The diffraction-weighted dose (DWD) values (Zeldin, Brock­hauser et al., 2013) are given in Supplementary Table S1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>14178</offset><text>Data processing and model refinement   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>14219</offset><text>Each data set was integrated using iMosflm (Leslie &amp; Powell, 2007) and was scaled using AIMLESS (Evans &amp; Murshudov, 2013; Winn et al., 2011) using the same 5% R free set of test reflections for each data set. To phase the structure obtained from the first data set, molecular replacement was carried out with Phaser (McCoy et al., 2007), using an identical TRAP–RNA structure (PDB entry 1gtf; resolution 1.75 Å; Hopcroft et al., 2002) as a search model. The resulting TRAP–RNA structure (TR1) was refined using REFMAC5 (Murshudov et al., 2011), initially using rigid-body refinement, followed by repeated cycles of restrained, TLS and isotropic B-factor refinement, coupled with visual inspection in Coot (Emsley et al., 2010). TR1 was refined to 1.98 Å resolution, with a dimeric assembly of non-RNA-bound and RNA-bound TRAP rings within the asymmetric unit. Consistent with previous structures of the TRAP–RNA complex, the RNA sequence termini were not observed within the 2F o − F c map; the first spacer (U4) was then modelled at all 11 repeats around the TRAP ring and the second spacer (U5) was omitted from the final refined structure. For the later data sets, the observed structure-factor amplitudes from each separately scaled data set (output from AIMLESS) were combined with the phases of TR1 and the resulting higher-dose model was refined with phenix.refine (Adams et al., 2010) using only rigid-body and isotropic B-factor refinement. During this refinement, the TRAP–RNA complex and nonbound TRAP ring were treated as two separate rigid bodies within the asymmetric unit. Supplementary Table S1 shows the relevant summary statistics.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>15885</offset><text> D loss metric calculation   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>15916</offset><text>The CCP4 program CAD was used to create a series of nine merged .mtz files combining observed structure-factor amplitudes for the first data set F obs(d 1) with each later data set F obs(d n) (for n = 2, …, 10). All later data sets were scaled against the initial low-dose data set in SCALEIT. For each data set an atom-tagged .map file was generated using the ATMMAP mode in SFALL (Winn et al., 2011). A full set of nine Fourier difference maps F obs(d n) − F obs(d 1) were calculated using FFT (Ten Eyck, 1973) over the full TRAP–RNA unit-cell dimensions, with the same grid-sampling dimensions as the atom-tagged .map file. All maps were cropped to the TRAP asymmetric unit in MAPMASK. Comparing the atom-tagged .map file and F obs(d n) − F obs(d 1) difference map at each dose, each refined atom was assigned a set of density-change values X. The maximum density-loss metric, D loss (units of e Å−3), was calculated to quantify the per-atom electron-density decay at each dose, assigned as the absolute magnitude of the most negative Fourier difference map voxel value in a local volume around each atom as defined by the set X.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>17062</offset><text>Model system calculation   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>17091</offset><text>Model calculations were run for the simple amino acids glutamate and aspartate. In order to avoid decarboxylation at the C-terminus instead of the side chain on the Cα atom, the C-terminus of each amino acid was methylated. While the structures of the closed shell acids are well known, the same is not true of those in the oxidized state. The quantum-chemical calculations employed were chosen to provide a satisfactory description of the structure of such radical species and also provide a reliable estimation of the relative C—C(O2) bond strengths, which are otherwise not available.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>17683</offset><text>Structures of methyl-terminated (at the N- and C-termini) carboxylates were determined using analytic energy gradients with density functional theory (B3LYP functional; Becke, 1993) and a flexible basis set of polarized valence triple-zeta size with diffuse functions on the non-H atoms [6-311+G(d,p)] in the Gaussian 09 computational chemistry package (Frisch et al., 2009). The stationary points obtained were characterized as at least local minima by examination of the associated analytic Hessian. Effects of the medium were modelled using a dielectric cavity approach (Tomasi et al., 1999) parameterized for water.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>18303</offset><text>Results   </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>18315</offset><text>Per-atom quantification of electron density   </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18363</offset><text>To quantify the exact effects of nucleic acid binding to a protein on SRD susceptibility, a high-throughput and automated pipeline was created to systematically calculate the electron-density change for every refined atom within the TRAP–RNA structure as a function of dose. This provides an atom-specific quantification of density–dose dynamics, which was previously lacking within the field. Previous studies have characterized SRD sites by reporting magnitudes of F obs(d n) − F obs(d 1) Fourier difference map peaks in terms of the sigma (σ) contour level (the number of standard deviations from the mean map electron-density value) at which peaks become visible. However, these σ levels depend on the standard deviation values of the map, which can deviate between data sets, and are thus unsuitable for quantitative comparison of density between different dose data sets. Instead, we use here a maximum density-loss metric (D loss), which is the per-atom equivalent of the magnitude of these negative Fourier difference map peaks in units of e Å−3. Large positive D loss values indicate radiation-induced atomic disordering reproducibly throughout the unit cells with respect to the initial low-dose data set.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19592</offset><text>For each TRAP–RNA data set, the D loss metric successfully identified the recognized forms of protein SRD (Fig. 2 ▸ a), with clear Glu and Asp side-chain decarboxylation even in the first difference map calculated (3.9 MGy; Fig. 3 ▸ a). The main sequence of TRAP does not contain any Trp and Cys residues (and thus contains no disulfide bonds). The substrate Trp amino-acid ligands also exhibited disordering of the free terminal carboxyl groups at higher doses (Fig. 2 ▸ a); however, no clear Fourier difference peaks could be observed visually. Even for radiation-insensitive residues (e.g. Gly) the average D loss increases with dose: this is the effect of global radiation damage, since as dose increases the electron density associated with each refined atom becomes weaker as the atomic occupancy decreases (Fig. 2 ▸ b). Only Glu and Asp residues exhibit a rate of D loss increase that consistently exceeds the average decay (Fig. 2 ▸ b, dashed line) at each dose. Additionally, the density surrounding ordered solvent molecules was determined to significantly diminish with increasing dose (Fig. 2 ▸ b). The rate of D loss (attributed to side-chain decarboxylation) was consistently larger for Glu compared with Asp residues over the large dose range (Fig. 2 ▸ b and Supplementary Fig. S3); this observation is consistent with our calculations on model systems (see above) that suggest that, without considering differential hydrogen-bonding environments, CO2 loss is more exothermic by around 8 kJ mol−1 from oxidized Glu residues than from their Asp counterparts.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>21189</offset><text>RNA is less susceptible to electron-density loss than protein within the TRAP–RNA complex   </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21285</offset><text>Visual inspection of Fourier difference maps illustrated the clear lack of RNA electron-density degradation with increasing dose compared with the obvious protein damage manifestations (Figs. 3 ▸ b and 3 ▸ c). Only at the highest doses investigated (&gt;20 MGy) was density loss observed at the RNA phosphate and C—O bonds of the phosphodiester backbone. However, the median D loss was lower by a factor of &gt;2 for RNA P atoms than for Glu and Asp side-chain groups at 25.0 MGy (Supplementary Fig. S4), and furthermore could not be numerically distinguished from Gly Cα atoms within TRAP, which are not radiation-sensitive at the doses tested here (Supplementary Fig. S3).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>21965</offset><text>RNA binding protects radiation-sensitive residues   </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22019</offset><text>For the large number of acidic residues per TRAP ring (four Asp and six Glu residues per protein monomer), a strong dependence of decarboxylation susceptibility on local environment was observed (Fig. 4 ▸). For each Glu Cδ or Asp Cγ atom, D loss provided a direct measure of the rate of side-chain carboxyl-group disordering and subsequent decarboxylation. For acidic residues with no differing interactions between nonbound and bound TRAP (Fig. 4 ▸ a), similar damage was apparent between the two rings within the asymmetric unit, as expected. However, TRAP residues directly on the RNA-binding interfaces exhibited greater damage accumulation in nonbound TRAP (Fig. 4 ▸ b), and for residues at the ring–ring interfaces (where crystal contacts were detected) bound TRAP exhibited enhanced SRD accumulation (Fig. 4 ▸ c).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22856</offset><text>Three acidic residues (Glu36, Asp39 and Glu42) are involved in RNA interactions within each of the 11 TRAP ring subunits, and Fig. 5 ▸ shows their density changes with increasing dose. Hotelling’s T-squared test (the multivariate counterpart of Student’s t-test) was used to reject the null hypothesis that the means of the D loss metric were equal for the bound and nonbound groups in Fig. 5 ▸.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23260</offset><text>A significant reduction in D loss is seen for Glu36 in RNA-bound compared with nonbound TRAP, indicative of a lower rate of side-chain decarboxylation (Fig. 5 ▸ a; p = 6.06 × 10−5). For each TRAP ring subunit, the Glu36 side-chain carboxyl group accepts a pair of hydrogen bonds from the two N atoms of the G3 RNA base. In our analysis, Asp39 in the TRAP–(GAGUU)10GAG structure appears to exhibit two distinct hydrogen bonds to the G1 base within each of the 11 TRAP–RNA interfaces, as does Glu36 to G3; however, the reduction in density disordering upon RNA binding is far less significant for Asp39 than for Glu36 (Fig. 5 ▸ b, p = 0.0925).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>23913</offset><text>RNA binding reduces radiation-induced disorder on the atomic scale   </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23984</offset><text>One oxygen (O∊1) of Glu42 appears to form a hydrogen bond to a nearby water within each TRAP RNA-binding pocket, with the other (O∊2) being involved in a salt-bridge interaction with Arg58 (Hopcroft et al., 2002; Antson et al., 1999). Salt-bridge interactions have previously been suggested to reduce the glutamate decarboxylation rate within the large (∼62.4 kDa) myrosinase protein structure (Burmeister, 2000). A significant difference was observed between the D loss dynamics for the nonbound/bound Glu42 O∊1 atoms (Fig. 5 ▸ c; p = 0.007) but not for the Glu42 O∊2 atoms (Fig. 5 ▸ d; p = 0.239), indicating that the stabilizing strength of this salt-bridge interaction was conserved upon RNA binding and that the water-mediated hydrogen bond had a greater relative susceptibility to atomic disordering in the absence of RNA. The density-change dynamics were statistically indistinguishable between bound and nonbound TRAP for each Glu42 carboxyl group Cδ atom (p = 0.435), indicating that upon RNA binding the conserved salt-bridge interaction ultimately dictated the overall Glu42 decarboxylation rate.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>25115</offset><text>The RNA-stabilizing effect was not restricted to radiation-sensitive acidic residues. The side chain of Phe32 stacks against the G3 base within the 11 TRAP RNA-binding interfaces (Antson et al., 1999). With increasing dose, the D loss associated with the Phe32 side chain was significantly reduced upon RNA binding (Fig. 5 ▸ e; Phe32 Cζ; p = 0.0014), an indication that radiation-induced conformation disordering of Phe32 had been reduced. The extended aliphatic Lys37 side chain stacks against the nearby G1 base, making a series of nonpolar contacts within each RNA-binding interface. The D loss for Lys37 side-chain atoms was also reduced when stacked against the G1 base (Fig. 5 ▸ f; p = 0.0243 for Lys37 C∊ atoms). Representative Phe32 and Lys37 atoms were selected to illustrate these trends.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>25925</offset><text>Discussion   </text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>25940</offset><text>Here, MX radiation-induced specific structural changes within the large TRAP–RNA assembly over a large dose range (1.3–25.0 MGy) have been analysed using a high-throughput quantitative approach, providing a measure of the electron-density distribution for each refined atom with increasing dose, D loss. Compared with previous studies, the results provide a further step in the detailed characterization of SRD effects in MX. Our method­ology, which eliminated tedious and error-prone visual inspection, permitted the determination on a per-atom basis of the most damaged sites, as characterized by F obs(d n) − F obs(d 1) Fourier difference map peaks between successive data sets collected from the same crystal. Here, it provided the precision required to quantify the role of RNA in the damage susceptibilities of equivalent atoms between RNA-bound and nonbound TRAP, but it is applicable to any MX SRD study.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>26862</offset><text>The RNA was found to be substantially more radiation-resistant than the protein, even at the highest doses investigated (∼25.0 MGy), which is in strong concurrence with our previous SRD investigation of the C.Esp1396I protein–DNA complex (Bury et al., 2015). Consistent with that study, at high doses of above ∼20 MGy, F obs(d n) − F obs(d 1) map density was detected around P, O3′ and O5′ atoms of the RNA backbone, with no significant difference density localized to RNA ribose and basic subunits. RNA backbone disordering thus appears to be the main radiation-induced effect in RNA, with the protein–base interactions maintained even at high doses (&gt;20 MGy). The U4 phosphate exhibited marginally larger D loss values above 20 MGy than G1, A2 and G3 (Supplementary Fig. S4). Since U4 is the only refined nucleotide not to exhibit significant base–protein interactions around TRAP (with a water-mediated hydrogen bond detected in only three of the 11 subunits and a single Arg58 hydrogen bond suggested in a further four subunits), this increased U4 D loss can be explained owing to its greater flexibility. At 25.0 MGy, the magnitude of the RNA backbone D loss was of the same order as for the radiation-insensitive Gly Cα atoms and on average less than half that of the acidic residues of the protein (Supplementary Fig. S3). Consequently, no clear single-strand breaks could be located, and since RNA-binding within the current TRAP–(GAGUU)10GAG complex is mediated predominantly through base–protein interactions, the biological integrity of the RNA complex was dictated by the rate at which protein decarboxylation occurred.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>28527</offset><text>RNA interacting with TRAP was shown to offer significant protection against radiation-induced structural changes. Both Glu36 and Asp39 bind directly to RNA, each through two hydrogen bonds to guanine bases (G3 and G1, respectively). However, compared with Asp39, Glu36 is strikingly less decarboxylated when bound to RNA (Fig. 4 ▸). This is in good agreement with previous mutagenesis and nucleoside analogue studies (Elliott et al., 2001), which indicated that the G1 nucleotide does not bind to TRAP as strongly as do A2 and G3, and plays little role in the high RNA-binding affinity of TRAP (K d ≃ 1.1 ± 0.4 nM). For Glu36 and Asp39, no direct quantitative correlation could be established between hydrogen-bond length and D loss (linear R 2 of &lt;0.23 for all doses; Supplementary Fig. S5). Thus, another factor must be responsible for this clear reduction in Glu36 CO2 decarboxyl­ation in RNA-bound TRAP. The Glu36 carboxyl side chain also potentially forms hydrogen bonds to His34 and Lys56, but since these interactions are conserved irrespective of G3 nucleotide binding, this cannot directly account for the stabilization effect on Glu36 in RNA-bound TRAP. Radiation-induced decarboxylation has been proposed to be mediated by preferential positive-hole migration to the side-chain carboxyl group, with rapid proton transfer trapping the hole at the carboxyl group (Burmeister, 2000; Symons, 1997):where the forward rate is K 1 and the backward rate is K −1, where the forward rate is K 2.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>30034</offset><text>When bound to RNA, the average solvent-accessible area of the Glu36 side-chain O atoms is reduced from ∼15 to 0 Å2. We propose that with no solvent accessibility Glu36 decarboxylation is inhibited, since the CO2-formation rate K 2 is greatly reduced, and suggest that steric hindrance prevents each radicalized Glu36 CO2 group from achieving the planar conformation required for complete dissociation from TRAP. The electron-recombination rate K −1 remains high, however, owing to rapid electron migration through the protein–RNA complex to refill the Glu36 positive hole (the precursor for Glu decarboxylation). Upon RNA binding, the Asp39 side-chain carboxyl group solvent-accessible area changes from ∼75 to 35 Å2, still allowing a high CO2-formation rate K 2.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>30812</offset><text>Previous studies have reported inconsistent results concerning the dependence of the acidic residue decarboxylation rate on solvent accessibility (Weik et al., 2000; Fioravanti et al., 2007; Gerstel et al., 2015). The prevalence of radical attack from solvent channels surrounding the protein in the crystal is a questionable cause, considering previous observations indicating that the strongly oxidizing hydroxyl radical is immobile at 100 K (Allan et al., 2013; Owen et al., 2012). Furthermore, the suggested electron hole-trapping mechanism which induces decarboxylation within proteins at 100 K has no clear mechanistic dependence on the solvent-accessible area of each carboxyl group. By comparing equivalent acidic residues with and without RNA, we have now deconvoluted the role of solvent accessibility from other local protein environment factors, and thus propose a suitable mechanism by which exceptionally low solvent accessibility can reduce the rate of decarboxylation. Overall, no direct correlation between solvent accessibility and decarboxylation susceptibility was observed, but it is very clear that inaccessible residues are protected.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>31974</offset><text>Apart from these RNA-binding interfaces, RNA binding was seen to enhance decarboxylation for residues Glu50, Glu71 and Glu73, all of which are involved in crystal contacts between TRAP rings (Fig. 4 ▸ c). However, for each of these residues the exact crystal contacts are not preserved between bound and nonbound TRAP or even between monomers within one TRAP ring. For example, in bound TRAP, Glu73 hydrogen-bonds to a nearby lysine on each of the 11 subunits, whereas in nonbound TRAP no such interaction exists and Glu73 interacts with a variable number of refined waters in each subunit. Thus, the dependence of decarboxylation rates on these interactions could not be established.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>32661</offset><text>Radiation-induced side-chain conformational changes have been poorly characterized in previous SRD investigations owing to their strong dependence on packing density and geometric strain. Such structural changes are known to have significant roles within enzymatic pathways, and experimenters must be aware of these possible confounding factors when assigning true functional mechanisms using MX. Our results show that RNA binding to TRAP physically stabilizes non-acidic residues within the TRAP macromolecule, most notably Lys37 and Phe32, which stack against the G1 and G3 bases, respectively. It has been suggested (Burmeister, 2000) that Tyr residues can lose their aromatic –OH group owing to radiation-induced effects; however, no energetically favourable pathway for –OH cleavage exists and this has not been detected in aqueous radiation-chemistry studies. In TRAP, D loss increased at a similar rate for both the Tyr O atoms and aromatic ring atoms, suggesting that full ring conformational disordering is more likely. Indeed, no convincing reproducible Fourier difference peaks above the background map noise were observed around any Tyr terminal –OH groups.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>33837</offset><text>The RNA-stabilization effects on protein are observed at short ranges and are restricted to within the RNA-binding interfaces around the TRAP ring. For example, Asp17 is located ∼6.8 Å from the G1 base, outside the RNA-binding interfaces, and has indistinguishable Cγ atom D loss dose-dynamics between RNA-bound and nonbound TRAP (p &gt; 0.9). An increase in the dose at which functionally important residues remain intact has biological ramifications for understanding the mechanisms at which ionizing radiation damage is mitigated within naturally forming DNA–protein and RNA–protein complexes. Observations of lower protein radiation-sensitivity in DNA-bound forms have been recorded in solution at RT at much lower doses (∼1 kGy) than those used for typical MX experiments [e.g. an oestrogen response element–receptor complex (Stísová et al., 2006) and a DNA glycosylase and its abasic DNA target site (Gillard et al., 2004)]. In these studies, the main damaging species is predicted to be the oxidizing hydroxyl radical produced through solvent irradiation, which is known to add to double covalent bonds within both DNA and RNA bases to induce strand breaks and base modification (Spotheim-Maurizot &amp; Davídková, 2011; Chance et al., 1997). It was suggested that physical screening of DNA by protein shielded the DNA–protein interaction sites from radical damage, yielding an extended life-dose for the nucleoprotein complex compared with separate protein and DNA constituents at RT.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>35345</offset><text>However, in the current MX study at 100 K, the main damaging species are believed to be migrating LEEs and holes produced directly within the protein–RNA components or in closely associated solvent. The results presented here suggest that biologically relevant nucleoprotein complexes also exhibit prolonged life-doses under the effect of LEE-induced structural changes, involving direct physical protection of key RNA-binding residues. Such reduced radiation-sensitivity in this case ensures that the interacting protein remains bound long enough to the RNA to complete its function, even whilst exposed to ionizing radiation. Within the nonbound TRAP macromolecule, the acidic residues within the unoccupied RNA-binding interfaces (Asp39, Glu36, Glu42) are notably amongst the most susceptible residues within the asymmetric unit (Fig. 4 ▸). When exposed to X-rays, these residues will be preferentially damaged by X-rays and subsequently reduce the affinity with which TRAP binds to RNA. Within the cellular environment, this mechanism could reduce the risk that radiation-damaged proteins might bind to RNA, thus avoiding the detrimental introduction of incorrect DNA-repair, transcriptional and base-modification pathways.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>36579</offset><text>The Python scripts written to calculate the per atom D loss metric are available from the authors on request.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>36689</offset><text>Related literature   </text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>36712</offset><text>The following references are cited in the Supporting Information for this article: Chen et al. (2010).</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>36815</offset><text>Supplementary Material</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>36838</offset><text>References</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>36849</offset><text>Adams, P. D. et al. (2010). Acta Cryst. D66, 213–221.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>36905</offset><text>Alizadeh, E. &amp; Sanche, L. (2014). Eur. Phys. J. D, 68, 97.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>36964</offset><text>Allan, E. G., Kander, M. C., Carmichael, I. &amp; Garman, E. F. (2013). J. Synchrotron Rad. 20, 23–36.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>37065</offset><text>Antson, A. A., Dodson, E. J., Dodson, G., Greaves, R. B., Chen, X. &amp; Gollnick, P. (1999). Nature (London), 401, 235–242.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>37188</offset><text>Becke, A. D. (1993). J. Chem. Phys. 98, 5648–5652.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>37241</offset><text>Bourenkov, G. P. &amp; Popov, A. N. (2010). Acta Cryst. D66, 409–419.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>37309</offset><text>Burmeister, W. P. (2000). Acta Cryst. D56, 328–341.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>37363</offset><text>Bury, C., Garman, E. F., Ginn, H. M., Ravelli, R. B. G., Carmichael, I., Kneale, G. &amp; McGeehan, J. E. (2015). J. Synchrotron Rad. 22, 213–224.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>37508</offset><text>Chance, M. R., Sclavi, B., Woodson, S. A. &amp; Brenowitz, M. (1997). Structure, 5, 865–869.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>37599</offset><text>Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S. &amp; Richardson, D. C. (2010). Acta Cryst. D66, 12–21.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>37777</offset><text>Dubnovitsky, A. P., Ravelli, R. B. G., Popov, A. N. &amp; Papageorgiou, A. C. (2005). Protein Sci. 14, 1498–1507.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>37889</offset><text>Elliott, M. B., Gottlieb, P. A. &amp; Gollnick, P. (2001). RNA, 7, 85–93.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>37961</offset><text>Emsley, P., Lohkamp, B., Scott, W. G. &amp; Cowtan, K. (2010). Acta Cryst. D66, 486–501.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>38048</offset><text>Evans, P. R. &amp; Murshudov, G. N. (2013). Acta Cryst. D69, 1204–1214.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>38118</offset><text>Fioravanti, E., Vellieux, F. M. D., Amara, P., Madern, D. &amp; Weik, M. (2007). J. Synchrotron Rad. 14, 84–91.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>38228</offset><text>Frisch, M. J. et al. (2009). Gaussian 09. Gaussian Inc., Wallingford, Connecticut, USA.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>38316</offset><text>Garman, E. F. (2010). Acta Cryst. D66, 339–351.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>38366</offset><text>Gerstel, M., Deane, C. M. &amp; Garman, E. F. (2015). J. Synchrotron Rad. 22, 201–212.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>38451</offset><text>Gillard, N., Begusova, M., Castaing, B. &amp; Spotheim-Maurizot, M. (2004). Radiat. Res. 162, 566–571.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>38552</offset><text>Holton, J. M. (2007). J. Synchrotron Rad. 14, 51–72.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>38607</offset><text>Holton, J. M. (2009). J. Synchrotron Rad. 16, 133–142.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>38664</offset><text>Hopcroft, N. H., Wendt, A. L., Gollnick, P. &amp; Antson, A. A. (2002). Acta Cryst. D58, 615–621.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>38760</offset><text>Jones, G. D., Lea, J. S., Symons, M. C. &amp; Taiwo, F. A. (1987). Nature (London), 330, 772–773.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>38856</offset><text>Leslie, A. G. W. &amp; Powell, H. R. (2007). Evolving Methods for Macromolecular Crystallography, edited by R. J. Read &amp; J. L. Sussman, pp. 41–51. Dordrecht: Springer.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>39022</offset><text>Liebschner, D., Rosenbaum, G., Dauter, M. &amp; Dauter, Z. (2015). Acta Cryst. D71, 772–778.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>39113</offset><text>Matsui, Y., Sakai, K., Murakami, M., Shiro, Y., Adachi, S., Okumura, H. &amp; Kouyama, T. (2002). J. Mol. Biol. 324, 469–481.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>39237</offset><text>McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. &amp; Read, R. J. (2007). J. Appl. Cryst. 40, 658–674.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>39371</offset><text>McGeehan, J. E., Streeter, S. D., Thresh, S. J., Ball, N., Ravelli, R. B. G. &amp; Kneale, G. G. (2008). Nucleic Acids Res. 36, 4778–4787.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>39508</offset><text>Murray, J. &amp; Garman, E. (2002). J. Synchrotron Rad. 9, 347–354.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>39574</offset><text>Murshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F. &amp; Vagin, A. A. (2011). Acta Cryst. D67, 355–367.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>39741</offset><text>O’Neill, P., Stevens, D. L. &amp; Garman, E. (2002). J. Synchrotron Rad. 9, 329–332.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>39826</offset><text>Owen, R. L., Axford, D., Nettleship, J. E., Owens, R. J., Robinson, J. I., Morgan, A. W., Doré, A. S., Lebon, G., Tate, C. G., Fry, E. E., Ren, J., Stuart, D. I. &amp; Evans, G. (2012). Acta Cryst. D68, 810–818.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>40037</offset><text>Owen, R. L., Rudiño-Piñera, E. &amp; Garman, E. F. (2006). Proc. Natl Acad. Sci. USA, 103, 4912–4917.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>40139</offset><text>Ptasińska, S. &amp; Sanche, L. (2007). Phys. Rev. E, 75, 031915.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>40201</offset><text>Ravelli, R. B. G. &amp; McSweeney, S. M. (2000). Structure, 8, 315–328.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>40271</offset><text>Shimizu, N., Hirata, K., Hasegawa, K., Ueno, G. &amp; Yamamoto, M. (2007). J. Synchrotron Rad. 14, 4–10.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>40374</offset><text>Simons, J. (2006). Acc. Chem. Res. 39, 772–779.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>40424</offset><text>Spotheim-Maurizot, M. &amp; Davídková, M. (2011). Mutat. Res. 711, 41–48.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>40498</offset><text>Stísová, V., Goffinont, S., Spotheim-Maurizot, M. &amp; Davídková, M. (2006). Radiat. Prot. Dosimetry, 122, 106–109.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>40617</offset><text>Symons, M. C. R. (1997). Free Radical Biol. Med. 22, 1271–1276.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>40683</offset><text>Ten Eyck, L. F. (1973). Acta Cryst. A29, 183–191.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>40735</offset><text>Tomasi, J., Mennucci, B. &amp; Cancès, E. (1999). J. Mol. Struct. 464, 211–226.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>40814</offset><text>Weik, M., Ravelli, R. B. G., Kryger, G., McSweeney, S., Raves, M. L., Harel, M., Gros, P., Silman, I., Kroon, J. &amp; Sussman, J. L. (2000). Proc. Natl Acad. Sci. USA, 97, 623–628.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>40994</offset><text>Winn, M. D. et al. (2011). Acta Cryst. D67, 235–242.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>41049</offset><text>Yano, J., Kern, J., Irrgang, K. D., Latimer, M. J., Bergmann, U., Glatzel, P., Pushkar, Y., Biesiadka, J., Loll, B., Sauer, K., Messinger, J., Zouni, A. &amp; Yachandra, V. K. (2005). Proc. Natl Acad. Sci. USA, 102, 12047–12052.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>41276</offset><text>Zeldin, O. B., Brockhauser, S., Bremridge, J., Holton, J. M. &amp; Garman, E. F. (2013). Proc. Natl Acad. Sci. USA, 110, 20551–20556.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>41408</offset><text>Zeldin, O. B., Gerstel, M. &amp; Garman, E. F. (2013). J. Appl. Cryst. 46, 1225–1230.</text></passage><passage><infon key="file">d-72-00648-fig1.jpg</infon><infon key="id">fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>41492</offset><text>The TRAP–(GAGUU)10GAG complex asymmetric unit (PDB entry 1gtf; Hopcroft et al., 2002). Bound tryptophan ligands are represented as coloured spheres. RNA is shown is yellow.</text></passage><passage><infon key="file">d-72-00648-fig2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>41667</offset><text>(a) Electron-density loss sites as indicated by D
+loss in the TRAP–RNA complex crystal by residue/nucleotide type for five doses [sites determined above the 4× average D
+loss threshold, calculated over the TRAP–RNA structure for the first difference map: F
+obs(d
+2) − F
+obs(d
+1)]. Cumulative frequencies are normalized to both the total number of non-H atoms per residue/nucleotide and the total number of that residue/nucleotide type present. (b) Average D
+loss for each residue/nucleotide type with respect to the DWD (diffraction-weighted dose; Zeldin, Brock­hauser et al., 2013). 95% confidence intervals (CI) are shown. Only a subset of key TRAP residue types are included. The average D
+loss (calculated over the whole TRAP asymmetric unit) is shown at each dose (dashed line).</text></passage><passage><infon key="file">d-72-00648-fig3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>42459</offset><text>
+F
+obs(d
+n) − F
+obs(d
+1) Fourier difference maps for (a) n = 2 (3.9 MGy), (b) n = 3 (6.5 MGy) and (c) n = 7 (16.7 MGy) contoured at ±4σ (a) and ±3.5σ (b, c). In (a) clear difference density is observed around the Glu42 carboxyl side chain in chain H, within the lowest dose difference map at d
+2 = 3.9 MGy. Radiation-induced protein disordering is evident across the large dose range (b, c); in comparison, no clear deterioration of the RNA density was observed.</text></passage><passage><infon key="file">d-72-00648-fig4.jpg</infon><infon key="id">fig4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>42938</offset><text>
+D
+loss calculated for all side-chain carboxyl group Glu Cδ and Asp Cγ atoms within the TRAP–RNA complex for a dose of 19.3 MGy (d
+8). Residues have been grouped by amino-acid number, and split into bound and nonbound groupings, with each bar representing the mean calculated over 11 equivalent atoms around a TRAP ring. Whiskers indicate 95% CI. The D
+loss behaviour shown here was consistently exhibited across the entire investigated dose range.</text></passage><passage><infon key="file">d-72-00648-fig5.jpg</infon><infon key="id">fig5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>43397</offset><text>
+D
+loss against dose for (a) Glu36 Cδ, (b) Asp39 Cγ, (c) Glu42 O∊1, (d) Glu42 O∊2, (e) Phe32 Cζ and (f) Lys37 C∊ atoms. 95% CI are included for each set of 11 equivalent atoms grouped as bound/nonbound. RNA-binding interface interactions are shown for TRAP chain N, with the F
+obs(d
+7) − F
+obs(d
+1) Fourier difference map (dose 16.7 MGy) overlaid and contoured at a ±4σ level.</text></passage></document></collection>
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+<collection><source>PMC</source><date>20201221</date><key>pmc.key</key><document><id>4871749</id><infon key="license">NO-CC CODE</infon><passage><infon key="article-id_doi">10.1038/nchembio.2065</infon><infon key="article-id_manuscript">NIHMS769551</infon><infon key="article-id_pmc">4871749</infon><infon key="article-id_pmid">27089029</infon><infon key="fpage">396</infon><infon key="issue">6</infon><infon key="kwd">YEATS domain crotonylated lysine chromatin Taf14 histone PTM</infon><infon key="license">Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
+</infon><infon key="lpage">398</infon><infon key="name_0">surname:Andrews;given-names:Forest H.</infon><infon key="name_1">surname:Shinsky;given-names:Stephen A.</infon><infon key="name_2">surname:Shanle;given-names:Erin K.</infon><infon key="name_3">surname:Bridgers;given-names:Joseph B.</infon><infon key="name_4">surname:Gest;given-names:Anneliese</infon><infon key="name_5">surname:Tsun;given-names:Ian K.</infon><infon key="name_6">surname:Krajewski;given-names:Krzysztof</infon><infon key="name_7">surname:Shi;given-names:Xiaobing</infon><infon key="name_8">surname:Strahl;given-names:Brian D.</infon><infon key="name_9">surname:Kutateladze;given-names:Tatiana G.</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">12</infon><infon key="year">2016</infon><offset>0</offset><text>The Taf14 YEATS domain is a reader of histone crotonylation</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>60</offset><text>The discovery of new histone modifications is unfolding at startling rates, however, the identification of effectors capable of interpreting these modifications has lagged behind. Here we report the YEATS domain as an effective reader of histone lysine crotonylation – an epigenetic signature associated with active transcription. We show that the Taf14 YEATS domain engages crotonyllysine via a unique π-π-π-stacking mechanism and that other YEATS domains have crotonyllysine binding activity.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>560</offset><text>Crotonylation of lysine residues (crotonyllysine, Kcr) has emerged as one of the fundamental histone post-translational modifications (PTMs) found in mammalian chromatin. This epigenetic PTM is widespread and enriched at active gene promoters and potentially enhancers. The crotonyllysine mark on histone H3K18 is produced by p300, a histone acetyltransferase also responsible for acetylation of histones. Owing to some differences in their genomic distribution, the crotonyllysine and acetyllysine (Kac) modifications have been linked to distinct functional outcomes. p300-catalyzed histone crotonylation, which is likely metabolically regulated, stimulates transcription to a greater degree than p300-catalyzed acetylation. The discovery of individual biological roles for the crotonyllysine and acetyllysine marks suggests that these PTMs can be read by distinct readers. While a number of acetyllysine readers have been identified and characterized, a specific reader of the crotonyllysine mark remains unknown (reviewed in). A recent survey of bromodomains (BDs) demonstrates that only one BD associates very weakly with a crotonylated peptide, however it binds more tightly to acetylated peptides, inferring that bromodomains do not possess physiologically relevant crotonyllysine binding activity.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1865</offset><text>The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. The acetyllysine binding function of the AF9 YEATS domain is essential for the recruitment of the histone methyltransferase DOT1L to H3K9ac-containing chromatin and for DOT1L-mediated H3K79 methylation and transcription. Similarly, activation of a subset of genes and DNA damage repair in yeast require the acetyllysine binding activity of the Taf14 YEATS domain. Consistent with its role in gene regulation, Taf14 was identified as a core component of the transcription factor complexes TFIID and TFIIF. However, Taf14 is also found in a number of chromatin-remodeling complexes (i.e., INO80, SWI/SNF and RSC) and the histone acetyltransferase complex NuA3, indicating a multifaceted role of Taf14 in transcriptional regulation and chromatin biology. In this study, we identified the Taf14 YEATS domain as a reader of crotonyllysine that binds to histone H3 crotonylated at lysine 9 (H3K9cr) via a distinctive binding mechanism. We found that H3K9cr is present in yeast and is dynamically regulated.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3072</offset><text>To elucidate the molecular basis for recognition of the H3K9cr mark, we obtained a crystal structure of the Taf14 YEATS domain in complex with H3K9cr5-13 (residues 5–13 of H3) peptide (Fig. 1, Supplementary Results, Supplementary Fig. 1 and Supplementary Table 1). The Taf14 YEATS domain adopts an immunoglobin-like β sandwich fold containing eight anti-parallel β strands linked by short loops that form a binding site for H3K9cr (Fig. 1b). The H3K9cr peptide lays in an extended conformation in an orientation orthogonal to the β strands and is stabilized through an extensive network of direct and water-mediated hydrogen bonds and a salt bridge (Fig. 1c).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3741</offset><text>The most striking feature of the crotonyllysine recognition mechanism is the unique coordination of crotonylated lysine residue. The fully extended side chain of K9cr transverses the narrow tunnel, crossing the β sandwich at right angle in a corkscrew-like manner (Fig. 1b and Supplementary Figure 1b). The planar crotonyl group is inserted between Trp81 and Phe62 of the protein, the aromatic rings of which are positioned strictly parallel to each other and at equal distance from the crotonyl group, yielding a novel aromatic-amide/aliphatic-aromatic π-π-π-stacking system that, to our knowledge, has not been reported previously for any protein-protein interaction (Fig. 1d and Supplementary Fig. 1c). The side chain of Trp81 appears to adopt two conformations, one of which provides maximum π-stacking with the alkene functional group while the other rotamer affords maximum π-stacking with the amide π electrons (Supplementary Fig. 1c). The dual conformation of Trp81 is likely due to the conjugated nature of the C=C and C=O π-orbitals within the crotonyl functional group.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4832</offset><text>In addition to π-π-π stacking, the crotonyl group is stabilized by a set of hydrogen bonds and electrostatic interactions. The π bond conjugation of the crotonyl group gives rise to a dipole moment of the alkene moiety, resulting in a partial positive charge on the β-carbon (Cβ) and a partial negative charge on the α-carbon (Cα). This provides the capability for the alkene moiety to form electrostatic contacts, as Cα and Cβ lay within electrostatic interaction distances of the carbonyl oxygen of Gln79 and of the hydroxyl group of Thr61, respectively. The hydroxyl group of Thr61 also participates in a hydrogen bond with the amide nitrogen of the K9cr side chain (Fig. 1d). The fixed position of the Thr61 hydroxyl group, which facilitates interactions with both the amide and Cα of K9cr, is achieved through a hydrogen bond with imidazole ring of His59. Extra stabilization of K9cr is attained by a hydrogen bond formed between its carbonyl oxygen and the backbone nitrogen of Trp81, as well as a water-mediated hydrogen bond with the backbone carbonyl group of Gly82 (Fig 1d). This distinctive mechanism was corroborated through mapping the Taf14 YEATS-H3K9cr binding interface in solution using NMR chemical shift perturbation analysis (Supplementary Fig. 2a, b).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6134</offset><text>Binding of the Taf14 YEATS domain to H3K9cr is robust. The dissociation constant (Kd) for the Taf14 YEATS-H3K9cr5-13 complex was found to be 9.5 μM, as measured by fluorescence spectroscopy (Supplementary Fig. 2c). This value is in the range of binding affinities exhibited by the majority of histone readers, thus attesting to the physiological relevance of the H3K9cr recognition by Taf14.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6527</offset><text>To determine whether H3K9cr is present in yeast, we generated whole cell extracts from logarithmically growing yeast cells and subjected them to Western blot analysis using antibodies directed towards H3K9cr, H3K9ac and H3 (Fig. 2a, b, Supplementary Fig. 3 and Supplementary Table 2). Both H3K9cr and H3K9ac were detected in yeast histones; to our knowledge, this is the first report of H3K9cr occurring in yeast. We next asked if H3K9cr is regulated by the actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Towards this end, we probed extracts derived from yeast cells in which major yeast HATs (HAT1, Gcn5, and Rtt109) or HDACs (Rpd3, Hos1, and Hos2) were deleted. As shown in Figure 2a, b and Supplementary Fig. 3e, H3K9cr levels were abolished or reduced considerably in the HAT deletion strains, whereas they were dramatically increased in the HDAC deletion strains. Furthermore, fluctuations in the H3K9cr levels were more substantial than fluctuations in the corresponding H3K9ac levels. Together, these results reveal that H3K9cr is a dynamic mark of chromatin in yeast and suggest an important role for this modification in transcription as it is regulated by HATs and HDACs.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>7741</offset><text>We have previously shown that among acetylated histone marks, the Taf14 YEATS domain prefers acetylated H3K9 (also see Supplementary Fig. 3b), however it binds to H3K9cr tighter. The selectivity of Taf14 towards crotonyllysine was substantiated by 1H,15N HSQC experiments, in which either H3K9cr5-13 or H3K9ac5-13 peptide was titrated into the 15N-labeled Taf14 YEATS domain (Fig. 2c and Supplementary Fig. 4a, b). Binding of H3K9cr induced resonance changes in slow exchange regime on the NMR time scale, indicative of strong interaction. In contrast, binding of H3K9ac resulted in an intermediate exchange, which is characteristic of a weaker association. Furthermore, crosspeaks of Gly80 and Trp81 of the YEATS domain were uniquely perturbed by H3K9cr and H3K9ac, indicating a different chemical environment in the respective crotonyllysine and acetyllysine binding pockets (Supplementary Fig. 4a). These differences support our model that Trp81 adopts two conformations upon complex formation with the H3K9cr mark as compared to H3K9ac (Supplementary Figs. 1c, d and 4c). One of the conformations, characterized by the π stacking involving two aromatic residues and the alkene group, is observed only in the YEATS-H3K9cr complex.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>8976</offset><text>To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1–20 of H3). As shown in Figure 2d and Supplementary Fig. 5a, the Taf14 YEATS domain binds more strongly to H3K9cr1-20, as compared to other acylated histone peptides. The preference for H3K9cr over H3K9ac, H3K9pr and H3K9bu was supported by 1H,15N HSQC titration experiments. Addition of H3K9ac1-20, H3K9pr1-20, and H3K9bu1-20 peptides caused chemical shift perturbations in the Taf14 YEATS domain in intermediate exchange regime, implying that these interactions are weaker compared to the interaction with the H3K9cr1-20 peptide (Supplementary Fig. 5b). We concluded that H3K9cr is the preferred target of this domain. From comparative structural analysis of the YEATS complexes, Gly80 emerged as candidate residue potentially responsible for the preference for crotonyllysine. In attempt to generate a mutant capable of accommodating a short acetyl moiety but discriminating against a longer, planar crotonyl moiety, we mutated Gly80 to more bulky residues, however all mutants of Gly80 lost their binding activities towards either acylated peptide, suggesting that Gly80 is absolutely required for the interaction. In contrast, mutation of Val24, a residue located on another side of Trp81, had no effect on binding (Fig. 2d and Supplementary Fig. 5a, c).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>10484</offset><text>To determine if the binding to crotonyllysine is conserved, we tested human YEATS domains by pull-down experiments using singly and multiply acetylated, propionylated, butyrylated, and crotonylated histone peptides (Supplementary Fig. 6). We found that all YEATS domains tested are capable of binding to crotonyllysine peptides, though they display variable preferences for the acyl moieties. While YEATS2 and ENL showed selectivity for the crotonylated peptides, GAS41 and AF9 bound acylated peptides almost equally well.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>11007</offset><text>Unlike the YEATS domain, a known acetyllysine reader, bromodomain, does not recognize crotonyllysine. We assayed a large set of BDs in pull-down experiments and found that this module is highly specific for acetyllysine and propionyllysine containing peptides (Supplementary Fig. 7). However, bromodomains did not interact (or associated very weakly) with longer acyl modifications, including crotonyllysine, as in the case of BDs of TAF1 and BRD2, supporting recent reports. These results demonstrate that the YEATS domain is currently the sole reader of crotonyllysine.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>11579</offset><text>In conclusion, we have identified the YEATS domain of Taf14 as the first reader of histone crotonylation. The unique and previously unobserved aromatic-amide/aliphatic-aromatic π-π-π-stacking mechanism facilitates the specific recognition of the crotonyl moiety. We further demonstrate that H3K9cr exists in yeast and is dynamically regulated by HATs and HDACs. As we previously showed the importance of acyllysine binding by the Taf14 YEATS domain for the DNA damage response and gene transcription, it will be essential in the future to define the physiological role of crotonyllysine recognition and to differentiate the activities of Taf14 that are due to binding to crotonyllysine and acetyllysine modifications. Furthermore, the functional significance of crotonyllysine recognition by other YEATS proteins will be of great importance to elucidate and compare.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>12449</offset><text>ONLINE METHODS</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>12464</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>12500</offset><text>The Taf14 YEATS constructs (residues 1–132 or 1–137) were expressed in E. coli BL21 (DE3) RIL in either Luria Broth or M19 minimal media supplemented with 15NH4Cl and purified as N-terminal GST fusion proteins. Cells were harvested by centrifugation and resuspended in 50 mM HEPES (pH 7.5) supplemented with 150 mM NaCl and 1 mM TCEP. Cells are lysed by freeze-thaw followed by sonication. Proteins were purified on glutathione Sepharose 4B beads and the GST tag was cleaved with PreScission protease.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>13006</offset><text>X-ray data collection and structure determination</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>13056</offset><text>Taf14 YEATS (residues 1–137) was concentrated to 9 mg/mL in 25 mM MES (pH 6.5) and incubated with 2 molar equivalence of the H3K9cr5-13 at RT for 30 mins prior to crystallization. Crystals were obtain via sitting drop diffusion method at 18°C by mixing 800 nL of protein/peptide solution with 800 nL of well solution composed of 44% PEG600 (v/v) and 0.2 M citric acid (pH 6.0). X-ray diffraction data was collected at a wavelength of 1.54 Å at 100 K from a single crystal on the UC Denver Biophysical Core home source composed of a Rigaku Micromax 007 high frequency microfocus X-ray generator with a Pilatus 200K 2D area detector. HKL3000 was used for indexing, scaling, and data reduction. Solution was solved via molecular replacement with Phaser using the Taf14 YEATS domain (PDB 5D7E) as search model with waters, ligands, and peptide removed. Phenix was used for refinement of structure and waters were manually placed by inception of difference maps in Coot. Ramachandran plot indicates good stereochemistry of the three-dimensional structure with 100% of all residues falling within the favored (98%) and allowed (2%) regions. The crystallographic statistics are shown in Supplementary Table 1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>14263</offset><text>NMR spectroscopy</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>14280</offset><text>NMR spectroscopy was carried out on a Varian INOVA 600 MHz spectrometer outfitted with a cryogenic probe. Chemical shift perturbation (CSP) analysis was performed using uniformly 15N-labeled Taf14 (1–132). 1H,15N heteronuclear single quantum coherence (HSQC) spectra of the Taf14 YEATS domain were collected in the presence of increasing concentrations of either H3K9cr5-13, H3K9ac5-13, H3K9cr1-20, H3K9ac1-20 H3K9pr1-20, H3K9bu1-20 or free Kcr in PBS buffer pH 6.8, 8% D2O.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>14757</offset><text>Fluorescence binding assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>14785</offset><text>Tryptophan fluorescence measurements were performed on a Fluorolog spectrofluorometer at room temperature as described. The samples containing 2 μM of Taf14 YEATS in PBS (pH 7.4) and increasing concentrations of H3K9cr5-13 were excited at 295 nm. Emission spectra were recorded from 310 to 340 nm with a 1 nm step size and a 0.5 sec integration time. The Kd value was determined using a nonlinear least-squares analysis and the equation:   where [L] is the concentration of the peptide, [P] is the concentration of the protein, ΔI is the observed change of signal intensity, and ΔImax is the difference in signal intensity of the free and bound states. The Kd values were averaged over 3 separate experiments, with error calculated as the standard deviation (SD).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>15558</offset><text>Peptide pull-downs</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>15577</offset><text>YEATS domains in pGEX vectors were expressed in SoluBL21 cells (Amsbio) by induction with 1 mM IPTG at 16–18°C overnight with shaking. Cells were lysed by freeze-thaw and sonication then purified over glutathione agarose (Pierce) in a buffer containing 50 mM Tris pH 8.0, 500 mM NaCl, 20% glycerol (v/v) and 1 mM dithiothreitol (DTT). Peptide pull-downs were performed essentially as described except that the assay buffer contained 50 mM Tris pH 8.0, 500 mM NaCl, and 0.1% NP-40, and 500 pmols of biotinylated histone peptides were loaded onto streptavidin coated magnetic beads before incubation with 40 pmols of protein. Bound proteins were detected with rabbit GST antibody (Sigma, G7781). Point mutants were generated by site-directed mutagenesis and purified/assayed as described above. The YEATS domains of Taf14, AF9, ENL, and GAS41 were previously described.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>16448</offset><text>Western blotting</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>16465</offset><text>Yeast cultures were grown in YPD media at 30°C to mid-log phase and extracts were prepared as previously described. Proteins from cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane. Anti-H3K9ac (Millipore, 07-352) and anti-H3K9cr (PTM Biolabs, PTM-516) were diluted to 1:2000 and 1:1000, respectively, in 1x Superblock (ThermoScientific). An HRP-conjugated anti-rabbit (GE Healthcare) was used for detection. Bands were quantified using the ImageJ program.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>16947</offset><text>Dot blotting</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>16960</offset><text>Increasing concentrations of biotinylated histone peptides (0.06–1.5 μg) were spotted onto a PVDF membrane then probed with the anti-H3K9ac (Millipore, 07-352) or H3K9cr (PTM Biolabs, PTM-516) at 1:2000 in a 5% non-fat milk solution and detected with an HRP-conjugated anti-rabbit by enhanced chemiluminesence (ECL).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>17280</offset><text>Bromodomains pull-downs</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>17304</offset><text>cDNAs of GST-fused bromodomains were obtained either from EpiCypher Inc. or as a kind gift from Katrin Chua (Stanford University). GST fusions were expressed as described above except that the preparation buffer contained 50 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol (v/v), and 1 mM DTT. Pull-down assays were preformed as described above except that the assay buffer contained 50 mM Tris (pH 8.0), 300 mM NaCl, and 0.1% NP-40.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>17734</offset><text>Supplementary Material</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>17757</offset><text>Accession codes. Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5IOK.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>17881</offset><text>Author contributions</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>17902</offset><text>F.H.A., S.A.S., E.K.S., J.B.B., A.G., I.K.T and K.K. performed experiments and together with X.S., B.D.S and T.G.K. analyzed the data. F.H.A., S.A.S., B.D.S. and T.G.K. wrote the manuscript with input from all authors.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>18121</offset><text>Competing Financial Interest</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>18150</offset><text>The authors declare no competing financial interests.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>18204</offset><text>Additional information</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>18227</offset><text>Any supplementary information is available in the online version of this paper.</text></passage><passage><infon key="fpage">1016</infon><infon key="lpage">28</infon><infon key="name_0">surname:Tan;given-names:M</infon><infon key="pub-id_pmid">21925322</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">146</infon><infon key="year">2011</infon><offset>18307</offset><text>Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification</text></passage><passage><infon key="fpage">203</infon><infon key="lpage">15</infon><infon key="name_0">surname:Sabari;given-names:BR</infon><infon key="pub-id_pmid">25818647</infon><infon key="section_type">REF</infon><infon key="source">Mol Cell</infon><infon key="type">ref</infon><infon key="volume">58</infon><infon key="year">2015</infon><offset>18413</offset><text>Intracellular Crotonyl-CoA Stimulates Transcription through p300-Catalyzed Histone Crotonylation</text></passage><passage><infon key="fpage">947</infon><infon key="lpage">60</infon><infon key="name_0">surname:Lin;given-names:H</infon><infon key="name_1">surname:Su;given-names:X</infon><infon key="name_2">surname:He;given-names:B</infon><infon key="pub-id_pmid">22571489</infon><infon key="section_type">REF</infon><infon key="source">ACS Chem Biol</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2012</infon><offset>18510</offset><text>Protein lysine acylation and cysteine succination by intermediates of energy metabolism</text></passage><passage><infon key="name_0">surname:Bao;given-names:X</infon><infon key="section_type">REF</infon><infon key="source">Elife</infon><infon key="type">ref</infon><infon key="volume">3</infon><infon key="year">2014</infon><offset>18598</offset><text>Identification of ‘erasers’ for lysine crotonylated histone marks using a chemical proteomics approach</text></passage><passage><infon key="fpage">1218</infon><infon key="lpage">27</infon><infon key="name_0">surname:Musselman;given-names:CA</infon><infon key="name_1">surname:Lalonde;given-names:ME</infon><infon key="name_2">surname:Cote;given-names:J</infon><infon key="name_3">surname:Kutateladze;given-names:TG</infon><infon key="pub-id_pmid">23211769</infon><infon key="section_type">REF</infon><infon key="source">Nat Struct Mol Biol</infon><infon key="type">ref</infon><infon key="volume">19</infon><infon key="year">2012</infon><offset>18705</offset><text>Perceiving the epigenetic landscape through histone readers</text></passage><passage><infon key="fpage">627</infon><infon key="lpage">43</infon><infon key="name_0">surname:Rothbart;given-names:SB</infon><infon key="name_1">surname:Strahl;given-names:BD</infon><infon key="pub-id_pmid">24631868</infon><infon key="section_type">REF</infon><infon key="source">Biochim Biophys Acta</infon><infon key="type">ref</infon><infon key="volume">1839</infon><infon key="year">2014</infon><offset>18765</offset><text>Interpreting the language of histone and DNA modifications</text></passage><passage><infon key="fpage">1801</infon><infon key="lpage">14</infon><infon key="name_0">surname:Flynn;given-names:EM</infon><infon key="pub-id_pmid">26365797</infon><infon key="section_type">REF</infon><infon key="source">Structure</infon><infon key="type">ref</infon><infon key="volume">23</infon><infon key="year">2015</infon><offset>18824</offset><text>A Subset of Human Bromodomains Recognizes Butyryllysine and Crotonyllysine Histone Peptide Modifications</text></passage><passage><infon key="fpage">214</infon><infon key="lpage">31</infon><infon key="name_0">surname:Filippakopoulos;given-names:P</infon><infon key="pub-id_pmid">22464331</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">149</infon><infon key="year">2012</infon><offset>18929</offset><text>Histone recognition and large-scale structural analysis of the human bromodomain family</text></passage><passage><infon key="fpage">65</infon><infon key="lpage">75</infon><infon key="name_0">surname:Schulze;given-names:JM</infon><infon key="name_1">surname:Wang;given-names:AY</infon><infon key="name_2">surname:Kobor;given-names:MS</infon><infon key="pub-id_pmid">19234524</infon><infon key="section_type">REF</infon><infon key="source">Biochem Cell Biol</infon><infon key="type">ref</infon><infon key="volume">87</infon><infon key="year">2009</infon><offset>19017</offset><text>YEATS domain proteins: a diverse family with many links to chromatin modification and transcription</text></passage><passage><infon key="fpage">558</infon><infon key="lpage">71</infon><infon key="name_0">surname:Li;given-names:Y</infon><infon key="pub-id_pmid">25417107</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">159</infon><infon key="year">2014</infon><offset>19117</offset><text>AF9 YEATS domain links histone acetylation to DOT1L-mediated H3K79 methylation</text></passage><passage><infon key="fpage">1795</infon><infon key="lpage">800</infon><infon key="name_0">surname:Shanle;given-names:EK</infon><infon key="pub-id_pmid">26341557</infon><infon key="section_type">REF</infon><infon key="source">Genes Dev</infon><infon key="type">ref</infon><infon key="volume">29</infon><infon key="year">2015</infon><offset>19196</offset><text>Association of Taf14 with acetylated histone H3 directs gene transcription and the DNA damage response</text></passage><passage><infon key="fpage">398</infon><infon key="lpage">403</infon><infon key="name_0">surname:Kabani;given-names:M</infon><infon key="name_1">surname:Michot;given-names:K</infon><infon key="name_2">surname:Boschiero;given-names:C</infon><infon key="name_3">surname:Werner;given-names:M</infon><infon key="pub-id_pmid">15896708</infon><infon key="section_type">REF</infon><infon key="source">Biochem Biophys Res Commun</infon><infon key="type">ref</infon><infon key="volume">332</infon><infon key="year">2005</infon><offset>19299</offset><text>Anc1 interacts with the catalytic subunits of the general transcription factors TFIID and TFIIF, the chromatin remodeling complexes RSC and INO80, and the histone acetyltransferase complex NuA3</text></passage><passage><infon key="fpage">401</infon><infon key="lpage">12</infon><infon key="name_0">surname:Shen;given-names:X</infon><infon key="pub-id_pmid">14979041</infon><infon key="section_type">REF</infon><infon key="source">Methods Enzymol</infon><infon key="type">ref</infon><infon key="volume">377</infon><infon key="year">2004</infon><offset>19493</offset><text>Preparation and analysis of the INO80 complex</text></passage><passage><infon key="fpage">3308</infon><infon key="lpage">16</infon><infon key="name_0">surname:Cairns;given-names:BR</infon><infon key="name_1">surname:Henry;given-names:NL</infon><infon key="name_2">surname:Kornberg;given-names:RD</infon><infon key="pub-id_pmid">8668146</infon><infon key="section_type">REF</infon><infon key="source">Mol Cell Biol</infon><infon key="type">ref</infon><infon key="volume">16</infon><infon key="year">1996</infon><offset>19539</offset><text>TFG/TAF30/ANC1, a component of the yeast SWI/SNF complex that is similar to the leukemogenic proteins ENL and AF-9</text></passage><passage><infon key="fpage">1196</infon><infon key="lpage">208</infon><infon key="name_0">surname:John;given-names:S</infon><infon key="pub-id_pmid">10817755</infon><infon key="section_type">REF</infon><infon key="source">Genes Dev</infon><infon key="type">ref</infon><infon key="volume">14</infon><infon key="year">2000</infon><offset>19654</offset><text>The something about silencing protein, Sas3, is the catalytic subunit of NuA3, a yTAF(II)30-containing HAT complex that interacts with the Spt16 subunit of the yeast CP (Cdc68/Pob3)-FACT complex</text></passage><passage><infon key="fpage">14</infon><infon key="lpage">20</infon><infon key="name_0">surname:Andrews;given-names:FH</infon><infon key="name_1">surname:Shanle;given-names:EK</infon><infon key="name_2">surname:Strahl;given-names:BD</infon><infon key="name_3">surname:Kutateladze;given-names:TG</infon><infon key="pub-id_pmid">26934307</infon><infon key="section_type">REF</infon><infon key="source">Transcription</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2016</infon><offset>19849</offset><text>The essential role of acetyllysine binding by the YEATS domain in transcriptional regulation</text></passage><passage><infon key="fpage">658</infon><infon key="lpage">674</infon><infon key="name_0">surname:McCoy;given-names:AJ</infon><infon key="pub-id_pmid">19461840</infon><infon key="section_type">REF</infon><infon key="source">J Appl Crystallogr</infon><infon key="type">ref</infon><infon key="volume">40</infon><infon key="year">2007</infon><offset>19942</offset><text>Phaser crystallographic software</text></passage><passage><infon key="fpage">213</infon><infon key="lpage">21</infon><infon key="name_0">surname:Adams;given-names:PD</infon><infon key="pub-id_pmid">20124702</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>19975</offset><text>PHENIX: a comprehensive Python-based system for macromolecular structure solution</text></passage><passage><infon key="fpage">486</infon><infon key="lpage">501</infon><infon key="name_0">surname:Emsley;given-names:P</infon><infon key="name_1">surname:Lohkamp;given-names:B</infon><infon key="name_2">surname:Scott;given-names:WG</infon><infon key="name_3">surname:Cowtan;given-names:K</infon><infon key="pub-id_pmid">20383002</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>20057</offset><text>Features and development of Coot</text></passage><passage><infon key="fpage">11296</infon><infon key="lpage">301</infon><infon key="name_0">surname:Ali;given-names:M</infon><infon key="pub-id_pmid">23798402</infon><infon key="section_type">REF</infon><infon key="source">Proc Natl Acad Sci U S A</infon><infon key="type">ref</infon><infon key="volume">110</infon><infon key="year">2013</infon><offset>20090</offset><text>Molecular basis for chromatin binding and regulation of MLL5</text></passage><passage><infon key="fpage">1155</infon><infon key="lpage">60</infon><infon key="name_0">surname:Rothbart;given-names:SB</infon><infon key="pub-id_pmid">23022729</infon><infon key="section_type">REF</infon><infon key="source">Nat Struct Mol Biol</infon><infon key="type">ref</infon><infon key="volume">19</infon><infon key="year">2012</infon><offset>20151</offset><text>Association of UHRF1 with methylated H3K9 directs the maintenance of DNA methylation</text></passage><passage><infon key="fpage">1795</infon><infon key="lpage">800</infon><infon key="name_0">surname:Shanle;given-names:EK</infon><infon key="pub-id_pmid">26341557</infon><infon key="section_type">REF</infon><infon key="source">Genes Dev</infon><infon key="type">ref</infon><infon key="volume">29</infon><infon key="year">2015</infon><offset>20236</offset><text>Association of Taf14 with acetylated histone H3 directs gene transcription and the DNA damage response</text></passage><passage><infon key="fpage">660</infon><infon key="lpage">5</infon><infon key="name_0">surname:Keogh;given-names:MC</infon><infon key="pub-id_pmid">16543219</infon><infon key="section_type">REF</infon><infon key="source">Genes Dev</infon><infon key="type">ref</infon><infon key="volume">20</infon><infon key="year">2006</infon><offset>20339</offset><text>The Saccharomyces cerevisiae histone H2A variant Htz1 is acetylated by NuA4</text></passage><passage><infon key="fpage">497</infon><infon key="lpage">501</infon><infon key="name_0">surname:Keogh;given-names:MC</infon><infon key="pub-id_pmid">16299494</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">439</infon><infon key="year">2006</infon><offset>20415</offset><text>A phosphatase complex that dephosphorylates gammaH2AX regulates DNA damage checkpoint recovery</text></passage><passage><infon key="file">nihms769551f1.jpg</infon><infon key="id">F1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>20510</offset><text>The structural mechanism for the recognition of H3K9cr</text></passage><passage><infon key="file">nihms769551f1.jpg</infon><infon key="id">F1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>20565</offset><text>(a) Chemical structure of crotonyllysine. (b) The crystal structure of the Taf14 YEATS domain (wheat) in complex with the H3K9cr5-13 peptide (green). (c) H3K9cr is stabilized via an extensive network of intermolecular electrostatic and polar interactions with the Taf14 YEATS domain. (d) The π-π-π stacking mechanism involving the alkene moiety of crotonyllysine.</text></passage><passage><infon key="file">nihms769551f2.jpg</infon><infon key="id">F2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>20932</offset><text>H3K9cr is a selective target of the Taf14 YEATS domain</text></passage><passage><infon key="file">nihms769551f2.jpg</infon><infon key="id">F2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>20987</offset><text>(a, b) Western blot analysis comparing the levels of H3K9cr and H3K9ac in wild type (WT), HAT deletion, or HDAC deletion yeast strains. Total H3 was used as a loading control. (c) Superimposed 1H,15N HSQC spectra of Taf14 YEATS recorded as H3K9cr5-13 and H3K9ac5-13 peptides were titrated in. Spectra are color coded according to the protein:peptide molar ratio. (d) Western blot analyses of peptide pull-down assays using wild-type and mutated Taf14 YEATS domains and indicated peptides.</text></passage></document></collection>
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+<collection><source>PMC</source><date>20201219</date><key>pmc.key</key><document><id>4872110</id><infon key="license">CC BY-NC</infon><passage><infon key="article-id_doi">10.1093/nar/gkw244</infon><infon key="article-id_pmc">4872110</infon><infon key="article-id_pmid">27084949</infon><infon key="fpage">4304</infon><infon key="issue">9</infon><infon key="license">This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com</infon><infon key="lpage">4316</infon><infon key="name_0">surname:Meyer;given-names:Britta</infon><infon key="name_1">surname:Wurm;given-names:Jan Philip</infon><infon key="name_2">surname:Sharma;given-names:Sunny</infon><infon key="name_3">surname:Immer;given-names:Carina</infon><infon key="name_4">surname:Pogoryelov;given-names:Denys</infon><infon key="name_5">surname:Kötter;given-names:Peter</infon><infon key="name_6">surname:Lafontaine;given-names:Denis L. J.</infon><infon key="name_7">surname:Wöhnert;given-names:Jens</infon><infon key="name_8">surname:Entian;given-names:Karl-Dieter</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">44</infon><infon key="year">2016</infon><offset>0</offset><text>Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>133</offset><text>The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive. Here we identify the cytoplasmic ribosome biogenesis protein Tsr3 as the responsible enzyme in yeast and human cells. In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation. The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode. This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate. Structurally, Tsr3 therefore represents a novel class of acp transferase enzymes.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1127</offset><text>INTRODUCTION</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1140</offset><text>Eukaryotic ribosome biogenesis is highly complex and requires a large number of non-ribosomal proteins and small non-coding RNAs in addition to ribosomal RNAs (rRNAs) and proteins. An increasing number of diseases—so called ribosomopathies—are associated with disturbed ribosome biogenesis.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1435</offset><text>During eukaryotic ribosome biogenesis several dozens of rRNA nucleotides become chemically modified. The most abundant rRNA modifications are methylations at the 2′-OH ribose moieties and isomerizations of uridine residues to pseudouridine, catalyzed by small nucleolar ribonucleoprotein particles (snoRNPs). In addition, 18S and 25S (yeast)/ 28S (humans) rRNAs contain several base modifications catalyzed by site-specific and snoRNA-independent enzymes. In Saccharomyces cerevisiae 18S rRNA contains four base methylations, two acetylations and a single 3-amino-3-carboxypropyl (acp) modification, whereas six base methylations are present in the 25S rRNA. While in humans the 18S rRNA base modifications are highly conserved, only three of the yeast base modifications catalyzed by ScRrp8/HsNML, ScRcm1/HsNSUN5 and ScNop2/HsNSUN1 are preserved in the corresponding human 28S rRNA.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2321</offset><text>Ribosomal RNA modifications have been suggested to optimize ribosome function, although in most cases this remains to be clearly established. They might contribute to increased RNA stability by providing additional hydrogen bonds (pseudouridines), improved base stacking (pseudouridines and base methylations) or an increased resistance against hydrolysis (ribose methylations). Most modified rRNA nucleotides cluster in the vicinity of the decoding or the peptidyl transferase center, suggesting an influence on ribosome functionality and stability. Defects of rRNA modification enzymes often lead to disturbed ribosome biogenesis or functionally impaired ribosomes, although the lack of individual rRNA modifications often has no or only a slight influence on the cell. Importantly, malfunctions of several base modifying enzymes are linked to developmental diseases, aging or tumorigenesis.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3215</offset><text>The chemically most complex modification is located in the loop capping helix 31 of 18S rRNA (Supplementary Figure S1B). There a uridine (U1191 in yeast) is modified to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ, Figure 1A). This base modification was first described in 1968 for hamster cells and is conserved in eukaryotes. This hypermodified nucleotide, which is located at the P-site tRNA, is synthesized in three steps beginning with the snR35 H/ACA snoRNP guided conversion of uridine into pseudouridine. In a second step, the essential SPOUT-class methyltransferase Nep1/Emg1 modifies the pseudouridine to N1-methylpseudouridine. Methylation can only occur once pseudouridylation has taken place, as the latter reaction generates the substrate for the former. The final acp modification leading to N1-methyl-N3-aminocarboxypropyl-pseudouridine occurs late during 40S biogenesis in the cytoplasm, while the two former reactions are taking place in the nucleolus and nucleus, and is independent from pseudouridylation or methylation. Both the methyl and the acp group are derived from S-adenosylmethionine (SAM), but the enzyme responsible for acp modification remained elusive for more than 40 years.</text></passage><passage><infon key="file">gkw244fig1.jpg</infon><infon key="id">F1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>4441</offset><text>Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3Ψ is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3. The asterisk indicates the C1-atom labeled in the 14C-incorporation assay. (B) RP-HPLC elution profile of yeast 18S rRNA nucleosides. Hypermodified m1acp3Ψ elutes at 7.4 min (wild type, left profile) and is missing in Δtsr3 (middle profile) and Δnep1 Δnop6 mutants (right profile). (C) 14C-acp labeling of 18S rRNAs. Wild type (WT) and plasmid encoded 18S rRNA (U1191U) show the 14C-acp signal, whereas the 14C-acp signal is missing in the U1191A mutant plasmid encoded 18S rRNA (U1191A) and Δtsr3 mutants (Δtsr3). Upper lanes show the ethidium bromide staining of the 18S rRNAs for quantification. All samples were loaded on the gel with two different amounts of 5 and 10 μl. (D) Primer extension analysis of acp modification in yeast 18S rRNA (right gel) including a sequencing ladder (left gel). The primer extension stop at nucleotide 1191 is missing exclusively in Δtsr3 mutants and Δtsr3 Δsnr35 recombinants. (E) Primer extension analysis of human 18S rRNA after siRNA knockdown of HsNEP1/EMG1 (541, 542 and 543) and HsTSR3 (544 and 545) (right gel), including a sequencing ladder (left gel). The primer extension arrest is reduced in HTC116 cells transfected with siRNAs 544 and 545. The efficiency of siRNA mediated HsTSR3 repression correlates with the primer extension signals (see Supplementary Figure S2A). As a loading control, a structural stop is shown (asterisks).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6133</offset><text>Only a few acp transferring enzymes have been characterized until now. During the biosynthesis of wybutosine, a tricyclic nucleoside present in eukaryotic and archaeal phenylalanine tRNA, Tyw2 (Trm12 in yeast) transfers an acp group from SAM to an acidic carbon atom. Archaeal Tyw2 has a structure very similar to Rossmann-fold (class I) RNA-methyltransferases, but its distinctive SAM-binding mode enables the transfer of the acp group instead of the methyl group of the cofactor. Another acp modification has been described in the diphtamide biosynthesis pathway, where an acp group is transferred from SAM to the carbon atom of a histidine residue of eukaryotic translation elongation factor 2 by use of a radical mechanism.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6861</offset><text>In a recent bioinformatic study, the uncharacterized yeast gene YOR006c was predicted to be involved in ribosome biogenesis. It is highly conserved among eukaryotes and archaea (Supplementary Figure S1A) and its deletion leads to an accumulation of the 20S pre-rRNA precursor of 18S rRNA, suggesting an influence on D-site cleavage during the maturation of the small ribosomal subunit. On this basis, YOR006C was renamed ‘Twenty S rRNA accumulation 3′ (TSR3). However, its function remained unclear although recently a putative nuclease function during 18S rRNA maturation was predicted.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>7453</offset><text>Here, we identify Tsr3 as the long-sought acp transferase that catalyzes the last step in the biosynthesis of the hypermodified nucleotide m1acp3Ψ in yeast and human cells. Furthermore using catalytically defective mutants of yeast Tsr3 we demonstrated that the acp modification is required for 18S rRNA maturation. Surprisingly, the crystal structures of archaeal homologs revealed that Tsr3 is structurally similar to the SPOUT-class RNA methyltransferases. In contrast, the only other structurally characterized acp transferase enzyme Tyw2 belongs to the Rossmann-fold class of methyltransferase proteins. Interestingly, the two structurally very different enzymes use similar strategies in binding the SAM-cofactor in order to ensure that in contrast to methyltransferases the acp and not the methyl group of SAM is transferred to the substrate.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>8305</offset><text>MATERIALS AND METHODS</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>8327</offset><text>Genetic constructions, growth conditions and yeast media</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>8384</offset><text>Detailed descriptions are available in Supplementary Data.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>8443</offset><text>Cell culture</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>8456</offset><text>HCT116(+/+) cells (CCL-247; ATCC) were grown at 37°C in a humidified incubator under 5% CO2 in the McCoy's 5a modified (Sigma-Aldrich)/10% FBS media. All media were supplemented with 50 U/ml penicillin and 50 μg/ml streptomycin (Life Technologies).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>8707</offset><text>DsiRNA inactivation and RT-qPCR</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>8739</offset><text>Reverse transfection of HCT116 cells, DsiRNA inactivation and RT-qPCR using total human RNA are described in Supplementary Data.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>8868</offset><text>Sucrose gradient analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>8894</offset><text>Detailed descriptions for analytical or preparative separations of ribosomal subunits or polysome gradients are provided in Supplementary Data.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>9038</offset><text>HPLC analysis of 18S rRNA nucleosides</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>9076</offset><text>40S subunits from 200 ml yeast culture were isolated by sucrose gradient centrifugation in a SW28 rotor as described above, and precipitated with 2.5 vol of 100% ethanol (−20°C over night). Precipitated 40S subunits were dissolved in water and the 18S rRNA was purified via spin columns (Ambion PureLink RNA Mini Kit). RNA fragments were hydrolysed and dephosphorylated as described by Gehrke and Kuo. HPLC analysis of rRNA nucleoside composition was performed using a Supelcosil LC-18S column (Sigma; 250 × 4.6 mm, 5 μm) with a pre-column (4.6 × 20 mm) as previously described.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>9661</offset><text>14C labeling of 18S rRNA nucleotide Ψ(U)1191</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>9708</offset><text>To enhance 14C-labeling, mutants of interest were recombined with a Δmet13 deletion. Resulting strains were cultivated with l-[1-14C]-methionine (Hartmann Analytic, 0.1 mCi/ml, 54 mCi/mmol) as described before. From isotope labeled cells total RNA was isolated with the PureLink RNA Mini Kit (Ambion) after enzymatic cell lysis with zymolyase. Ribosomal RNAs were separated on a 4% denaturing polyacrylamide gel. After ethidium bromide straining gels were dried and analyzed by autoradiography for 3–5 days using a storage phosphor screen. Signals were visualized with the Typhoon 9100 (GE Healthcare).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>10315</offset><text>Northern blot analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>10338</offset><text>5 μg of total yeast RNAs extracted with phenol/chloroform were separated on 1.2% agarose gels in BPTE buffer for 16 h at 60V and afterwards transferred to a Biodyne B membrane by vacuum blotting. Oligonucleotides D/A2 or +1-A0 were radiolabeled using γ-[32P]-ATP and T4-polynucleotide kinase and hybridized to the membrane at 37°C. Signals were visualized by phosphoimaging with the Typhoon 9100 (GE Healthcare). RNA extraction from human cells, gel-electrophoresis and northern blotting were performed as described before.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>10867</offset><text>Primer extension</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>10884</offset><text>20 pmol of oligonucleotide PE-1191 complementary to yeast 18S rRNA nucleotides 1247–1228 were labeled with 50 μCi γ-[32P]-ATP using T4-polynucleotide kinase, purified via Sephadex G-25 and annealed to 500 ng of 18S rRNA. Primer annealing and reverse transcription were carried out as described by Sharma et al.. After precipitation with ethanol and 3 M NaAc pH 5.2 pellets were washed with 70% ethanol, dried and dissolved in 12 μl formamide loading dye. 2 μl of primer extension samples were separated on sequencing or mini gels which were dried after running and exposed on a storage phosphor screen. Signals were visualized with the Typhoon 9100 (GE Healthcare).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>11557</offset><text>Primer extension on human RNA was performed using 5 μg of total RNA with AMV Reverse Transcriptase (Promega) and oligonucleotide PE_1248. Following alkaline hydrolysis, cDNAs were precipitated with ethanol, resuspended in acrylamide loading buffer and separated on a 6% (v/v) denaturing acrylamide gel in 0.5× TBE at 80 W for 1.5 h. After migration, the gels were dried and exposed to Fuji Imaging plates (Fujifilm). The signal was acquired with a Phosphor imager (FLA-7000, Fujifilm).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>12045</offset><text>Protein detection and localization</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>12080</offset><text>A description of the western blot detection of HA-fused Tsr3 in yeast crude extracts or sucrose gradients fractions is provided in Supplementary Data. For cellular localization Tsr3 was expressed as N-terminal fusion with yEGFP in a yeast strain encoding for ScNop56-mRFP. Protein localization in exponentially growing cells was visualized using a Leica TCS SP5.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>12443</offset><text>In vitro SAM binding</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>12464</offset><text>Purified SsTsr3 protein in 25 mM Tris–HCl pH 7.8 250 mM NaCl was mixed with S-[methyl-14C]-adenosyl-l-methionine (PerkinElmer; 20 μCi/ml, 58 mCi/mmol) and 0–10 mM non-labeled SAM in a binding buffer (50 mM Tris–HCl pH 7.8, 250 mM NaCl) in a total volume of 50 μl and incubated at 30°C for 10 min. Samples were passed over HAWP02500 membrane filters (Millipore) and unbound 14C-SAM was removed by washing three times with 5 ml buffer using a vacuum filtering equipment. Filter bound 14C-SAM was measured by liquid scintillation spectrometry in a Wallac 1401 scintillation counter.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>13053</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>13089</offset><text>Genes coding for archaeal Tsr3 homologs without any tags were obtained commercially (Genscript) in pET11a vectors and overexpressed in Escherichia coli BL21(DE3). Proteins were purified by a combination of heat shock and appropriate column chromatography steps as described in detail in the Supplementary Data.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>13400</offset><text>Crystallization, X-ray data collection, structure calculation and refinement</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>13477</offset><text>Initial hits for VdTsr3 and SsTsr3 were obtained using the Morpheus Screen (Molecular dimensions) and further refined as described in the Supplementary Data. Diffraction data were collected at the Swiss Light Source (Paul Scherer Institut). The structure of VdTsr3 was determined at 1.6 Å by SAD using a selenomethionine derivative. The structure of SsTsr3 was determined at 2.25 Å by molecular replacement using VdTsr3 as the search model. A detailed description of the data collection, processing, structure calculation and refinement procedures can be found in the Supplementary Data and in Supplementary Table S1. Structures were deposited in the Protein Data Bank as entries 5APG (VdTsr3) and 5AP8 (SsTsr3).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>14192</offset><text>Analytical gel filtration</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>14218</offset><text>For analytical gel filtration experiments a Sephadex S75 10/300 GL column (GE Healthcare) was used. 100 μl protein samples (25 mM Tris–HCl pH 7.8, 250 mM NaCl, 2 mM β-mercaptoethanol) with a protein concentration of 150 μM were used. The flow rate was 0.5 ml/min. The column was calibrated using the marker proteins of the LMW gel filtration calibration kit (GE Healthcare). Protein elution was followed by recording the adsorption at a wavelength of λ = 280 nm.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>14691</offset><text>Fluorescence quenching and fluorescence anisotropy measurements</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>14755</offset><text>Fluorescence quenching and fluorescence anisotropy measurements were carried out in triplicates at 25°C on a Fluorolog 3 spectrometer (Horiba Jobin Yvon) equipped with polarizers. For fluorescence quenching with SAM, SAH and 5′-methylthioadenosine experiments the tryptophan fluorescence of SsTsr3 (200 nM in 25 mM Tris–HCl pH 7.8, 250 mM NaCl, 2 mM β-mercaptoethanol) was excited at 295 nm and emission spectra were recorded from 250 to 450 nm for each titration step. The fluorescence intensity at 351 nm for each titration step was normalized with regard to the fluorescence of the free protein and was used for deriving binding curves. KD's were derived by nonlinear regression with Origin 8.0 (Origin Labs) using Equation (1):  (F is the normalized fluorescence intensity, a is the change in fluorescence intensity, c is the ligand concentration and KD is the dissociation constant).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>15650</offset><text>5′-Fluoresceine labeled RNAs for fluorescence anisotropy measurements were obtained commercially (Dharmacon), deprotected according to the manufacturer's protocol and the RNA concentration adjusted to 50 nM in 25 mM Tris–HCl pH 7.8, 250 mM NaCl. Fluoresceine fluorescence was excited at 492 nm and emission was recorded at 516 nm. The data were fitted to Equation (1) (F is the normalized fluorescence anisotropy, a is the change in fluorescence anisotropy).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>16113</offset><text>RESULTS</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>16121</offset><text>Tsr3 is the enzyme responsible for 18S rRNA acp modification in yeast and humans</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16202</offset><text>The S. cerevisiae 18S rRNA acp transferase was identified in a systematic genetic screen where numerous deletion mutants from the EUROSCARF strain collection (www.euroscarf.de) were analyzed by HPLC for alterations in 18S rRNA base modifications.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16449</offset><text>For the Δtsr3 deletion strain the HPLC elution profile of 18S rRNA nucleosides (Figure 1B) was very similar to that of the pseudouridine-N1 methyltransferase mutant Δnep1, where a shoulder at ∼ 7.4 min elution time was missing in the elution profile. As previously reported this shoulder was identified by ESI-MS as corresponding to m1acp3Ψ. In order to directly analyze the presence of the acp modification of nucleotide 1191 we used an in vivo14C incorporation assay with 1-14C-methionine. Whereas the acp labeling of 18S rRNA was clearly present in the wild type strain no radioactive labeling could be observed in a Δtsr3 strain (Figure 1C). No radioactive labeling was detected in the 18S U1191A mutant which served as a control for the specificity of the 14C-aminocarboxypropyl incorporation.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17262</offset><text>As previously shown, only the acp but none of the other modifications at U1191 of yeast 18S rRNA blocks reverse transcriptase activity. Therefore the presence of the acp modification can be directly assessed by primer extension. Indeed, in wild-type yeast a strong primer extension stop signal occurred at position 1192. In contrast, in a Δtsr3 mutant no primer extension stop signal was present at this position. As expected, in a Δsnr35 deletion preventing pseudouridylation and N1-methylation (resulting in acp3U) as well as in a Δnep1 deletion strain where pseudouridine is not methylated (resulting in acp3Ψ) a primer extension stop signal of similar intensity as in the wild type was observed. In a Δtsr3 Δsnr35 double deletion strain the 18S rRNA contains an unmodified U and the primer extension stop signal was missing (Figure 1D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18125</offset><text>The Tsr3 protein is highly conserved in yeast and humans (50% identity). Human 18S rRNA has also been shown to contain m1acp3Ψ in the 18S rRNA at position 1248. After siRNA-mediated depletion of Tsr3 in human colon carcinoma HCT116(+/+) cells the acp primer extension arrest was reduced in comparison to cells transfected with a non-targeting scramble siRNA control (Figure 1E, compare lanes 544 and scramble). The efficiency of siRNA-mediated depletion was established by RT-qPCR and found to be very high with siRNA 544 (Supplementary Figure S2A, remaining TSR3 mRNA level of 2%). By comparison, treating cells with siRNA 545, which only reduced the TSR3 mRNA to 20%, did not markedly reduced the acp signal. This suggests that low residual levels of HsTsr3 are sufficient to modify the RNA. As a control for loading, a structural stop is shown (asterisk, Figure 1E). Thus, HsTsr3 is also responsible for the acp modification of 18S rRNA nucleotide Ψ1248 in helix 31. Similar to yeast, siRNA-mediated depletion of the Ψ1248 N1-methyltransferase Nep1/Emg1 had no influence on the primer extension arrest (Figure 1E).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>19249</offset><text>Phenotypic characterization of Δtsr3 mutants</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19298</offset><text>Although the acp modification of 18S rRNA is highly conserved in eukaryotes, yeast Δtsr3 mutants showed only a minor growth defect. However, the Δtsr3 deletion was synthetic sick with a Δsnr35 deletion preventing pseudouridylation and Nep1-catalyzed methylation of nucleotide 1191 (Figure 2A). Interestingly, no increased growth defect could be observed for Δtsr3 Δnep1 recombinants containing the nep1 suppressor mutation Δnop6 as well as for Δtsr3 Δsnr35 Δnep1 recombinants with unmodified U1191 (Supplementary Figure S2D and E).</text></passage><passage><infon key="file">gkw244fig2.jpg</infon><infon key="id">F2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>19866</offset><text>Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. The Δtsr3 deletion is synthetic sick with a Δsnr35 deletion preventing U1191 pseudouridylation. (B) In agar diffusion assays the yeast Δtsr3 deletion mutant shows a hypersensitivity against paromomycin and hygromycin B which is further increased by recombination with Δsnr35. (C) Northern blot analysis with an ITS1 hybridization probe after siRNA depletion of HsTSR3 (siRNAs 544 and 545) and a scrambled siRNA as control. The accumulation of 18SE and 47S and/or 45S pre-RNAs is enforced upon HsTSR3 depletion. Right gel: Ethidium bromide staining showing 18S and 28S rRNAs. (D) Cytoplasmic localization of yeast Tsr3 shown by fluorescence microscopy of GFP-fused Tsr3. From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). The TSR3 gene was genetically modified at its native locus, resulting in a C-terminal fusion of Tsr3 with a 3xHA epitope expressed by the native promotor in yeast strain CEN.BM258-5B.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21519</offset><text>The influence of the acp modification of nucleotide 1191 on ribosome function was analyzed by treating Δtsr3 mutants with protein synthesis inhibitors. Similar to a temperature-sensitive nep1 mutant, the Δtsr3 deletion caused hypersensitivity to paromomycin and, to a lesser extent, to hygromycin B (Figure 2B), but not to G418 or cycloheximide (data not shown). In accordance with the synthetic sick growth phenotype the paromomycin and hygromycin B hypersensitivity further increased in a Δtsr3 Δsnr35 recombination strain (Figure 2B).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22073</offset><text>In a yeast Δtsr3 strain as well as in the Δtsr3 Δsnr35 recombinant 20S pre-rRNA accumulated significantly and the level of mature 18S rRNA was reduced (Supplementary Figures S2C and S3D), as reported previously. A minor effect on 20S rRNA accumulation was also observed for Δsnr35, but - probably due to different strain backgrounds – to a weaker extent than described earlier. In human cells, the depletion of HsTsr3 in HCT116(+/+) cells caused an accumulation of the human 20S pre-rRNA equivalent 18S-E suggesting an evolutionary conserved role of Tsr3 in the late steps of 18S rRNA processing (Figure 2C and Supplementary Figure S2B). Surprisingly, early nucleolar processing reactions were also inhibited, and this was observed in both yeast Δtsr3 cells (see accumulation of 35S in Supplementary Figure S2C) and Tsr3 depleted human cells (see 47S/45S accumulation in Figure 2C and Northern blot quantification in Supplementary Figure S2B).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23037</offset><text>Consistent with its role in late 18S rRNA processing, TSR3 deletion leads to a ribosomal subunit imbalance with a reduced 40S to 60S ratio of 0.81 (σ = 0.024) which was further increased in a Δtsr3 Δsnr35 recombinant to 0.73 (σ = 0.023) (Supplementary Figure S2F). In polysome profiles, a reduced level of 80S ribosomes and a strong signal for free 60S subunits was observed in line with the 40S subunit deficiency (Supplementary Figure S2G).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>23496</offset><text>Cellular localization of Tsr3 in S. cerevisiae</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23543</offset><text>Fluorescence microscopy of GFP-tagged Tsr3 localized the fusion protein in the cytoplasm of yeast cells and no co-localization with the nucleolar marker protein Nop56 could be observed (Figure 2D). This agrees with previous biochemical data suggesting that the acp modification of 18S rRNA occurs late during 40S subunit biogenesis in the cytoplasm, and makes an additional nuclear localization as reported in a previous large-scale analysis unlikely. After polysome gradient separation C-terminally epitope-labeled Tsr3-3xHA was exclusively detectable in the low-density fraction (Figure 2E). Such distribution on a density gradient suggests that Tsr3 only interacts transiently with pre-40S subunits, which presumably explains why it was not characterized in pre-ribosome affinity purifications.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>24341</offset><text>Structure of Tsr3</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24359</offset><text>Searches for sequence homologs of S. cerevisiae Tsr3 (ScTsr3) by us and others revealed that the genomes of many archaea contain genes encoding Tsr3-like proteins. However, these archaeal homologs are significantly smaller than ScTsr3 (∼190 aa in archaea vs. 313 aa in yeast) due to shortened N- and C-termini (Supplementary Figure S1A).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24699</offset><text>To locate the domains most important for Tsr3 activity, ScTsr3 fragments of different lengths containing the highly conserved central part were expressed in a Δtsr3 mutant (Figure 3A) and analyzed by primer extension (Figure 3B) and Northern blotting (Figure 3C). N-terminal truncations of up to 45 aa and C-terminal truncations of up to 76 aa mediated acp modification as efficiently as the full-length protein and no significant increased levels of 20S pre-RNA were detected. Even a Tsr3 fragment with a 90 aa C-terminal truncation showed a residual primer extension stop, whereas N-terminal truncations exceeding 46 aa almost completely abolished the primer extension arrest (Figure 3B).</text></passage><passage><infon key="file">gkw244fig3.jpg</infon><infon key="id">F3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>25394</offset><text>Domain characterization of yeast Tsr3 and correlation of acp modification with late 18S rRNA processing steps. (A) Scheme of the TSR3 gene with truncation positions in the open reading frame. TSR3 fragments of different length were expressed under the native promotor from multicopy plasmids in a Δtsr3 deletion strain. (B) Primer extension analysis of 18S rRNA acp modification in yeast cells expressing the indicated TSR3 fragments. N-terminal deletions of 36 or 45 amino acids and C-terminal deletions of 43 or 76 residues show a primer extension stop comparable to the wild type. Tsr3 fragments 37–223 or 46–223 cause a nearly complete loss of the arrest signal. The box highlights the shortest Tsr3 fragment (aa 46–270) with wild type activity (strong primer extension block). (C) Northern blot analysis of 20S pre-rRNA accumulation. A weak 20S rRNA signal, indicating normal processing, is observed for Tsr3 fragment 46–270 (highlighted in a box) showing its functionality. Strong 20S rRNA accumulation similar to that of the Δtsr3 deletion is observed for Tsr3 fragments 37–223 or 46–223.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>26504</offset><text>Thus, the archaeal homologs correspond to the functional core of Tsr3. In order to define the structural basis for Tsr3 function, homologs from thermophilic archaea were screened for crystallization. We focused on archaeal species containing a putative Nep1 homolog suggesting that these species are in principle capable of synthesizing N1-methyl-N3-acp-pseudouridine. Well diffracting crystals were obtained for Tsr3 homologs from the two crenarchaeal species Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) which share 36% (VdTsr3) and 38% (SsTsr3) identity with the ScTsr3 core region (ScTsr3 aa 46–223). While for S. solfataricus the existence of a modified nucleotide of unknown chemical composition in the loop capping helix 31 of its 16S rRNA has been demonstrated, no information regarding rRNA modifications is yet available for V. distributa.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>27381</offset><text>Crystals of VdTsr3 diffracted to a resolution of 1.6 Å whereas crystals of SsTsr3 diffracted to 2.25 Å. Serendipitously, VdTsr3 was purified and crystallized in complex with endogenous (E. coli) SAM (Supplementary Figure S4) while SsTsr3 crystals contained the protein in the apo state. The structure of VdTsr3 was solved ab initio, by single-wavelength anomalous diffraction phasing (Se-SAD) with Se containing derivatives (selenomethionine and seleno-substituted SAM). The structure of SsTsr3 was solved by molecular replacement using VdTsr3 as a search model (see Supplementary Table S1 for data collection and refinement statistics). The structure of VdTsr3 can be divided into two domains (Figure 4A). The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. The loop connecting β2 and β3 contains a single turn of a 310-helix. Helices α1 and α2 are located on one side of the five-stranded β-sheet while α3 packs against the opposite β-sheet surface. The C-terminal domain (aa 93–184) has a globular all α-helical structure comprising α-helices α4 to α9. Both domains are tightly packed against each other. Remarkably, the entire C-terminal domain (92 aa) of the protein is threaded through the loop which connects β-strand β3 and α-helix α2 of the N-terminal domain. Thus, the VdTsr3 structure contains a deep trefoil knot. The structure of SsTsr3 in the apo state is very similar to that of VdTsr3 (Figure 4C) with an RMSD for equivalent Cα atoms of 1.1 Å. The only significant difference in the global structure of the two proteins is the presence of an extended α-helix α8 and the absence of α-helix α9 in SsTsr3.</text></passage><passage><infon key="file">gkw244fig4.jpg</infon><infon key="id">F4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>29350</offset><text>Tsr3 has a fold similar to SPOUT-class RNA methyltransferases. (A) Cartoon representation of the X-ray structure of VdTsr3 in two orientations. β-strands are colored in crimson whereas α-helices in the N-terminal domain are colored light blue and α-helices in the C-terminal domain are colored dark blue. The bound S-adenosylmethionine is shown in a stick representation and colored by atom type. A red arrow marks the location of the topological knot in the structure. (B) Secondary structure representation of the VdTsr3 structure. The color coding is the same as in (A). (C) Structural superposition of the X-ray structures of VdTsr3 in the SAM-bound state (red) and SsTsr3 (blue) in the apo state. The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. Vertical lines indicate the elution volumes of molecular weight markers. Vd, Vulcanisaeta distributa; Ss, Sulfolobus solfataricus.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>30665</offset><text>Structure predictions suggested that Tsr3 might contain a so-called RLI domain which contains a ‘bacterial like’ ferredoxin fold and binds two iron-sulfur clusters through eight conserved cysteine residues. However, no structural similarity to an RLI-domain was detectable. This is in accordance with the functional analysis of alanine replacement mutations of cysteine residues in ScTsr3 (Supplementary Figure S3).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>31085</offset><text>The β-strand topology and the deep C-terminal trefoil knot of archaeal Tsr3 are the structural hallmarks of the SPOUT-class RNA-methyltransferase fold. The closest structural homolog identified in a DALI search is the tRNA methyltransferase Trm10 (DALI Z-score 6.8) which methylates the N1 nitrogen of G9/A9 in many archaeal and eukaryotic tRNAs by using SAM as the methyl group donor. In comparison to Tsr3 the central β-sheet element of Trm10 is extended by one additional β-strand pairing to β2. Furthermore, the trefoil knot of Trm10 is not as deep as that of Tsr3 (Figure 4D). Interestingly, Nep1—the enzyme preceding Tsr3 in the biosynthetic pathway for the synthesis of m1acp3Ψ—also belongs to the SPOUT-class of RNA methyltransferases. However, the structural similarities between Nep1 and Tsr3 (DALI Z-score 4.4) are less pronounced than between Tsr3 and Trm10. Most SPOUT-class RNA-methyltransferases are homodimers. A notable exception is Trm10. Gel filtration experiments with both VdTsr3 and SsTsr3 (Figure 4E) showed that both proteins are monomeric in solution thereby extending the structural similarities to Trm10.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>32231</offset><text>So far, structural information is only available for one other enzyme that transfers the acp group from SAM to an RNA nucleotide. This enzyme, Tyw2, is part of the biosynthesis pathway of wybutosine nucleotides in tRNAs. However, there are no structural similarities between Tsr3 and Tyw2, which contains an all-parallel β-sheet of a different topology and no knot structure. Instead, Tyw2 has a fold typical for the class-I-or Rossmann-fold class of methyltransferases (Supplementary Figure S5B).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>32732</offset><text>Cofactor binding of Tsr3</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>32757</offset><text>The SAM-binding site of Tsr3 is located in a deep crevice between the N- and C-terminal domains in the vicinity of the trefoil knot as typical for SPOUT-class RNA-methyltransferases (Figure 4A). The adenine base of the cofactor is recognized by hydrogen bonds between its N1 nitrogen and the backbone amide of L93 directly preceding β5 as well as between its N6-amino group and the backbone carbonyl group of Y108 located in the loop connecting β5 in the N-terminal and α4 in the C-terminal domain (Figure 5A). Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. The ribose 2′ and 3′ hydroxyl groups of SAM are hydrogen bonded to the backbone carbonyl group of I69. The acp side chain of SAM is fixed in position by hydrogen bonding of its carboxylate group to the backbone amide and the side chain hydroxyl group of T19 in α1 as well as the backbone amide group of T112 in α4 (C-terminal domain). Most importantly, the methyl group of SAM is buried in a hydrophobic pocket formed by the sidechains of W73 and A76 both located in α3 (Figure 5A and B). W73 is highly conserved in all known Tsr3 proteins, whereas A76 can be replaced by other hydrophobic amino acids. Consequently, the accessibility of this methyl group for a nucleophilic attack is strongly reduced in comparison with RNA-methyltransferases such as Trm10 (Figure 5B, C). In contrast, the acp side chain of SAM is accessible for reactions in the Tsr3-bound state (Figure 5B).</text></passage><passage><infon key="file">gkw244fig5.jpg</infon><infon key="id">F5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>34453</offset><text>SAM-binding by Tsr3. (A) Close-up view of the SAM-binding pocket of VdTsr3. Nitrogen atoms are dark blue, oxygen atoms red, sulfur atoms orange, carbon atoms of the protein light blue and carbon atoms of SAM yellow. Hydrogen bonds are indicated by dashed lines. (B) Solvent accessibility of the acp group of SAM bound to VdTsr3. The solvent accessible surface of the protein is shown in semitransparent gray whereas SAM is show in a stick representation. Atoms are colored as in (A). A red arrow indicates the reactive CH2-moiety of the acp group. (C) Solvent accessibility of the SAM methyl group for SAM bound to the RNA methyltransferase Trm10. Bound SAM was modelled based on the X-ray structure of the Trm10/SAH-complex (pdb4jwf). A red arrow indicates the SAM methyl group. (D) Binding of SAM analogs to SsTsr3. Tryptophan fluorescence quenching curves upon addition of SAM (blue), 5′-methyl-thioadenosine (red) and SAH (black). (E) Binding of 14C-labeled SAM to SsTsr3. Radioactively labeled SAM is retained on a filter in the presence of SsTsr3. Addition of unlabeled SAM competes with the binding of labeled SAM. A W66A-mutant of SsTsr3 (W73 in VdTsr3) does not bind SAM. (F) Primer extension (upper left) shows a strongly reduced acp modification of yeast 18S rRNA in Δtsr3 cells expressing Tsr3-S62D, -E111A or –W114A. This correlates with a 20S pre-rRNA accumulation comparable to the Δtsr3 deletion (right: northern blot). 3xHA tagged Tsr3 mutants are expressed comparable to the wild type as shown by western blot (lower left).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>36003</offset><text>Binding affinities for SAM and its analogs 5′-methylthioadenosin and SAH to SsTsr3 were measured using tryptophan fluorescence quenching. VdTsr3 could not be used in these experiments since we could not purify it in a stable SAM-free form. SsTsr3 bound SAM with a KD of 6.5 μM, which is similar to SAM-KD's reported for several SPOUT-class methyltransferases. 5′-methylthioadenosin—the reaction product after the acp-transfer—binds only ∼2.5-fold weaker (KD = 16.7 μM) compared to SAM. S-adenosylhomocysteine which lacks the methyl group of SAM binds with significantly lower affinity (KD = 55.5 μM) (Figure 5D). This suggests that the hydrophobic interaction between SAM's methyl group and the hydrophobic pocket of Tsr3 is thermodynamically important for the interaction. On the other hand, the loss of hydrogen bonds between the acp sidechain carboxylate group and the protein appears to be thermodynamically less important but these hydrogen bonds might play a crucial role for the proper orientation of the cofactor side chain in the substrate binding pocket.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>37081</offset><text>Accordingly, a W66A-mutation (W73 in VdTsr3) of SsTsr3 significantly diminished SAM-binding in a filter binding assay compared to the wild type (Figure 5E). Furthermore, a W to A mutation at the equivalent position W114 in ScTsr3 strongly reduced the in vivo acp transferase activity (Figure 5F). The side chain hydroxyl group of T19 seems of minor importance for SAM binding since mutations of T17 (T19 in VdTsr3) to either A or D did not significantly influence the SAM-binding affinity of SsTsr3 (KD's = 3.9 or 11.2 mM, respectively). Nevertheless, a mutation of the equivalent position S62 of ScTsr3 to D, but not to A, resulted in reduced acp modification in vivo, as shown by primer extension analysis (Figure 5F).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>37802</offset><text>The acp-transfer reaction catalyzed by Tsr3 most likely requires the presence of a catalytic base in order to abstract a proton from the N3 imino group of the modified pseudouridine. The side chain of D70 (VdTsr3) located in β4 is ∼5 Å away from the SAM sulfur atom. This residue is conserved as D or E both in archaeal and eukaryotic Tsr3 homologs. Mutations of the corresponding residue in SsTsr3 to A (D63) does not significantly alter the SAM-binding affinity of the protein (KD = 11.0 μM). However, the mutation of the corresponding residue of ScTsr3 (E111A) leads to a significant decrease of the acp transferase activity in vivo (Figure 5F).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>38456</offset><text>RNA-binding of Tsr3</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>38476</offset><text>Analysis of the electrostatic surface properties of VdTsr3 clearly identified positively charged surface patches in the vicinity of the SAM-binding site suggesting a putative RNA-binding site (Figure 6A). Furthermore, a negatively charged MES-ion is found in the crystal structure of VdTsr3 complexed to the side chain of K22 in helix α1. Its negatively charged sulfate group might mimic an RNA backbone phosphate. Helix α1 contains two more positively charged amino acids K17 and R25 as does the loop preceding it (R9). A second cluster of positively charged residues is found in or near helix α3 (K74, R75, K82, R85 and K87). Some of these amino acids are conserved between archaeal and eukaryotic Tsr3 (Supplementary Figure S1A). In the C-terminal domain, the surface exposed α-helices α5 and α7 carry a significant amount of positively charged amino acids. A triple mutation of the conserved positively charged residues R60, K65 and R131 to A in ScTsr3 resulted in a protein with a significantly impaired acp transferase activity in vivo (Figure 6D) in line with an important functional role for these positively charged residues.</text></passage><passage><infon key="file">gkw244fig6.jpg</infon><infon key="id">F6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>39635</offset><text>RNA-binding of Tsr3. (A) Electrostatic charge distribution on the surface of VdTsr3. Surface areas colored in blue are positively charged whereas red areas are negatively charged. SAM is shown in a stick representation. Also shown in stick representation is a negatively charged MES ion. Conserved basic amino acids are labeled. (B) Comparison of the secondary structures of helix 31 from the small ribosomal subunit rRNAs in S. cerevisiae and S. solfataricus with the location of the hypermodified nucleotide indicated in red. For S. solfataricus the chemical identity of the hypermodified nucleotide is not known but the existence of NEP1 and TSR3 homologs suggest that it is indeed N1-methyl-N3-acp-pseudouridine. (C) Binding of SsTsr3 to RNA. 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). Oligo-U9-RNA was used for comparison (black). The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. Combination of the three point mutations (R60A/K65A/R131A) leads to a strongly reduced acp modification of 18S rRNA.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>41195</offset><text>In order to explore the RNA-ligand specificity of Tsr3 we titrated SsTsr3 prepared in RNase-free form with 5′-fluoresceine-labeled RNA and determined the affinity by fluorescence anisotropy measurements. SsTsr3 in the apo state bound a 20mer RNA corresponding to helix 31 of S. solfataricus 16S rRNA (Figure 6B) with a KD of 1.9 μM and to a version of this hairpin stabilized by additional GC base pairs (20mer-GC) with a KD of 0.6 μM (Figure 6C). A single stranded oligoU-RNA bound with a 10-fold-reduced affinity (6.0 μM). The presence of saturating amounts of SAM (2 mM) did not have a significant influence on the RNA-affinity of SsTsr3 (KD of 1.7 μM for the 20mer-GC-RNA) suggesting no cooperativity in substrate binding.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>41928</offset><text>DISCUSSION</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>41939</offset><text>U1191 is the only hypermodified base in the yeast 18S rRNA and is strongly conserved in eukaryotes. The formation of 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ) is very complex requiring three successive modification reactions involving one H/ACA snoRNP (snR35) and two protein enzymes (Nep1/Emg1 and Tsr3). This makes it unique in eukaryotic rRNA modification. The m1acp3Ψ base is located at the tip of helix 31 on the 18S rRNA (Supplementary Figure S1B) which, together with helices 18, 24, 34 and 44, contribute to building the decoding center of the small ribosomal subunit. A similar modification (acp3U) was identified in Haloferax volcanii and corresponding modified nucleotides were also shown to occur in other archaea.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>42689</offset><text>As shown here TSR3 encodes the transferase catalyzing the acp modification as the last step in the biosynthesis of m1acp3Ψ in yeast and human cells. Unexpectedly, archaeal Tsr3 has a structure similar to SPOUT-class RNA methyltransferases, and it is the first example for an enzyme of this class transferring an acp group, due to a modified SAM-binding pocket that exposes the acp instead of the methyl group of SAM to its RNA substrate. Similar to the structurally unrelated Rossmann-fold Tyw2 acp transferase, the SAM methyl group of Tsr3 is bound in an inaccessible hydrophobic pocket whereas the acp side chain becomes accessible for a nucleophilic attack by the N3 of pseudouridine. In contrast, in the structurally closely related RNA methyltransferase Trm10 the methyl group of the cofactor SAM is accessible whereas its acp side chain is buried inside the protein. This suggests that enzymes with a SAM-dependent acp transferase activity might have evolved from SAM-dependent methyltransferases by slight modifications of the SAM-binding pocket. Thus, additional examples for acp transferase enzymes might be found with similarities to other structural classes of methyltransferases. In contrast to Nep1, the enzyme preceding Tsr3 in the m1acp3Ψ biosynthesis pathway, Tsr3 binds rather weakly and with little specificity to its isolated substrate RNA. This suggests that Tsr3 is not stably incorporated into pre-ribosomal particles and that its binding to the nascent ribosomal subunit possibly requires additional interactions with other pre-ribosomal components. Consistently, in sucrose gradient analysis, Tsr3 was found in low-molecular weight fractions rather than with pre-ribosome containing high-molecular weight fractions.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>44433</offset><text>In contrast to several enzymes that catalyze base specific modifications in rRNAs Tsr3 is not an essential protein. Typically, other small subunit rRNA methyltransferases as Dim1, Bud23 and Nep1/Emg1 carry dual functions, in ribosome biogenesis and rRNA modification, and it is their involvement in pre-RNA processing that is essential rather than their RNA-methylating activity (, discussed in 7). In contrast, for several Tsr3 mutants (SAM-binding and cysteine mutations) we found a systematic correlation between the loss of acp modification and the efficiency of 18S rRNA maturation. This demonstrates that, unlike the other small subunit rRNA base modifications, the acp modification is required for efficient pre-rRNA processing.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>45169</offset><text>Recently, structural, functional, and CRAC (cross-linking and cDNA analysis) experiments of late assembly factors involved in cytoplasmic processing of 40S subunits, along with cryo-EM studies of the late pre-40S subunits have provided important insights into late pre-40S processing. Apart from most of the ribosomal proteins, cytoplasmic pre-40S particles contain 20S rRNA and at least seven non-ribosomal proteins including the D-site endonuclease Nob1 as well as Tsr1, a putative GTPase and Rio2 which block the mRNA channel and the initiator tRNA binding site, respectively, thus preventing translation initiation.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>45789</offset><text>After structural changes, possibly driven by GTP hydrolysis, which go together with the formation of the decoding site, the 20S pre-rRNA becomes accessible for Nob1 cleavage at site D. This also involves joining of pre-40S and 60S subunits to 80S-like particles in a translation-like cycle promoted by eIF5B. The cleavage step most likely acts as a quality control check that ensures the proper 40S subunit assembly with only completely processed precursors. Finally, termination factor Rli1, an ATPase, promotes the dissociation of assembly factors and the 80S-like complex dissociates and releases the mature 40S subunit.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>46413</offset><text>Interestingly, differences in the level of acp modification were demonstrated for different steps of the cytoplasmic pre-40S subunit maturation after analyzing purified 20S pre-rRNAs using different purification bait proteins. Early cytoplasmic pre-40S subunits still containing the ribosome assembly factors Tsr1, Ltv1, Enp1 and Rio2 were not or only partially acp modified. In contrast, late pre-40S subunits containing Nob1 and Rio1 or already associated with 60S subunits in 80S-like particles showed acp modification levels comparable to mature 40S subunits. Thus, the acp transfer to m1Ψ1191 occurs during the step at which Rio2 leaves the pre-40S particle.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>47079</offset><text>These data and the finding that a missing acp modification hinders pre-20S rRNA processing, suggest that the acp modification together with the release of Rio2 promotes the formation of the decoding site and thus D-site cleavage by Nob1. The interrelation between acp modification and Rio2 release is also supported by CRAC analysis showing that Rio2 binds to helix 31 next to the Ψ1191 residue that receives the acp modification. Therefore, Rio2 either blocks the access of Tsr3 to helix 31, and acp modification can only occur after Rio2 is released, or the acp modification of m1Ψ1191 and putative subsequent conformational changes of 20S rRNA weaken the binding of Rio2 to helix 31 and support its release from the pre-rRNA.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>47812</offset><text>In summary, by identifying Tsr3 as the enzyme responsible for introducing the acp group to the hypermodified m1acp3Ψ nucleotide at position 1191 (yeast)/ 1248 (humans) of 18S rRNA we added one of the last remaining pieces to the puzzle of eukaryotic small ribosomal subunit rRNA modifications. The current data together with the finding that acp modification takes place at the very last step in pre-40S subunit maturation indicate that the acp modification probably supports the formation of the decoding site and efficient 20S pre-rRNA D-site cleavage. Furthermore, our structural data unravelled how the regioselectivity of SAM-dependent group transfer reactions can be tuned by distinct small evolutionary adaptions of the ligand binding pocket of SAM-binding enzymes.</text></passage><passage><infon key="section_type">KEYWORD</infon><infon key="type">title_1</infon><offset>48587</offset><text>ACCESSION NUMBERS</text></passage><passage><infon key="section_type">KEYWORD</infon><infon key="type">paragraph</infon><offset>48605</offset><text>Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes PDB 5APG (VdTsr3/SAM-complex) and PDB 5AP8 (SsTsr3).</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>48759</offset><text>Supplementary Material</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>48782</offset><text>SUPPLEMENTARY DATA</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">paragraph</infon><offset>48801</offset><text>Supplementary Data are available at NAR Online.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">title_1</infon><offset>48849</offset><text>FUNDING</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>48857</offset><text>DFG grant [En134/9-1]; SFB 902 (Molecular Principles of RNA-based Regulation); DFG SPP1784 (Chemical Biology of Native Nucleic Acid Modifications, DFG grants) [En134/13-1, Wo 901/5-1]; European Community's Seventh Framework Programme [FP7/2007-2013] under BioStruct-X [283570]; Goethe University and the State of Hesse; EMBO long-term fellowship [ALTF 644-2014 to S.S.]; Research in the Lab of DLJL is supported by the Université Libre de Bruxelles (ULB); Fonds National de la Recherche (F.R.S./FNRS); Walloon Region [DGO6]; Fédération Wallonie-Bruxelles; European Research Development Fund (ERDF). Funding for open access charge: DFG SPP1784 (Chemical Biology of Native Nucleic Acid Modifications, DFG grants) [En134/13-1, Wo 901/5-1].</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>49597</offset><text>Conflict of interest statement. None declared.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>49644</offset><text>REFERENCES</text></passage><passage><infon key="fpage">643</infon><infon key="lpage">681</infon><infon key="name_0">surname:Woolford;given-names:J.L.;suffix:Jr</infon><infon key="name_1">surname:Baserga;given-names:S.J.</infon><infon key="pub-id_pmid">24190922</infon><infon key="section_type">REF</infon><infon key="source">Genetics</infon><infon key="type">ref</infon><infon key="volume">195</infon><infon key="year">2013</infon><offset>49655</offset><text>Ribosome biogenesis in the yeast Saccharomyces cerevisiae</text></passage><passage><infon key="fpage">1491</infon><infon key="lpage">1500</infon><infon key="name_0">surname:Armistead;given-names:J.</infon><infon key="name_1">surname:Triggs-Raine;given-names:B.</infon><infon key="pub-id_pmid">24657617</infon><infon key="section_type">REF</infon><infon key="source">FEBS Lett.</infon><infon key="type">ref</infon><infon key="volume">588</infon><infon key="year">2014</infon><offset>49713</offset><text>Diverse diseases from a ubiquitous process: the ribosomopathy paradox</text></passage><passage><infon key="fpage">849</infon><infon key="lpage">850</infon><infon key="name_0">surname:McCann;given-names:K.L.</infon><infon key="name_1">surname:Baserga;given-names:S.J.</infon><infon key="section_type">REF</infon><infon key="source">Science (New York, N.Y.)</infon><infon key="type">ref</infon><infon key="volume">341</infon><infon key="year">2013</infon><offset>49783</offset><text>Genetics. Mysterious ribosomopathies</text></passage><passage><infon key="fpage">758</infon><infon key="lpage">764</infon><infon key="name_0">surname:Sondalle;given-names:S.B.</infon><infon key="name_1">surname:Baserga;given-names:S.J.</infon><infon key="pub-id_pmid">24240090</infon><infon key="section_type">REF</infon><infon key="source">Biochim. Biophys. Acta</infon><infon key="type">ref</infon><infon key="volume">1842</infon><infon key="year">2014</infon><offset>49820</offset><text>Human diseases of the SSU processome</text></passage><passage><infon key="fpage">1077</infon><infon key="lpage">1088</infon><infon key="name_0">surname:Kiss-Laszlo;given-names:Z.</infon><infon key="name_1">surname:Henry;given-names:Y.</infon><infon key="name_2">surname:Bachellerie;given-names:J.P.</infon><infon key="name_3">surname:Caizergues-Ferrer;given-names:M.</infon><infon key="name_4">surname:Kiss;given-names:T.</infon><infon key="pub-id_pmid">8674114</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">85</infon><infon key="year">1996</infon><offset>49857</offset><text>Site-specific ribose methylation of preribosomal RNA. a novel function for small nucleolar RNAs</text></passage><passage><infon key="fpage">799</infon><infon key="lpage">809</infon><infon key="name_0">surname:Ganot;given-names:P.</infon><infon key="name_1">surname:Bortolin;given-names:M.L.</infon><infon key="name_2">surname:Kiss;given-names:T.</infon><infon key="pub-id_pmid">9182768</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">89</infon><infon key="year">1997</infon><offset>49953</offset><text>Site-specific pseudouridine formation in preribosomal RNA is guided by small nucleolar RNAs</text></passage><passage><infon key="fpage">560</infon><infon key="lpage">575</infon><infon key="name_0">surname:Sharma;given-names:S.</infon><infon key="name_1">surname:Lafontaine;given-names:D.L.J.</infon><infon key="pub-id_pmid">26410597</infon><infon key="section_type">REF</infon><infon key="source">Trends Biochem. Sci.</infon><infon key="type">ref</infon><infon key="volume">40</infon><infon key="year">2015</infon><offset>50045</offset><text>'View from a bridge': a new perspective on eukaryotic rRNA base modification</text></passage><passage><infon key="fpage">1151</infon><infon key="lpage">1163</infon><infon key="name_0">surname:Peifer;given-names:C.</infon><infon key="name_1">surname:Sharma;given-names:S.</infon><infon key="name_2">surname:Watzinger;given-names:P.</infon><infon key="name_3">surname:Lamberth;given-names:S.</infon><infon key="name_4">surname:Kötter;given-names:P.</infon><infon key="name_5">surname:Entian;given-names:K.-D.</infon><infon key="pub-id_pmid">23180764</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">41</infon><infon key="year">2013</infon><offset>50122</offset><text>Yeast Rrp8p, a novel methyltransferase responsible for m1A 645 base modification of 25S rRNA</text></passage><passage><infon key="fpage">9062</infon><infon key="lpage">9076</infon><infon key="name_0">surname:Sharma;given-names:S.</infon><infon key="name_1">surname:Yang;given-names:J.</infon><infon key="name_2">surname:Watzinger;given-names:P.</infon><infon key="name_3">surname:Kotter;given-names:P.</infon><infon key="name_4">surname:Entian;given-names:K.-D.</infon><infon key="pub-id_pmid">23913415</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">41</infon><infon key="year">2013</infon><offset>50215</offset><text>Yeast Nop2 and Rcm1 methylate C2870 and C2278 of the 25S rRNA, respectively</text></passage><passage><infon key="fpage">6158</infon><infon key="name_0">surname:Schosserer;given-names:M.</infon><infon key="name_1">surname:Minois;given-names:N.</infon><infon key="name_2">surname:Angerer;given-names:T.B.</infon><infon key="name_3">surname:Amring;given-names:M.</infon><infon key="name_4">surname:Dellago;given-names:H.</infon><infon key="name_5">surname:Harreither;given-names:E.</infon><infon key="name_6">surname:Calle-Perez;given-names:A.</infon><infon key="name_7">surname:Pircher;given-names:A.</infon><infon key="name_8">surname:Gerstl;given-names:M.P.</infon><infon key="name_9">surname:Pfeifenberger;given-names:S.</infon><infon key="pub-id_pmid">25635753</infon><infon key="section_type">REF</infon><infon key="source">Nat. Commun.</infon><infon key="type">ref</infon><infon key="volume">6</infon><infon key="year">2015</infon><offset>50291</offset><text>Methylation of ribosomal RNA by NSUN5 is a conserved mechanism modulating organismal lifespan</text></passage><passage><infon key="fpage">721</infon><infon key="lpage">733</infon><infon key="name_0">surname:Helm;given-names:M.</infon><infon key="pub-id_pmid">16452298</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">34</infon><infon key="year">2006</infon><offset>50385</offset><text>Post-transcriptional nucleotide modification and alternative folding of RNA</text></passage><passage><infon key="fpage">610</infon><infon key="lpage">619</infon><infon key="name_0">surname:Chow;given-names:C.S.</infon><infon key="name_1">surname:Lamichhane;given-names:T.N.</infon><infon key="name_2">surname:Mahto;given-names:S.K.</infon><infon key="pub-id_pmid">17894445</infon><infon key="section_type">REF</infon><infon key="source">ACS Chem. Biol.</infon><infon key="type">ref</infon><infon key="volume">2</infon><infon key="year">2007</infon><offset>50461</offset><text>Expanding the nucleotide repertoire of the ribosome with post-transcriptional modifications</text></passage><passage><infon key="fpage">330</infon><infon key="lpage">339</infon><infon key="name_0">surname:Ishitani;given-names:R.</infon><infon key="name_1">surname:Yokoyama;given-names:S.</infon><infon key="name_2">surname:Nureki;given-names:O.</infon><infon key="pub-id_pmid">18539024</infon><infon key="section_type">REF</infon><infon key="source">Curr. Opin. Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">2008</infon><offset>50553</offset><text>Structure, dynamics, and function of RNA modification enzymes</text></passage><passage><infon key="fpage">344</infon><infon key="lpage">351</infon><infon key="name_0">surname:Decatur;given-names:W.A.</infon><infon key="name_1">surname:Fournier;given-names:M.J.</infon><infon key="pub-id_pmid">12114023</infon><infon key="section_type">REF</infon><infon key="source">Trends Biochem. Sci.</infon><infon key="type">ref</infon><infon key="volume">27</infon><infon key="year">2002</infon><offset>50615</offset><text>rRNA modifications and ribosome function</text></passage><passage><infon key="fpage">e174</infon><infon key="name_0">surname:Baxter-Roshek;given-names:J.L.</infon><infon key="name_1">surname:Petrov;given-names:A.N.</infon><infon key="name_2">surname:Dinman;given-names:J.D.</infon><infon key="pub-id_pmid">17245450</infon><infon key="section_type">REF</infon><infon key="source">PLoS One</infon><infon key="type">ref</infon><infon key="volume">2</infon><infon key="year">2007</infon><offset>50656</offset><text>Optimization of ribosome structure and function by rRNA base modification</text></passage><passage><infon key="fpage">1716</infon><infon key="lpage">1728</infon><infon key="name_0">surname:Liang;given-names:X.H.</infon><infon key="name_1">surname:Liu;given-names:Q.</infon><infon key="name_2">surname:Fournier;given-names:M.J.</infon><infon key="pub-id_pmid">19628622</infon><infon key="section_type">REF</infon><infon key="source">RNA</infon><infon key="type">ref</infon><infon key="volume">15</infon><infon key="year">2009</infon><offset>50730</offset><text>Loss of rRNA modifications in the decoding center of the ribosome impairs translation and strongly delays pre-rRNA processing</text></passage><passage><infon key="fpage">425</infon><infon key="lpage">435</infon><infon key="name_0">surname:King;given-names:T.H.</infon><infon key="name_1">surname:Liu;given-names:B.</infon><infon key="name_2">surname:McCully;given-names:R.R.</infon><infon key="name_3">surname:Fournier;given-names:M.J.</infon><infon key="pub-id_pmid">12620230</infon><infon key="section_type">REF</infon><infon key="source">Mol. Cell</infon><infon key="type">ref</infon><infon key="volume">11</infon><infon key="year">2003</infon><offset>50856</offset><text>Ribosome structure and activity are altered in cells lacking snoRNPs that form pseudouridines in the peptidyl transferase center</text></passage><passage><infon key="fpage">3196</infon><infon key="lpage">3205</infon><infon key="name_0">surname:Narla;given-names:A.</infon><infon key="name_1">surname:Ebert;given-names:B.L.</infon><infon key="pub-id_pmid">20194897</infon><infon key="section_type">REF</infon><infon key="source">Blood</infon><infon key="type">ref</infon><infon key="volume">115</infon><infon key="year">2010</infon><offset>50985</offset><text>Ribosomopathies: human disorders of ribosome dysfunction</text></passage><passage><infon key="fpage">11</infon><infon key="lpage">19</infon><infon key="name_0">surname:Lafontaine;given-names:D.L.J.</infon><infon key="pub-id_pmid">25565028</infon><infon key="section_type">REF</infon><infon key="source">Nat. Struct. Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">22</infon><infon key="year">2015</infon><offset>51042</offset><text>Noncoding RNAs in eukaryotic ribosome biogenesis and function</text></passage><passage><infon key="fpage">2721</infon><infon key="lpage">2727</infon><infon key="name_0">surname:Sato;given-names:G.</infon><infon key="name_1">surname:Saijo;given-names:Y.</infon><infon key="name_2">surname:Uchiyama;given-names:B.</infon><infon key="name_3">surname:Kumano;given-names:N.</infon><infon key="name_4">surname:Sugawara;given-names:S.</infon><infon key="name_5">surname:Fujimura;given-names:S.</infon><infon key="name_6">surname:Sato;given-names:M.</infon><infon key="name_7">surname:Sagawa;given-names:M.</infon><infon key="name_8">surname:Ohkuda;given-names:K.</infon><infon key="name_9">surname:Koike;given-names:K.</infon><infon key="pub-id_pmid">10561346</infon><infon key="section_type">REF</infon><infon key="source">J. Clin. Oncol.</infon><infon key="type">ref</infon><infon key="volume">17</infon><infon key="year">1999</infon><offset>51104</offset><text>Prognostic value of nucleolar protein p120 in patients with resected lung adenocarcinoma</text></passage><passage><infon key="fpage">319</infon><infon key="lpage">322</infon><infon key="name_0">surname:Enger;given-names:M.D.</infon><infon key="name_1">surname:Saponara;given-names:A.G.</infon><infon key="pub-id_pmid">5646651</infon><infon key="section_type">REF</infon><infon key="source">J. Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">33</infon><infon key="year">1968</infon><offset>51193</offset><text>Incorporation of 14C from [2-14C]methionine into 18 s but not 28 s RNA of Chinese hamster cells</text></passage><passage><infon key="fpage">2548</infon><infon key="lpage">2554</infon><infon key="name_0">surname:Samarsky;given-names:D.A.</infon><infon key="name_1">surname:Balakin;given-names:A.G.</infon><infon key="name_2">surname:Fournier;given-names:M.J.</infon><infon key="pub-id_pmid">7630735</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">23</infon><infon key="year">1995</infon><offset>51289</offset><text>Characterization of three new snRNAs from Saccharomyces cerevisiae. snR34, snR35 and snR36</text></passage><passage><infon key="fpage">1542</infon><infon key="lpage">1554</infon><infon key="name_0">surname:Taylor;given-names:A.B.</infon><infon key="name_1">surname:Meyer;given-names:B.</infon><infon key="name_2">surname:Leal;given-names:B.Z.</infon><infon key="name_3">surname:Kötter;given-names:P.</infon><infon key="name_4">surname:Schirf;given-names:V.</infon><infon key="name_5">surname:Demeler;given-names:B.</infon><infon key="name_6">surname:Hart;given-names:P.J.</infon><infon key="name_7">surname:Entian;given-names:K.-D.</infon><infon key="name_8">surname:Wöhnert;given-names:J.</infon><infon key="pub-id_pmid">18208838</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">36</infon><infon key="year">2008</infon><offset>51380</offset><text>The crystal structure of Nep1 reveals an extended SPOUT-class methyltransferase fold and a pre-organized SAM-binding site</text></passage><passage><infon key="fpage">2387</infon><infon key="lpage">2398</infon><infon key="name_0">surname:Wurm;given-names:J.P.</infon><infon key="name_1">surname:Meyer;given-names:B.</infon><infon key="name_2">surname:Bahr;given-names:U.</infon><infon key="name_3">surname:Held;given-names:M.</infon><infon key="name_4">surname:Frolow;given-names:O.</infon><infon key="name_5">surname:Kotter;given-names:P.</infon><infon key="name_6">surname:Engels;given-names:J.W.</infon><infon key="name_7">surname:Heckel;given-names:A.</infon><infon key="name_8">surname:Karas;given-names:M.</infon><infon key="name_9">surname:Entian;given-names:K.D.</infon><infon key="pub-id_pmid">20047967</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">38</infon><infon key="year">2010</infon><offset>51502</offset><text>The ribosome assembly factor Nep1 responsible for Bowen-Conradi syndrome is a pseudouridine-N1-specific methyltransferase</text></passage><passage><infon key="fpage">1526</infon><infon key="lpage">1537</infon><infon key="name_0">surname:Meyer;given-names:B.</infon><infon key="name_1">surname:Wurm;given-names:J.P.</infon><infon key="name_2">surname:Kötter;given-names:P.</infon><infon key="name_3">surname:Leisegang;given-names:M.S.</infon><infon key="name_4">surname:Schilling;given-names:V.</infon><infon key="name_5">surname:Buchhaupt;given-names:M.</infon><infon key="name_6">surname:Held;given-names:M.</infon><infon key="name_7">surname:Bahr;given-names:U.</infon><infon key="name_8">surname:Karas;given-names:M.</infon><infon key="name_9">surname:Heckel;given-names:A.</infon><infon key="pub-id_pmid">20972225</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">39</infon><infon key="year">2011</infon><offset>51624</offset><text>The Bowen-Conradi syndrome protein Nep1 (Emg1) has a dual role in eukaryotic ribosome biogenesis, as an essential assembly factor and in the methylation of Ψ1191 in yeast 18S rRNA</text></passage><passage><infon key="fpage">71</infon><infon key="lpage">77</infon><infon key="name_0">surname:Brand;given-names:R.C.</infon><infon key="name_1">surname:Klootwijk;given-names:J.</infon><infon key="name_2">surname:Planta;given-names:R.J.</infon><infon key="name_3">surname:Maden;given-names:B.E.</infon><infon key="pub-id_pmid">629754</infon><infon key="section_type">REF</infon><infon key="source">Biochem J.</infon><infon key="type">ref</infon><infon key="volume">169</infon><infon key="year">1978</infon><offset>51806</offset><text>Biosynthesis of a hypermodified nucleotide in Saccharomyces carlsbergensis 17S and HeLa-cell 18S ribosomal ribonucleic acid</text></passage><passage><infon key="fpage">12138</infon><infon key="lpage">12154</infon><infon key="name_0">surname:Hector;given-names:R.D.</infon><infon key="name_1">surname:Burlacu;given-names:E.</infon><infon key="name_2">surname:Aitken;given-names:S.</infon><infon key="name_3">surname:Le Bihan;given-names:T.</infon><infon key="name_4">surname:Tuijtel;given-names:M.</infon><infon key="name_5">surname:Zaplatina;given-names:A.</infon><infon key="name_6">surname:Cook;given-names:A.G.</infon><infon key="name_7">surname:Granneman;given-names:S.</infon><infon key="pub-id_pmid">25200078</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">42</infon><infon key="year">2014</infon><offset>51930</offset><text>Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution</text></passage><passage><infon key="fpage">161</infon><infon key="lpage">170</infon><infon key="name_0">surname:Lin;given-names:H.</infon><infon key="pub-id_pmid">21762947</infon><infon key="section_type">REF</infon><infon key="source">Bioorg. Chem.</infon><infon key="type">ref</infon><infon key="volume">39</infon><infon key="year">2011</infon><offset>52040</offset><text>S-Adenosylmethionine-dependent alkylation reactions: when are radical reactions used?</text></passage><passage><infon key="fpage">2142</infon><infon key="lpage">2154</infon><infon key="name_0">surname:Noma;given-names:A.</infon><infon key="name_1">surname:Kirino;given-names:Y.</infon><infon key="name_2">surname:Ikeuchi;given-names:Y.</infon><infon key="name_3">surname:Suzuki;given-names:T.</infon><infon key="pub-id_pmid">16642040</infon><infon key="section_type">REF</infon><infon key="source">EMBO J.</infon><infon key="type">ref</infon><infon key="volume">25</infon><infon key="year">2006</infon><offset>52126</offset><text>Biosynthesis of wybutosine, a hyper-modified nucleoside in eukaryotic phenylalanine tRNA</text></passage><passage><infon key="fpage">15616</infon><infon key="lpage">15621</infon><infon key="name_0">surname:Umitsu;given-names:M.</infon><infon key="name_1">surname:Nishimasu;given-names:H.</infon><infon key="name_2">surname:Noma;given-names:A.</infon><infon key="name_3">surname:Suzuki;given-names:T.</infon><infon key="name_4">surname:Ishitani;given-names:R.</infon><infon key="name_5">surname:Nureki;given-names:O.</infon><infon key="pub-id_pmid">19717466</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl. Acad. Sci. U.S.A.</infon><infon key="type">ref</infon><infon key="volume">106</infon><infon key="year">2009</infon><offset>52215</offset><text>Structural basis of AdoMet-dependent aminocarboxypropyl transfer reaction catalyzed by tRNA-wybutosine synthesizing enzyme, TYW2</text></passage><passage><infon key="fpage">1213</infon><infon key="lpage">1226</infon><infon key="name_0">surname:Schaffrath;given-names:R.</infon><infon key="name_1">surname:Abdel-Fattah Mohamed;given-names:W.</infon><infon key="name_2">surname:Klassen;given-names:R.</infon><infon key="name_3">surname:Stark;given-names:M.J.</infon><infon key="pub-id_pmid">25352115</infon><infon key="section_type">REF</infon><infon key="source">Mol. Microbiol.</infon><infon key="type">ref</infon><infon key="volume">94</infon><infon key="year">2014</infon><offset>52344</offset><text>The diphthamide modification pathway from Saccharomyces cerevisiae - revisited</text></passage><passage><infon key="fpage">149</infon><infon key="lpage">154</infon><infon key="name_0">surname:Mattheakis;given-names:L.C.</infon><infon key="name_1">surname:Sor;given-names:F.</infon><infon key="name_2">surname:Collier;given-names:R.J.</infon><infon key="pub-id_pmid">8406038</infon><infon key="section_type">REF</infon><infon key="source">Gene</infon><infon key="type">ref</infon><infon key="volume">132</infon><infon key="year">1993</infon><offset>52423</offset><text>Diphthamide synthesis in Saccharomyces cerevisiae: structure of the DPH2 gene</text></passage><passage><infon key="fpage">9487</infon><infon key="lpage">9497</infon><infon key="name_0">surname:Liu;given-names:S.</infon><infon key="name_1">surname:Milne;given-names:G.T.</infon><infon key="name_2">surname:Kuremsky;given-names:J.G.</infon><infon key="name_3">surname:Fink;given-names:G.R.</infon><infon key="name_4">surname:Leppla;given-names:S.H.</infon><infon key="pub-id_pmid">15485916</infon><infon key="section_type">REF</infon><infon key="source">Mol. Cell. Biol.</infon><infon key="type">ref</infon><infon key="volume">24</infon><infon key="year">2004</infon><offset>52501</offset><text>Identification of the proteins required for biosynthesis of diphthamide, the target of bacterial ADP-ribosylating toxins on translation elongation factor 2</text></passage><passage><infon key="fpage">e1000213</infon><infon key="name_0">surname:Li;given-names:Z.</infon><infon key="name_1">surname:Lee;given-names:I.</infon><infon key="name_2">surname:Moradi;given-names:E.</infon><infon key="name_3">surname:Hung;given-names:N.-J.</infon><infon key="name_4">surname:Johnson;given-names:A.W.</infon><infon key="name_5">surname:Marcotte;given-names:E.M.</infon><infon key="pub-id_pmid">19806183</infon><infon key="section_type">REF</infon><infon key="source">PLoS Biol.</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2009</infon><offset>52657</offset><text>Rational extension of the ribosome biogenesis pathway using network-guided genetics</text></passage><passage><infon key="fpage">424</infon><infon key="name_0">surname:Burroughs;given-names:A.M.</infon><infon key="name_1">surname:Aravind;given-names:L.</infon><infon key="pub-id_pmid">25566315</infon><infon key="section_type">REF</infon><infon key="source">Front. Genet.</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2014</infon><offset>52741</offset><text>Analysis of two domains with novel RNA-processing activities throws light on the complex evolution of ribosomal RNA biogenesis</text></passage><passage><infon key="fpage">3</infon><infon key="lpage">36</infon><infon key="name_0">surname:Gehrke;given-names:C.W.</infon><infon key="name_1">surname:Kuo;given-names:K.C.</infon><infon key="pub-id_pmid">2670985</infon><infon key="section_type">REF</infon><infon key="source">J. Chromatogr.</infon><infon key="type">ref</infon><infon key="volume">471</infon><infon key="year">1989</infon><offset>52868</offset><text>Ribonucleoside analysis by reversed-phase high-performance liquid chromatography</text></passage><passage><infon key="name_0">surname:Sambrook;given-names:J.</infon><infon key="name_1">surname:Russell;given-names:D.</infon><infon key="section_type">REF</infon><infon key="source">Molecular Cloning. A Laboratory Manual</infon><infon key="type">ref</infon><infon key="year">2001</infon><offset>52949</offset></passage><passage><infon key="fpage">2242</infon><infon key="lpage">2258</infon><infon key="name_0">surname:Sharma;given-names:S.</infon><infon key="name_1">surname:Langhendries;given-names:J.-L.</infon><infon key="name_2">surname:Watzinger;given-names:P.</infon><infon key="name_3">surname:Kotter;given-names:P.</infon><infon key="name_4">surname:Entian;given-names:K.-D.</infon><infon key="name_5">surname:Lafontaine;given-names:D.L.J.</infon><infon key="pub-id_pmid">25653167</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">43</infon><infon key="year">2015</infon><offset>52950</offset><text>Yeast Kre33 and human NAT10 are conserved 18S rRNA cytosine acetyltransferases that modify tRNAs assisted by the adaptor Tan1/THUMPD1</text></passage><passage><infon key="fpage">3246</infon><infon key="lpage">3260</infon><infon key="name_0">surname:Sharma;given-names:S.</infon><infon key="name_1">surname:Yang;given-names:J.</infon><infon key="name_2">surname:Düttmann;given-names:S.</infon><infon key="name_3">surname:Watzinger;given-names:P.</infon><infon key="name_4">surname:Kötter;given-names:P.</infon><infon key="name_5">surname:Entian;given-names:K.-D.</infon><infon key="pub-id_pmid">24335083</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res</infon><infon key="type">ref</infon><infon key="volume">42</infon><infon key="year">2014</infon><offset>53084</offset><text>Identification of novel methyltransferases, Bmt5 and Bmt6, responsible for the m3U methylations of 25S rRNA in Saccharomyces cerevisiae</text></passage><passage><infon key="fpage">686</infon><infon key="lpage">691</infon><infon key="name_0">surname:Huh;given-names:W.K.</infon><infon key="name_1">surname:Falvo;given-names:J.V.</infon><infon key="name_2">surname:Gerke;given-names:L.C.</infon><infon key="name_3">surname:Carroll;given-names:A.S.</infon><infon key="name_4">surname:Howson;given-names:R.W.</infon><infon key="name_5">surname:Weissman;given-names:J.S.</infon><infon key="name_6">surname:O'Shea;given-names:E.K.</infon><infon key="pub-id_pmid">14562095</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">425</infon><infon key="year">2003</infon><offset>53220</offset><text>Global analysis of protein localization in budding yeast</text></passage><passage><infon key="fpage">60</infon><infon key="lpage">63</infon><infon key="name_0">surname:Maden;given-names:B.E.</infon><infon key="name_1">surname:Forbes;given-names:J.</infon><infon key="name_2">surname:de Jonge;given-names:P.</infon><infon key="name_3">surname:Klootwijk;given-names:J.</infon><infon key="pub-id_pmid">1225623</infon><infon key="section_type">REF</infon><infon key="source">FEBS Lett.</infon><infon key="type">ref</infon><infon key="volume">59</infon><infon key="year">1975</infon><offset>53277</offset><text>Presence of a hypermodified nucleotide in HeLa cell 18 S and Saccharomyces carlsbergensis 17 S ribosomal RNAs</text></passage><passage><infon key="fpage">771</infon><infon key="lpage">781</infon><infon key="name_0">surname:Buchhaupt;given-names:M.</infon><infon key="name_1">surname:Kötter;given-names:P.</infon><infon key="name_2">surname:Entian;given-names:K.D.</infon><infon key="pub-id_pmid">17425675</infon><infon key="section_type">REF</infon><infon key="source">FEMS Yeast Res.</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2007</infon><offset>53387</offset><text>Mutations in the nucleolar proteins Tma23 and Nop6 suppress the malfunction of the Nep1 protein</text></passage><passage><infon key="fpage">326</infon><infon key="lpage">338</infon><infon key="name_0">surname:Eschrich;given-names:D.</infon><infon key="name_1">surname:Buchhaupt;given-names:M.</infon><infon key="name_2">surname:Kötter;given-names:P.</infon><infon key="name_3">surname:Entian;given-names:K.D.</infon><infon key="pub-id_pmid">11935223</infon><infon key="section_type">REF</infon><infon key="source">Curr. Genet.</infon><infon key="type">ref</infon><infon key="volume">40</infon><infon key="year">2002</infon><offset>53483</offset><text>Nep1p (Emg1p), a novel protein conserved in eukaryotes and archaea, is involved in ribosome biogenesis</text></passage><passage><infon key="fpage">3</infon><infon key="name_0">surname:Armengaud;given-names:J.</infon><infon key="name_1">surname:Dedieu;given-names:A.</infon><infon key="name_2">surname:Solques;given-names:O.</infon><infon key="name_3">surname:Pellequer;given-names:J.-L.</infon><infon key="name_4">surname:Quemeneur;given-names:E.</infon><infon key="pub-id_pmid">15701177</infon><infon key="section_type">REF</infon><infon key="source">BMC Struct. Biol.</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2005</infon><offset>53586</offset><text>Deciphering structure and topology of conserved COG2042 orphan proteins</text></passage><passage><infon key="fpage">97</infon><infon key="lpage">105</infon><infon key="name_0">surname:Woese;given-names:C.R.</infon><infon key="name_1">surname:Gupta;given-names:R.</infon><infon key="name_2">surname:Hahn;given-names:C.M.</infon><infon key="name_3">surname:Zillig;given-names:W.</infon><infon key="name_4">surname:Tu;given-names:J.</infon><infon key="pub-id_pmid">11541975</infon><infon key="section_type">REF</infon><infon key="source">Syst. Appl. Microbiol.</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">1984</infon><offset>53658</offset><text>The phylogenetic relationships of three sulfur dependent archaebacteria</text></passage><passage><infon key="fpage">7962</infon><infon key="lpage">7971</infon><infon key="name_0">surname:Karcher;given-names:A.</infon><infon key="name_1">surname:Schele;given-names:A.</infon><infon key="name_2">surname:Hopfner;given-names:K.-P.</infon><infon key="pub-id_pmid">18160405</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">283</infon><infon key="year">2008</infon><offset>53730</offset><text>X-ray structure of the complete ABC enzyme ABCE1 from Pyrococcus abyssi</text></passage><passage><infon key="fpage">574</infon><infon key="lpage">585</infon><infon key="name_0">surname:Jackman;given-names:J.E.</infon><infon key="name_1">surname:Montange;given-names:R.K.</infon><infon key="name_2">surname:Malik;given-names:H.S.</infon><infon key="name_3">surname:Phizicky;given-names:E.M.</infon><infon key="pub-id_pmid">12702816</infon><infon key="section_type">REF</infon><infon key="source">RNA</infon><infon key="type">ref</infon><infon key="volume">9</infon><infon key="year">2003</infon><offset>53802</offset><text>Identification of the yeast gene encoding the tRNA m1G methyltransferase responsible for modification at position 9</text></passage><passage><infon key="fpage">509</infon><infon key="lpage">525</infon><infon key="name_0">surname:Shao;given-names:Z.</infon><infon key="name_1">surname:Yan;given-names:W.</infon><infon key="name_2">surname:Peng;given-names:J.</infon><infon key="name_3">surname:Zuo;given-names:X.</infon><infon key="name_4">surname:Zou;given-names:Y.</infon><infon key="name_5">surname:Li;given-names:F.</infon><infon key="name_6">surname:Gong;given-names:D.</infon><infon key="name_7">surname:Ma;given-names:R.</infon><infon key="name_8">surname:Wu;given-names:J.</infon><infon key="name_9">surname:Shi;given-names:Y.</infon><infon key="pub-id_pmid">24081582</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">42</infon><infon key="year">2014</infon><offset>53918</offset><text>Crystal structure of tRNA m1G9 methyltransferase Trm10: insight into the catalytic mechanism and recognition of tRNA substrate</text></passage><passage><infon key="fpage">1203</infon><infon key="lpage">1209</infon><infon key="name_0">surname:Ben-Shem;given-names:A.</infon><infon key="name_1">surname:Jenner;given-names:L.</infon><infon key="name_2">surname:Yusupova;given-names:G.</infon><infon key="name_3">surname:Yusupov;given-names:M.</infon><infon key="section_type">REF</infon><infon key="source">Science (New York, N.Y.)</infon><infon key="type">ref</infon><infon key="volume">330</infon><infon key="year">2010</infon><offset>54045</offset><text>Crystal structure of the eukaryotic ribosome</text></passage><passage><infon key="fpage">24484</infon><infon key="lpage">24489</infon><infon key="name_0">surname:Kowalak;given-names:J.A.</infon><infon key="name_1">surname:Bruenger;given-names:E.</infon><infon key="name_2">surname:Crain;given-names:P.F.</infon><infon key="name_3">surname:McCloskey;given-names:J.A.</infon><infon key="pub-id_pmid">10818097</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem.</infon><infon key="type">ref</infon><infon key="volume">275</infon><infon key="year">2000</infon><offset>54090</offset><text>Identities and phylogenetic comparisons of posttranscriptional modifications in 16 S ribosomal RNA from Haloferax volcanii</text></passage><passage><infon key="fpage">254</infon><infon key="lpage">261</infon><infon key="name_0">surname:Jones;given-names:W.</infon><infon key="name_1">surname:Leigh;given-names:J.</infon><infon key="name_2">surname:Mayer;given-names:F.</infon><infon key="name_3">surname:Woese;given-names:C.R.</infon><infon key="name_4">surname:Wolfe;given-names:R.</infon><infon key="section_type">REF</infon><infon key="source">Arch. Microbiol.</infon><infon key="type">ref</infon><infon key="volume">136</infon><infon key="year">1983</infon><offset>54213</offset><text>Methanococcus jannaschii sp. nov., an extremely thermophilic methanogen from a submarine hydrothermal vent</text></passage><passage><infon key="fpage">492</infon><infon key="lpage">497</infon><infon key="name_0">surname:Lafontaine;given-names:D.</infon><infon key="name_1">surname:Delcour;given-names:J.</infon><infon key="name_2">surname:Glasser;given-names:A.L.</infon><infon key="name_3">surname:Desgres;given-names:J.</infon><infon key="name_4">surname:Vandenhaute;given-names:J.</infon><infon key="pub-id_pmid">8064863</infon><infon key="section_type">REF</infon><infon key="source">J. Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">241</infon><infon key="year">1994</infon><offset>54320</offset><text>The DIM1 gene responsible for the conserved m6(2)Am6(2)A dimethylation in the 3'-terminal loop of 18 S rRNA is essential in yeast</text></passage><passage><infon key="fpage">3151</infon><infon key="lpage">3161</infon><infon key="name_0">surname:White;given-names:J.</infon><infon key="name_1">surname:Li;given-names:Z.</infon><infon key="name_2">surname:Sardana;given-names:R.</infon><infon key="name_3">surname:Bujnicki;given-names:J.M.</infon><infon key="name_4">surname:Marcotte;given-names:E.M.</infon><infon key="name_5">surname:Johnson;given-names:A.W.</infon><infon key="pub-id_pmid">18332120</infon><infon key="section_type">REF</infon><infon key="source">Mol. Cell. Biol.</infon><infon key="type">ref</infon><infon key="volume">28</infon><infon key="year">2008</infon><offset>54450</offset><text>Bud23 methylates G1575 of 18S rRNA and is required for efficient nuclear export of pre-40S subunits</text></passage><passage><infon key="fpage">2080</infon><infon key="lpage">2095</infon><infon key="name_0">surname:Zorbas;given-names:C.</infon><infon key="name_1">surname:Nicolas;given-names:E.</infon><infon key="name_2">surname:Wacheul;given-names:L.</infon><infon key="name_3">surname:Huvelle;given-names:E.</infon><infon key="name_4">surname:Heurgué-Hamard;given-names:V.</infon><infon key="name_5">surname:Lafontaine;given-names:D.L.J.</infon><infon key="pub-id_pmid">25851604</infon><infon key="section_type">REF</infon><infon key="source">Mol. Biol. Cell</infon><infon key="type">ref</infon><infon key="volume">26</infon><infon key="year">2015</infon><offset>54550</offset><text>The human 18S rRNA base methyltransferases DIMT1L and WBSCR22-TRMT112 but not rRNA modification are required for ribosome biogenesis</text></passage><passage><infon key="fpage">629</infon><infon key="lpage">639</infon><infon key="name_0">surname:Leulliot;given-names:N.</infon><infon key="name_1">surname:Bohnsack;given-names:M.T.</infon><infon key="name_2">surname:Graille;given-names:M.</infon><infon key="name_3">surname:Tollervey;given-names:D.</infon><infon key="name_4">surname:van Tilbeurgh;given-names:H.</infon><infon key="pub-id_pmid">18063569</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res.</infon><infon key="type">ref</infon><infon key="volume">36</infon><infon key="year">2008</infon><offset>54683</offset><text>The yeast ribosome synthesis factor Emg1 is a novel member of the superfamily of alpha/beta knot fold methyltransferases</text></passage><passage><infon key="fpage">2026</infon><infon key="lpage">2036</infon><infon key="name_0">surname:Granneman;given-names:S.</infon><infon key="name_1">surname:Petfalski;given-names:E.</infon><infon key="name_2">surname:Swiatkowska;given-names:A.</infon><infon key="name_3">surname:Tollervey;given-names:D.</infon><infon key="pub-id_pmid">20453830</infon><infon key="section_type">REF</infon><infon key="source">EMBO J.</infon><infon key="type">ref</infon><infon key="volume">29</infon><infon key="year">2010</infon><offset>54804</offset><text>Cracking pre-40S ribosomal subunit structure by systematic analyses of RNA-protein cross-linking</text></passage><passage><infon key="fpage">1449</infon><infon key="lpage">1453</infon><infon key="name_0">surname:Strunk;given-names:B.S.</infon><infon key="name_1">surname:Loucks;given-names:C.R.</infon><infon key="name_2">surname:Su;given-names:M.</infon><infon key="name_3">surname:Vashisth;given-names:H.</infon><infon key="name_4">surname:Cheng;given-names:S.</infon><infon key="name_5">surname:Schilling;given-names:J.</infon><infon key="name_6">surname:Brooks;given-names:C.L.</infon><infon key="name_7">surname:Karbstein;given-names:K.</infon><infon key="name_8">surname:Skiniotis;given-names:G.</infon><infon key="section_type">REF</infon><infon key="source">Science (New York, N.Y.)</infon><infon key="type">ref</infon><infon key="volume">333</infon><infon key="year">2011</infon><offset>54901</offset><text>Ribosome assembly factors prevent premature translation initiation by 40S assembly intermediates</text></passage><passage><infon key="fpage">15253</infon><infon key="lpage">15258</infon><infon key="name_0">surname:Hellmich;given-names:U.A.</infon><infon key="name_1">surname:Weis;given-names:B.L.</infon><infon key="name_2">surname:Lioutikov;given-names:A.</infon><infon key="name_3">surname:Wurm;given-names:J.P.</infon><infon key="name_4">surname:Kaiser;given-names:M.</infon><infon key="name_5">surname:Christ;given-names:N.A.</infon><infon key="name_6">surname:Hantke;given-names:K.</infon><infon key="name_7">surname:Kötter;given-names:P.</infon><infon key="name_8">surname:Entian;given-names:K.-D.</infon><infon key="name_9">surname:Schleiff;given-names:E.</infon><infon key="pub-id_pmid">24003121</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl. Acad. Sci. U.S.A.</infon><infon key="type">ref</infon><infon key="volume">110</infon><infon key="year">2013</infon><offset>54998</offset><text>Essential ribosome assembly factor Fap7 regulates a hierarchy of RNA-protein interactions during small ribosomal subunit biogenesis</text></passage><passage><infon key="fpage">744</infon><infon key="lpage">753</infon><infon key="name_0">surname:Lebaron;given-names:S.</infon><infon key="name_1">surname:Schneider;given-names:C.</infon><infon key="name_2">surname:van Nues;given-names:R.W.</infon><infon key="name_3">surname:Swiatkowska;given-names:A.</infon><infon key="name_4">surname:Walsh;given-names:D.</infon><infon key="name_5">surname:Böttcher;given-names:B.</infon><infon key="name_6">surname:Granneman;given-names:S.</infon><infon key="name_7">surname:Watkins;given-names:N.J.</infon><infon key="name_8">surname:Tollervey;given-names:D.</infon><infon key="pub-id_pmid">22751017</infon><infon key="section_type">REF</infon><infon key="source">Nat. Struct. Mol. Biol.</infon><infon key="type">ref</infon><infon key="volume">19</infon><infon key="year">2012</infon><offset>55130</offset><text>Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits</text></passage><passage><infon key="fpage">111</infon><infon key="lpage">121</infon><infon key="name_0">surname:Strunk;given-names:B.S.</infon><infon key="name_1">surname:Novak;given-names:M.N.</infon><infon key="name_2">surname:Young;given-names:C.L.</infon><infon key="name_3">surname:Karbstein;given-names:K.</infon><infon key="pub-id_pmid">22770215</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">150</infon><infon key="year">2012</infon><offset>55226</offset><text>A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits</text></passage></document></collection>
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+<collection><source>PMC</source><date>20201215</date><key>pmc.key</key><document><id>4880283</id><infon key="license">CC BY</infon><passage><infon key="alt-title">Crystal Structures of Putative Sugar Kinases</infon><infon key="article-id_doi">10.1371/journal.pone.0156067</infon><infon key="article-id_pmc">4880283</infon><infon key="article-id_pmid">27223615</infon><infon key="article-id_publisher-id">PONE-D-16-05184</infon><infon key="elocation-id">e0156067</infon><infon key="issue">5</infon><infon key="license">This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</infon><infon key="name_0">surname:Xie;given-names:Yuan</infon><infon key="name_1">surname:Li;given-names:Mei</infon><infon key="name_2">surname:Chang;given-names:Wenrui</infon><infon key="name_3">surname:Zeth;given-names:Kornelius</infon><infon key="name_4">surname:Chang;given-names:Wenrui</infon><infon key="name_5">surname:Li;given-names:Mei</infon><infon key="name_6">surname:Chang;given-names:Wenrui</infon><infon key="name_7">surname:Li;given-names:Mei</infon><infon key="notes">All structural files are available from the Protein Data Bank (accession numbers 5HTN, 5HTP, 5HUX, 5HV7, 5HTJ, 5HU2, 5HTY, 5HTR, 5HTV and 5HTX).</infon><infon key="section_type">TITLE</infon><infon key="title">Data Availability</infon><infon key="type">front</infon><infon key="volume">11</infon><infon key="year">2016</infon><offset>0</offset><text>Crystal Structures of Putative Sugar Kinases from Synechococcus Elongatus PCC 7942 and Arabidopsis Thaliana</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>108</offset><text>The genome of the Synechococcus elongatus strain PCC 7942 encodes a putative sugar kinase (SePSK), which shares 44.9% sequence identity with the xylulose kinase-1 (AtXK-1) from Arabidopsis thaliana. Sequence alignment suggests that both kinases belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. However, their exact physiological function and real substrates remain unknown. Here we solved the structures of SePSK and AtXK-1 in both their apo forms and in complex with nucleotide substrates. The two kinases exhibit nearly identical overall architecture, with both kinases possessing ATP hydrolysis activity in the absence of substrates. In addition, our enzymatic assays suggested that SePSK has the capability to phosphorylate D-ribulose. In order to understand the catalytic mechanism of SePSK, we solved the structure of SePSK in complex with D-ribulose and found two potential substrate binding pockets in SePSK. Using mutation and activity analysis, we further verified the key residues important for its catalytic activity. Moreover, our structural comparison with other family members suggests that there are major conformational changes in SePSK upon substrate binding, facilitating the catalytic process. Together, these results provide important information for a more detailed understanding of the cofactor and substrate binding mode as well as the catalytic mechanism of SePSK, and possible similarities with its plant homologue AtXK-1.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1612</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1625</offset><text>Carbohydrates are essential cellular compounds involved in the metabolic processes present in all organisms. Phosphorylation is one of the various pivotal modifications of carbohydrates, and is catalyzed by specific sugar kinases. These kinases exhibit considerable differences in their folding pattern and substrate specificity. Based on sequence analysis, they can be divided into four families, namely HSP 70_NBD family, FGGY family, Mer_B like family and Parm_like family. The FGGY family carbohydrate kinases contain different types of sugar kinases, all of which possess different catalytic substrates with preferences for short-chained sugar substrates, ranging from triose to heptose. These sugar substrates include L-ribulose, erythritol, L-fuculose, D-glycerol, D-gluconate, L-xylulose, D-ribulose, L-rhamnulose and D-xylulose. Structures reported in the Protein Data Bank of the FGGY family carbohydrate kinases exhibit a similar overall architecture containing two protein domains, one of which is responsible for the binding of substrate, while the second is used for binding cofactor ATP. While the binding pockets for substrates are at the same position, each FGGY family carbohydrate kinases uses different substrate-binding residues, resulting in high substrate specificity.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2917</offset><text>Synpcc7942_2462 from the cyanobacteria Synechococcus elongatus PCC 7942 encodes a putative sugar kinase (SePSK), and this kinase contains 426 amino acids. The At2g21370 gene product from Arabidopsis thaliana, xylulose kinase-1 (AtXK-1), whose mature form contains 436 amino acids, is located in the chloroplast (ChloroP 1.1 Server). SePSK and AtXK-1 display a sequence identity of 44.9%, and belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. Members of this sub-family are responsible for the phosphorylation of sugars similar to L-ribulose and D-ribulose. The sequence and the substrate specificity of ribulokinase-like carbohydrate kinases are different, but they share the common folding feature with two domains. Domain I exhibits a ribonuclease H-like folding pattern, and is responsible for the substrate binding, while domain II possesses an actin-like ATPase domain that binds cofactor ATP.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3870</offset><text>Two possible xylulose kinases (xylulose kinase-1: XK-1 and xylulose kinase-2: XK-2) from Arabidopsis thaliana were previously proposed. It was shown that XK-2 (At5g49650) located in the cytosol is indeed xylulose kinase. However, the function of XK-1 (At2g21370) inside the chloroplast stroma has remained unknown. SePSK from Synechococcus elongatus strain PCC 7942 is the homolog of AtXK-1, though its physiological function and substrates remain unclear. In order to obtain functional and structural information about these two proteins, here we reported the crystal structures of SePSK and AtXK-1. Our findings provide new details of the catalytic mechanism of SePSK and lay the foundation for future studies into its homologs in eukaryotes.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>4615</offset><text>Results and Discussion</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>4638</offset><text>Overall structures of apo-SePSK and apo-AtXK-1</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4685</offset><text>The attempt to solve the SePSK structure by molecular replacement method failed with ribulokinase from Bacillus halodurans (PDB code: 3QDK, 15.7% sequence identity) as an initial model. We therefore used single isomorphous replacement anomalous scattering method (SIRAS) for successful solution of the apo-SePSK structure at a resolution of 2.3 Å. Subsequently, the apo-SePSK structure was used as molecular replacement model to solve all other structures identified in this study.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5168</offset><text>Our structural analysis showed that apo-SePSK consists of one SePSK protein molecule in an asymmetric unit. The amino-acid residues were traced from Val2 to His419, except for the Met1 residue and the seven residues at the C-termini. Apo-SePSK contains two domains referred to further on as domain I and domain II (Fig 1A). Domain I consists of non-contiguous portions of the polypeptide chains (aa. 2–228 and aa. 402–419), exhibiting 11 α-helices and 11 β-sheets. Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. In addition, four β-sheets (β7, β10, β12 and β16) and five α-helices (α8, α9, α13, α14 and α15) flank the left side of the core region. Domain II is comprised of aa. 229–401 and classified into B2 (β31/β29/β22/β23/β25/β24) and A3 (α26/α27/α28/α30) (Fig 1A and S1 Fig). In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). The overall folding of SePSK resembles a clip, with A2 of domain I acting as a hinge region. As a consequence, a deep cleft is formed between the two domains.</text></passage><passage><infon key="file">pone.0156067.g001.jpg</infon><infon key="id">pone.0156067.g001</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>6599</offset><text>Overall structures of SePSK and AtXK-1.</text></passage><passage><infon key="file">pone.0156067.g001.jpg</infon><infon key="id">pone.0156067.g001</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>6639</offset><text>(A) Three-dimensional structure of apo-SePSK. The secondary structural elements are indicated (α-helix: cyan, β-sheet: yellow). (B) Three-dimensional structure of apo-AtXK-1. The secondary structural elements are indicated (α-helix: green, β-sheet: wheat).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6910</offset><text>Apo-AtXK-1 exhibits a folding pattern similar to that of SePSK in line with their high sequence identity (Fig 1B and S1 Fig). However, superposition of structures of AtXK-1 and SePSK shows some differences, especially at the loop regions. A considerable difference is found in the loop3 linking β3 and α4, which is stretched out in the AtXK-1 structure, while in the SePSK structure, it is bent back towards the inner part. The corresponding residues between these two structures (SePSK-Lys35 and AtXK-1-Lys48) have a distance of 15.4 Å (S3 Fig).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>7464</offset><text>Activity assays of SePSK and AtXK-1</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7500</offset><text>In order to understand the function of these two kinases, we performed structural comparison using Dali server. The structures most closely related to SePSK are xylulose kinase, glycerol kinase and ribulose kinase, implying that SePSK and AtXK-1 might function similarly to these kinases. We first tested whether both enzymes possessed ATP hydrolysis activity in the absence of substrates. As shown in Fig 2A, both SePSK and AtXK-1 exhibited ATP hydrolysis activity. This finding is in agreement with a previous result showing that xylulose kinase (PDB code: 2ITM) possessed ATP hydrolysis activity without adding substrate. To further identify the actual substrate of SePSK and AtXK-1, five different sugar molecules, including D-ribulose, L-ribulose, D-xylulose, L-xylulose and Glycerol, were used in enzymatic activity assays. As shown in Fig 2B, the ATP hydrolysis activity of SePSK greatly increased upon adding D-ribulose than adding other potential substrates, suggesting that it has D-ribulose kinase activity. In contrary, limited increasing of ATP hydrolysis activity was detected for AtXK-1 upon addition of D-ribulose (Fig 2C), despite its structural similarity with SePSK.</text></passage><passage><infon key="file">pone.0156067.g002.jpg</infon><infon key="id">pone.0156067.g002</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>8686</offset><text>The enzymatic activity assays of SePSK and AtXK-1.</text></passage><passage><infon key="file">pone.0156067.g002.jpg</infon><infon key="id">pone.0156067.g002</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>8737</offset><text>(A) The ATP hydrolysis activity of SePSK and AtXK-1. Both SePSK and AtXK-1 showed ATP hydrolysis activity in the absence of substrate. While the ATP hydrolysis activity of SePSK greatly increases upon addition of D-ribulose (DR). (B) The ATP hydrolysis activity of SePSK with addition of five different substrates. The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. Three single-site mutants of SePSK are D8A-SePSK, T11A-SePSK and D221A-SePSK. The ATP hydrolysis activity measured via luminescent ADP-Glo assay (Promega).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9478</offset><text>To understand the catalytic mechanism of SePSK, we performed structural comparisons among xylulose kinase, glycerol kinase, ribulose kinase and SePSK. Our results suggested that three conserved residues (D8, T11 and D221 of SePSK) play an important role in SePSK function. Mutations of the corresponding residue in xylulose kinase and glycerol kinase from Escherichia coli greatly reduced their activity. To identify the function of these three residues of SePSK, we constructed D8A, T11A and D221A mutants. Using enzymatic activity assays, we found that all of these mutants exhibit much lower activity of ATP hydrolysis after adding D-ribulose than that of wild type, indicating the possibility that these three residues are involved in the catalytic process of phosphorylation D-ribulose and are vital for the function of SePSK (Fig 2D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10319</offset><text>SePSK and AtXK-1 possess a similar ATP binding site</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10371</offset><text>To obtain more detailed information of SePSK and AtXK-1 in complex with ATP, we soaked the apo-crystals in the reservoir adding cofactor ATP, and obtained the structures of SePSK and AtXK-1 bound with ATP at the resolution of 2.3 Å and 1.8 Å, respectively. In both structures, a strong electron density was found in the conserved ATP binding pocket, but can only be fitted with an ADP molecule (S4 Fig). Thus the two structures were named ADP-SePSK and ADP-AtXK-1, respectively. The extremely weak electron densities of ATP γ-phosphate in both structures suggest that the γ-phosphate group of ATP is either flexible or hydrolyzed by SePSK and AtXK-1. This result was consistent with our enzymatic activity assays where SePSK and AtXK-1 showed ATP hydrolysis activity without adding any substrates (Fig 2A and 2C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11193</offset><text>To avoid hydrolysis of ATP, we soaked the crystals of apo-SePSK and apo-AtXK-1 into the reservoir adding AMP-PNP. However, we found that the electron densities of γ-phosphate group of AMP-PNP (AMP-PNP γ-phosphate) are still weak in the AMP-PNP-SePSK and AMP-PNP-AtXK-1 structures, suggesting high flexibility of ATP-γ-phosphate. The γ-phosphate group of ATP is transferred to the sugar substrate during the reaction process, so this flexibility might be important for the ability of these kinases. The overall structures as well as the coordination modes of ADP and AMP-PNP in the AMP-PNP-AtXK-1, ADP-AtXK-1, ADP-SePSK and AMP-PNP-SePSK structures are nearly identical (S5 Fig), therefore the structure of AMP-PNP-SePSK is used here to describe the structural details and to compare with those of other family members. As shown in Fig 3A, one SePSK protein molecule is in an asymmetric unit with one AMP-PNP molecule. The AMP-PNP is bound at the domain II, where it fits well inside a positively charged groove. The AMP-PNP binding pocket consists of four α-helices (α26, α28, α27 and α30) and forms a shape resembling a half-fist (Fig 3A and 3B). The head group of the AMP-PNP is embedded in a pocket surrounded by Trp383, Asn380, Gly376 and Gly377. The purine ring of AMP-PNP is positioned in parallel to the indole ring of Trp383. In addition, it is hydrogen-bonded with the side chain amide of Asn380 (Fig 3B). The tail of AMP-PNP points to the hinge region of SePSK, and its α-phosphate and β-phosphate groups are stabilized by Gly376 and Ser243, respectively. Together, this structure clearly shows that the AMP-PNP-β-phosphate is sticking out of the ATP binding pocket, thus the γ-phosphate group is at the empty space between domain I and domain II and is unconstrained in its movement by the protein.</text></passage><passage><infon key="file">pone.0156067.g003.jpg</infon><infon key="id">pone.0156067.g003</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>13053</offset><text>Structure of SePSK in complex with AMP-PNP.</text></passage><passage><infon key="file">pone.0156067.g003.jpg</infon><infon key="id">pone.0156067.g003</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>13097</offset><text>(A) The electron density of AMP-PNP. The SePSK structure is shown in the electrostatic potential surface mode. The AMP-PNP is depicted as sticks with its ǀFoǀ-ǀFcǀ map contoured at 3 σ shown as cyan mesh. (B) The AMP-PNP binding pocket. The head of AMP-PNP is sandwiched by four residues (Leu293, Gly376, Gly377 and Trp383). The protein skeleton is shown as cartoon (cyan). The four α-helices (α26, α28, α27 and α30) are labeled in red. The AMP-PNP and coordinated residues are shown as sticks. The interactions between them are represented as black dashed lines. The numerical note near the black dashed line indicates the distance (Å).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13759</offset><text>The potential substrate binding site in SePSK</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13805</offset><text>The results from our activity assays suggested that SePSK has D-ribulose kinase activity. To better understand the interaction pattern between SePSK and D-ribulose, the apo-SePSK crystals were soaked into the reservoir with 10 mM D-ribulose (RBL) and the RBL-SePSK structure was solved. As shown in S6 Fig, two residual electron densities are visible in domain I, which can be interpreted as two D-ribulose molecules with reasonable fit.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14243</offset><text>As shown in Fig 4A, the nearest distance between the carbon skeleton of two D-ribulose molecules are approx. 7.1 Å (RBL1-C4 and RBL2-C1). RBL1 is located in the pocket consisting of α21 and the loop between β6 and β7. The O4 and O5 of RBL1 are coordinated with the side chain carboxyl group of Asp221. Furthermore, the O2 of RBL1 interacts with the main chain amide nitrogen of Ser72 (Fig 4B). This pocket is at a similar position of substrate binding site of other sugar kinase, such as L-ribulokinase (PDB code: 3QDK) (S7 Fig). However, structural comparison shows that the substrate ligating residues between the two structures are not strictly conserved. Based on the structures, the ligating residues of RBL1 in RBL-SePSK structure are Ser72, Asp221 and Ser222, and the interacting residues of L-ribulose with L-ribulokinase are Ala96, Lys208, Asp274 and Glu329 (S7 Fig). Glu329 in 3QDK has no counterpart in RBL-SePSK structure. In addition, although Lys208 of L-ribulokinase has the corresponding residue (Lys163) in RBL-SePSK structure, the hydrogen bond of Lys163 is broken because of the conformational change of two α-helices (α9 and α13) of SePSK. These differences might account for their different substrate specificity.</text></passage><passage><infon key="file">pone.0156067.g004.jpg</infon><infon key="id">pone.0156067.g004</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>15500</offset><text>The binding of D-ribulose (RBL) with SePSK.</text></passage><passage><infon key="file">pone.0156067.g004.jpg</infon><infon key="id">pone.0156067.g004</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>15544</offset><text>(A) The electrostatic potential surface map of RBL-SePSK and a zoom-in view of RBL binding site. The RBL1 and RBL2 are depicted as sticks. (B) Interaction of two D-ribulose molecules (RBL1 and RBL2) with SePSK. The RBL molecules (carbon atoms colored yellow) and amino acid residues of SePSK (carbon atoms colored green) involved in RBL interaction are shown as sticks. The hydrogen bonds are indicated by the black dashed lines and the numbers near the dashed lines are the distances (Å). (C) The binding affinity assays of SePSK with D-ribulose. Single-cycle kinetic data are reflecting the interaction of SePSK and D8A-SePSK with D-ribulose. It shows two experimental sensorgrams after minus the empty sensorgrams. The original data is shown as black curve, and the fitted data is shown as different color (wild type SePSK: red curve, D8A-SePSK: green curve). Dissociation rate constant of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16499</offset><text>The binding pocket of RBL2 with relatively weak electron density is near the N-terminal region of SePSK and is negatively charged. The side chain of Asp8 interacts strongly with O3 and O4 of RBL2. The hydroxyl group of Ser12 coordinates with O2 of RBL2. The backbone amide nitrogens of Gly13 and Arg15 also keep hydrogen bonds with RBL2 (Fig 4B). Structural comparison of SePSK and AtXK-1 showed that while the RBL1 binding pocket is conserved, the RBL2 pocket is disrupted in AtXK-1 structure, despite the fact that the residues interacting with RBL2 are highly conserved between the two proteins. In the RBL-SePSK structure, a 2.6 Å hydrogen bond is present between RBL2 and Ser12 (Fig 4B), while in the AtXK-1 structure this hydrogen bond with the corresponding residue (Ser22) is broken. This break is probably induced by the conformational change of the two β-sheets (β1 and β2), with the result that the linking loop (loop 1) is located further away from the RBL2 binding site. This change might be the reason that AtXK-1 only shows limited increasing in its ATP hydrolysis ability upon adding D-ribulose as a substrate after comparing with SePSK (Fig 2C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17671</offset><text>Our SePSK structure shows that the Asp8 residue forms strong hydrogen bond with RBL2 (Fig 4B). In addition, our enzymatic assays indicated that Asp8 is important for the activity of SePSK (Fig 2D). To further verified this result, we measured the binding affinity for D-ribulose of both wild type (WT) and D8A mutant of SePSK using a surface plasmon resonance method. The results showed that the affinity of D8A-SePSK with D-ribulose is weaker than that of WT with a reduction of approx. two third (Fig 4C). Dissociation rate constant (Kd) of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively. The results implied that the second RBL binding site plays a role in the D-ribulose kinase function of SePSK. However, considering the high concentration of D-ribulose used for crystal soaking, as well as the relatively weak electron density of RBL2, it is also possible that the second binding site of D-ribulose in SePSK is an artifact.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>18614</offset><text>Simulated conformational change of SePSK during the catalytic process</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18684</offset><text>It was reported earlier that the crossing angle between the domain I and domain II in FGGY family carbohydrate kinases is different. In addition, this difference may be caused by the binding of substrates and/or ATP. As reported previously, members of the sugar kinase family undergo a conformational change to narrow the crossing angle between two domains and reduce the distance between substrate and ATP in order to facilitate the catalytic reaction of phosphorylation of sugar substrates. After comparing structures of apo-SePSK, RBL-SePSK and AMP-PNP-SePSK, we noticed that these structures presented here are similar. Superposing the structures of RBL-SePSK and AMP-PNP-SePSK, the results show that the nearest distance between AMP-PNP γ-phosphate and RBL1/RBL2 is 7.5 Å (RBL1-O5)/6.7 Å (RBL2-O1) (S8 Fig). This distance is too long to transfer the γ-phosphate group from ATP to the substrate. Since the two domains of SePSK are widely separated in this structure, we hypothesize that our structures of SePSK represent its open form, and that a conformational rearrangement must occur to switch to the closed state in order to facilitate the catalytic process of phosphorylation of sugar substrates.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19898</offset><text>For studying such potential conformational change, a simulation on the Hingeprot Server was performed to predict the movement of different SePSK domains. The results showed that domain I and domain II are closer to each other with Ala228 and Thr401 in A2 as Hinge-residues. Based on the above results, SePSK is divided into two rigid parts. The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). The results of superposition displayed different crossing angle between these two domains. After superposition, the distances of AMP-PNP γ-phosphate and the fifth hydroxyl group of RBL1 are 7.9 Å (superposed with AtXK-1), 7.4 Å (superposed with SePSK), 6.6 Å (superposed with 3LL3) and 6.1 Å (superposed with 1GLJ). Meanwhile, the distances of AMP-PNP γ-phosphate and the first hydroxyl group of RBL2 are 7.2 Å (superposed with AtXK-1), 6.7 Å (superposed with SePSK), 3.7 Å (superposed with 3LL3), until AMP-PNP γ-phosphate fully contacts RBL2 after superposition with 1GLJ (Fig 5). This distance between RBL2 and AMP-PNP-γ-phosphate is close enough to facilitate phosphate transferring. Together, our superposition results provided snapshots of the conformational changes at different catalytic stages of SePSK and potentially revealed the closed form of SePSK.</text></passage><passage><infon key="file">pone.0156067.g005.jpg</infon><infon key="id">pone.0156067.g005</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>21433</offset><text>Simulated conformational change of SePSK during the catalytic process.</text></passage><passage><infon key="file">pone.0156067.g005.jpg</infon><infon key="id">pone.0156067.g005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>21504</offset><text>The structures are shown as cartoon and the ligands are shown as sticks. Domain I from D-ribulose-SePSK (green) and Domain II from AMP-PNP-SePSK (cyan) are superposed with apo-AtXK-1 (1st), apo-SePSK (2nd), 3LL3 (3rd) and 1GLJ (4th), respectively. The numbers near the black dashed lines show the distances (Å) between two nearest atoms of RBL and AMP-PNP.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21862</offset><text>In summary, our structural and enzymatic analyses provide evidence that SePSK shows D-ribulose kinase activity, and exhibits the conserved features of FGGY family carbohydrate kinases. Three conserved residues in SePSK were identified to be essential for this function. Our results provide the detailed information about the interaction of SePSK with ATP and substrates. Moreover, structural superposition results enable us to visualize the conformational change of SePSK during the catalytic process. In conclusion, our results provide important information for a more detailed understanding of the mechanisms of SePSK and other members of FGGY family carbohydrate kinases.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>22537</offset><text>Materials and Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>22559</offset><text>Cloning, expression and purification of SePSK</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>22605</offset><text>The gene encoding SePSK was amplified by polymerase chain reaction (PCR) with forward primer 5' CATGCCATGGGCATGGTCGTTGCACTTGGCCTCG 3' containing an internal Nco I restriction site (underlined) and reverse primer 5' CCGCTCGAGGGTTCTCTTTAACCCCGCCG 3' including an internal Xho I restriction site (underlined) from Synechococcus elongatus PCC 7942 genomic DNA. The amplified PCR product was digested with Nco I and Xho I (Takara) and ligated into linearized pET28-a vector (Novagen) between Nco I and Xho I restriction sites with a C-terminal his6 tag. The recombinant plasmids were transformed into competent Escherichia coli Trans10 cells for DNA production and purification, and the final constructs were verified by sequencing. The recombinant vectors were transformed into Escherichia coli BL21 (DE3) to express the protein. After induction with the 1 mM IPTG at 289 K in Luria-Bertani medium until the cell density reached an OD 600 nm of 0.6–0.8, the cells were harvested by centrifugation at 6,000 g at 4°C for 15 min, re-suspended in buffer A (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole) and disrupted by sonication. After centrifuge 40,000 g for 30 min, the protein was purified by passage through a Ni2+ affinity column in buffer A, and then washed the unbound protein with buffer B (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 60 mM imidazole), and eluted the fraction with the buffer C (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 500 mM imidazole). After that, the protein was further purified by size exclusion chromatography with Superdex 200 10/300 GL (GE Healthcare) equilibrated with the buffer D (20 mM Tris-HCl, pH 8.0, 300 mM NaCl). The eluted major peak fraction was concentrated to 20 mg/mL protein using 10,000 MCWO centrifugal filter units (Millipore) and stored at -80°C for crystallization trials. The purified product was analyzed by SDS-PAGE with a single band visible only.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24505</offset><text>Cloning, expression and purification of AtXK-1</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24552</offset><text>The gene encoding AtXK-1 was amplified by PCR using a forward primer 5' TACTTCCAATCCAATGCTGTTATGAGTGGCAATAAAGGAACGA 3' and reverse primer 5' TTATCCACTTCCAATGTTACAAACCACTGTTCTGTTTTGCGCCC 3' from cDNA library of Arabidopsis thaliana. The underlined nucleotides were used for the ligation-independent cloning. The PCR product was treated by T4 DNA polymerase (LIC-qualified, Novagen) and then cloned into linearized pMCSG7 vector treated by T4 DNA polymerase (LIC-qualified, Novagen) with an N-terminal his6 tag though ligation-independent cloning method. The final construct was confirmed by DNA sequencing after amplified in competent Escherichia coli Trans10 cells. The recombinant vectors were transformed into Escherichia coli BL21 (DE3) for protein expression. After induction with 1 mM IPTG at 289 K in Luria-Bertani medium, cells were grown until the cell density reached an OD 600 nm of 0.6–0.8. Subsequent purification was identical to that used for SePSK, except that there was one additional step, during which tobacco etch virus protease was used to digest the crude AtXK-1 protein for removal of the N-terminal his6 tag following Ni2+ affinity purification. Ni2+ affinity column buffer contained extra 20% glycerol. The protein was further purified by size exclusion chromatography with Superdex 200 10/300 GL (GE Healthcare) in elution buffer consisting of 20 mM HEPES, pH 7.5, 100 mM NaCl. Finally, AtXK-1 protein was concentrated to 40 mg/mL protein using 10,000 MCWO centrifugal filter units (Millipore) and stored at -80°C prior to crystallization trials. Purity was verified by SDS-PAGE with a single band visible only.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>26192</offset><text>Site-directed mutagenesis of SePSK</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26227</offset><text>The gene of D8A and T11A mutations were amplified by PCR with the forward primer 5' CATGCCATGGGCATGGTCGTTGCACTTGGCCTCGCCTTCGGCAC 3' and forward primer 5' CATGCCATGGGCATGGTCGTTGCACTTGGCCTCGACTTCGGCGCCTCTGGAGCCC 3' (mismatched base pairs are underlined). The reverse primers of D8A and T11A mutants, the further constructions and purification procedures were identical with those used for wild type SePSK.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26631</offset><text>The N-terminal sequence of D221A was amplified with forward primer (T7 promoter primer) 5' TAATACGACTCACTATA 3' and reverse primer 5' AGCAGCAATGCTAGCCGTTGTACCG 3’, and the C-terminal sequence of D221A was amplified with forward primer 5' TGCCGGTACAACGGCTAGCATTGCT 3' and reverse primer (T7 terminator primer) CGATCAATAACGAGTCGCC (mismatched base pairs are underlined). The second cycle PCR used the above PCR products as templates, and the construction and purification procedures were identical to those used for wild type SePSK.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>27164</offset><text>Crystallization and data collection</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>27200</offset><text>Crystallization trials of SePSK and AtXK-1 were carried out at 281 K by mixing equal volume of 20 mg/ml protein and reservoir solution with the sitting-drop vapor diffusion method. The reservoir solution was PEG Rx I-35 (0.1 M BIS-TRIS pH 6.5, 20% w/v Polyethylene glycol monomethyl ether 5,000) (Hampton research). After 2 or 3 days, the rod-like crystals could be observed. For phasing, the high-quality apo-SePSK crystals were soaked in mother liquor containing 1 mM ethylmercuricthiosalicylic acid, sodium salt (Hampton research, heavy atom kit) overnight at 281 K. In order to get the complexes with ATP and AMP-PNP, the crystals of apo-SePSK and apo-AtXK-1 were incubated with the reservoir including 10 mM ATP and 20 mM MgCl2 as well as 10 mM AMP-PNP and 20 mM MgCl2, respectively. The apo-SePSK crystals were incubated with the reservoir including 10 mM D-ribulose in order to obtain the complex D-ribulose-bound SePSK (RBL-SePSK). The crystals of three mutants (D8A, T11A and D221A) grew in the same condition as that of the wild type SePSK. The crystals were dipped into reservoir solution supplemented with 15% glycerol and then flash frozen in a nitrogen gas stream at 100 K. All data sets were collected at Shanghai Synchrotron Radiation Facility, Photo Factory in Japan and Institute of Biophysics, Chinese Academy of Sciences. Diffraction data were processed using the HKL2000 package.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>28601</offset><text>Structure determination and refinement</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28640</offset><text>The initial phases of SePSK were obtained from the Hg-derivative crystals by single isomorphous replacement anomalous scattering (SIRAS) using AutoSol from the PHENIX suite. AutoBuild from the PHENIX suite was used to build 75% of the main chain of apo-SePSK, and the remaining residues were built manually by Coot. All other structures were solved by molecular replacement method using apo-SePSK as an initial model. The model was refined using phenix.refine and REFMAC5. The final model was checked with PROCHECK. All structural figures were prepared by PyMOL. The summary of the data-collection and structure-refinement statistics is shown in Table 1 and S1 Table. Atomic coordinates and structure factors in this article have been deposited in the Protein Data Bank. The deposited codes of all structures listed in the Table 1 and S1 Table.</text></passage><passage><infon key="file">pone.0156067.t001.xml</infon><infon key="id">pone.0156067.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>29485</offset><text>Data collection and refinement statistics.</text></passage><passage><infon key="file">pone.0156067.t001.xml</infon><infon key="id">pone.0156067.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;colgroup span=&quot;1&quot;&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;/colgroup&gt;&lt;thead&gt;&lt;tr&gt;&lt;th align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Data set&lt;/th&gt;&lt;th align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Hg-SePSK&lt;/th&gt;&lt;th align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;apo-SePSK&lt;/th&gt;&lt;th align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;AMP-PNP-SePSK&lt;/th&gt;&lt;th align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;RBL-SePSK&lt;/th&gt;&lt;th align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;apo-AtXK-1&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Data collection&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Space group&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;C 1 2 1&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;C 1 2 1&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;C 1 2 1&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;C 1 2 1&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;P21&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Wavelength (Å)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.54178&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.54178&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.54178&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.54178&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.54178&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Cell parameters&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;a/b/c(Å)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;103.1, 46.6, 88.3&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;110.2, 49.0, 86.9&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;103.5, 46.6, 88.0&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;102.6, 47.0, 88.7&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;49.7, 87.9, 53.6&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;α/β/γ(°)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90.0, 91.9, 90.0&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90.0, 110.3, 90.0&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90.0, 91.0, 90.0&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90.0, 91.4, 90.0&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90.0, 97.0, 90.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution (Å)&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t001fn001&quot;&gt;&lt;sup&gt;a&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;50.00–2.20(2.28–2.20)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;50.00–2.30(2.38–2.30)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;50.00–2.30(2.38–2.30)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;50.00–2.35(2.43–2.35)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;50.00–2.00(2.07–2.00)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;R merge&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t001fn002&quot;&gt;&lt;sup&gt;b&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.105(0.514)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.149(0.501)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.082(0.503)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.095(0.507)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.106(0.454)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;〈 I/σ(I)〉&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;28.89(4.07)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;13.85(4.10)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;10.18(1.79)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;19.4(4.6)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;12.91(4.08)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Completeness (%)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;92.3(99.2)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;96.1(94.2)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;98.9(99.8)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.8(100.0)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;97.1(94.5)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Redundancy&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.7(5.1)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;7.4(7.5)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.4(2.4)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.9(6.7)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;7.2(6.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Refinement statistics&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution (Å)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;32.501–2.301&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;24.707–2.300&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;24.475–2.344&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;23.771–1.998&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;R&lt;sub&gt;work&lt;/sub&gt;/ R&lt;sub&gt;free&lt;/sub&gt;&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t001fn003&quot;&gt;&lt;sup&gt;c&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1834/0.2276&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1975/0.2327&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.2336/0.2687&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1893/0.2161&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;No. atoms&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Protein&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3503&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3196&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3209&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3256&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ligand/ion&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;-&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;31&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;20&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;-&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Water&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;313&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;146&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;143&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;486&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;RMSD Bond lengths (Å)&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t001fn004&quot;&gt;&lt;sup&gt;d&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.003&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.005&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.003&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.003&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;RMSD Bond angles (°)&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t001fn004&quot;&gt;&lt;sup&gt;d&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.674&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.886&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.649&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.838&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Ramachandran plot (%)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;favoured&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;98.1&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;97.8&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;96.7&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;allowed&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.9&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.2&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.3&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.9&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;disallowed&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.0&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.0&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.0&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PDB code&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5HTN&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5HTP&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5HV7&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5HTR&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>29528</offset><text>Data set	Hg-SePSK	apo-SePSK	AMP-PNP-SePSK	RBL-SePSK	apo-AtXK-1	 	Data collection						 	Space group	C 1 2 1	C 1 2 1	C 1 2 1	C 1 2 1	P21	 	Wavelength (Å)	1.54178	1.54178	1.54178	1.54178	1.54178	 	Cell parameters						 	a/b/c(Å)	103.1, 46.6, 88.3	110.2, 49.0, 86.9	103.5, 46.6, 88.0	102.6, 47.0, 88.7	49.7, 87.9, 53.6	 	α/β/γ(°)	90.0, 91.9, 90.0	90.0, 110.3, 90.0	90.0, 91.0, 90.0	90.0, 91.4, 90.0	90.0, 97.0, 90.0	 	Resolution (Å)a	50.00–2.20(2.28–2.20)	50.00–2.30(2.38–2.30)	50.00–2.30(2.38–2.30)	50.00–2.35(2.43–2.35)	50.00–2.00(2.07–2.00)	 	R mergeb	0.105(0.514)	0.149(0.501)	0.082(0.503)	0.095(0.507)	0.106(0.454)	 	〈 I/σ(I)〉	28.89(4.07)	13.85(4.10)	10.18(1.79)	19.4(4.6)	12.91(4.08)	 	Completeness (%)	92.3(99.2)	96.1(94.2)	98.9(99.8)	99.8(100.0)	97.1(94.5)	 	Redundancy	6.7(5.1)	7.4(7.5)	2.4(2.4)	6.9(6.7)	7.2(6.9)	 	Refinement statistics						 	Resolution (Å)		32.501–2.301	24.707–2.300	24.475–2.344	23.771–1.998	 	Rwork/ Rfreec		0.1834/0.2276	0.1975/0.2327	0.2336/0.2687	0.1893/0.2161	 	No. atoms						 	Protein		3503	3196	3209	3256	 	ligand/ion		-	31	20	-	 	Water		313	146	143	486	 	RMSD Bond lengths (Å)d		0.003	0.005	0.003	0.003	 	RMSD Bond angles (°)d		0.674	0.886	0.649	0.838	 	Ramachandran plot (%)						 	favoured		98.1	97.8	96.7	99.1	 	allowed		1.9	2.2	3.3	0.9	 	disallowed		0.0	0.0	0.0	0.0	 	PDB code		5HTN	5HTP	5HV7	5HTR	 	</text></passage><passage><infon key="file">pone.0156067.t001.xml</infon><infon key="id">pone.0156067.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>30913</offset><text>a The values in parentheses correspond to the highest resolution shell.</text></passage><passage><infon key="file">pone.0156067.t001.xml</infon><infon key="id">pone.0156067.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>30985</offset><text>b Rmerge = ∑j∑h|Ij,h-&lt;Ih&gt;|/∑j∑h&lt;Ih&gt; where h are unique reflection indices and Ij,h are intensities of symmetry-related reflections and &lt;Ih&gt; is the mean intensity.</text></passage><passage><infon key="file">pone.0156067.t001.xml</infon><infon key="id">pone.0156067.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>31156</offset><text>c R-work and R-free were calculated as follows: R = Σ (|Fobs-Fcalc|)/Σ |Fobs| ×100, where Fobs and Fcalc are the observed and calculated structure factor amplitudes, respectively.</text></passage><passage><infon key="file">pone.0156067.t001.xml</infon><infon key="id">pone.0156067.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>31344</offset><text>d Root mean square deviations (r.m.s.d.) from standard values.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>31407</offset><text>ADP-Glo kinase assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31428</offset><text>ADP-Glo kinase assay was used according to the manufacturer’s instructions (Promega). Each reaction mixture system consisted of 8 uM enzyme, 100 uM ATP, 1 mM MgCl2, 20 mM HEPES (pH 7.4), 5 mM substrate. The reaction was initiated by adding the purified enzyme into the reaction system. After incubation at 298 K for different time, equal volume ADP-Glo™ reagent was added to terminate the kinase reaction and to deplete any remaining ATP. Subsequently, kinase detection reagent with double volume of reaction system was added to convert ADP to ATP and allowed the newly synthesized ATP to be measured using a luciferase/luciferin reaction which produced luminescence signal and could be recorded. After incubation at room temperature for another 60 min, luminescence was detected by Varioskan Flash Multimode Reader (Thermo). The reference experiment was carried out in the same reaction system without the enzyme. For each assay, at least three repeats were performed for the calculation of mean values and standard deviations (SDs). The purity of five substrates in the activity assays was ≥98% (D-ribulose, Santa cruz), 99.7% (L-ribulose, Carbosynth), 99.3% (D-xylulose, Carbosynth), 99.5% (L-xylulose, Carbosynth) and 99.0% (Glycerol, AMRESCO).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>32683</offset><text>Surface plasmon resonance</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>32709</offset><text>Surface plasmon resonance (SPR) was used to analyze the interaction of SePSK and D-ribulose. The SPR experiments were performed on a Biacore T100 system using series S CM5 sensor chips (GE Healthcare). All sensorgrams were recorded at 298 K. The proteins in buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCl, was diluted to 40 ug/ml by 10 mM sodium acetate buffer at pH 4.5. All flow cells on a CM5 sensor chip were activated with a freshly prepared solution of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.05 M N-hydroxysuccinimide (NHS) in a ratio of 1:1 at a constant flow rate of 10 ul/min for 420 s. Deactivation of the surface was performed with an injection of a 1 M solution of ethanolamine-HCl (pH 8.5) using the same flow rate and duration. Kinetic parameters were derived from data sets acquired in single-cycle mode. Each run consisted of five consecutive analytic injections at 125, 250, 500, 1000 and 2000 uM. Analytic injections lasted for 60 s, separated by 30 s dissociation periods. Each cycle was completed with an extended dissociation period of 300 s. The specific binding to a blank flow cell was subtracted to obtain corrected sensorgrams. Biacore data were analyzed using BiaEvaluation software (GE Healthcare) by fitting to a 1:1 Langmuir binding fitting model.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>34017</offset><text>Accession Codes</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34033</offset><text>Coordinates and structure factors for all the structures in this article have been deposited in the Protein Data Bank. These accession codes are 5HTN, 5HTP, 5HUX, 5HV7, 5HTJ, 5HU2, 5HTY, 5HTR, 5HTV and 5HTX. The corresponding-structures are apo-SePSK, AMP-PNP-SePSK, ADP-SePSK, RBL-SePSK, D8A-SePSK, T11A-SePSK, D221A-SePSK, apo-AtXK1, AMP-PNP-AtXK1 and ADP-AtXK1, respectively.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>34412</offset><text>Supporting Information</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>34435</offset><text>References</text></passage><passage><infon key="fpage">e1002318</infon><infon key="issue">12</infon><infon key="name_0">surname:Zhang;given-names:Y</infon><infon key="name_1">surname:Zagnitko;given-names:O</infon><infon key="name_2">surname:Rodionova;given-names:I</infon><infon key="name_3">surname:Osterman;given-names:A</infon><infon key="name_4">surname:Godzik;given-names:A</infon><infon key="pub-id_doi">10.1371/journal.pcbi.1002318</infon><infon key="pub-id_pmid">22215998</infon><infon key="section_type">REF</infon><infon key="source">PLoS computational biology</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2011</infon><offset>34446</offset><text>The FGGY carbohydrate kinase family: insights into the evolution of functional specificities</text></passage><passage><infon key="fpage">1643</infon><infon key="issue">3</infon><infon key="lpage">52</infon><infon key="name_0">surname:Bunker;given-names:RD</infon><infon key="name_1">surname:Bulloch;given-names:EM</infon><infon key="name_2">surname:Dickson;given-names:JM</infon><infon key="name_3">surname:Loomes;given-names:KM</infon><infon key="name_4">surname:Baker;given-names:EN</infon><infon key="pub-id_doi">10.1074/jbc.M112.427997</infon><infon key="pub-id_pmid">23179721</infon><infon key="section_type">REF</infon><infon key="source">Journal of Biological Chemistry</infon><infon key="type">ref</infon><infon key="volume">288</infon><infon key="year">2013</infon><offset>34539</offset><text>Structure and function of human xylulokinase, an enzyme with important roles in carbohydrate metabolism</text></passage><passage><infon key="fpage">261</infon><infon key="issue">1</infon><infon key="lpage">8</infon><infon key="name_0">surname:Agarwal;given-names:R</infon><infon key="name_1">surname:Burley;given-names:SK</infon><infon key="name_2">surname:Swaminathan;given-names:S</infon><infon key="pub-id_doi">10.1002/prot.23202</infon><infon key="pub-id_pmid">22072612</infon><infon key="section_type">REF</infon><infon key="source">Proteins</infon><infon key="type">ref</infon><infon key="volume">80</infon><infon key="year">2012</infon><offset>34643</offset><text>Structural insight into mechanism and diverse substrate selection strategy of L-ribulokinase</text></passage><passage><infon key="fpage">783</infon><infon key="issue">3</infon><infon key="lpage">98</infon><infon key="name_0">surname:Di Luccio;given-names:E</infon><infon key="name_1">surname:Petschacher;given-names:B</infon><infon key="name_2">surname:Voegtli;given-names:J</infon><infon key="name_3">surname:Chou;given-names:H-T</infon><infon key="name_4">surname:Stahlberg;given-names:H</infon><infon key="name_5">surname:Nidetzky;given-names:B</infon><infon key="pub-id_pmid">17123542</infon><infon key="section_type">REF</infon><infon key="source">Journal of molecular biology</infon><infon key="type">ref</infon><infon key="volume">365</infon><infon key="year">2007</infon><offset>34736</offset><text>Structural and Kinetic Studies of Induced Fit in Xylulose Kinase from Escherichia coli</text></passage><passage><infon key="fpage">978</infon><infon key="issue">5</infon><infon key="lpage">84</infon><infon key="name_0">surname:Emanuelsson;given-names:O</infon><infon key="name_1">surname:Nielsen;given-names:H</infon><infon key="name_2">surname:Von Heijne;given-names:G</infon><infon key="pub-id_pmid">10338008</infon><infon key="section_type">REF</infon><infon key="source">Protein Sci</infon><infon key="type">ref</infon><infon key="volume">8</infon><infon key="year">1999</infon><offset>34823</offset><text>ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites</text></passage><passage><infon key="fpage">441</infon><infon key="issue">2</infon><infon key="lpage">57</infon><infon key="name_0">surname:Hemmerlin;given-names:A</infon><infon key="name_1">surname:Tritsch;given-names:D</infon><infon key="name_2">surname:Hartmann;given-names:M</infon><infon key="name_3">surname:Pacaud;given-names:K</infon><infon key="name_4">surname:Hoeffler;given-names:J-F</infon><infon key="name_5">surname:van Dorsselaer;given-names:A</infon><infon key="pub-id_pmid">16920870</infon><infon key="section_type">REF</infon><infon key="source">Plant physiology</infon><infon key="type">ref</infon><infon key="volume">142</infon><infon key="year">2006</infon><offset>34931</offset><text>A cytosolic Arabidopsis D-xylulose kinase catalyzes the phosphorylation of 1-deoxy-D-xylulose into a precursor of the plastidial isoprenoid pathway</text></passage><passage><infon key="fpage">3508</infon><infon key="issue">12</infon><infon key="lpage">18</infon><infon key="name_0">surname:Bystrom;given-names:CE</infon><infon key="name_1">surname:Pettigrew;given-names:DW</infon><infon key="name_2">surname:Branchaud;given-names:BP</infon><infon key="name_3">surname:O'Brien;given-names:P</infon><infon key="name_4">surname:Remington;given-names:SJ</infon><infon key="pub-id_pmid">10090737</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry</infon><infon key="type">ref</infon><infon key="volume">38</infon><infon key="year">1999</infon><offset>35079</offset><text>Crystal structures of Escherichia coli glycerol kinase variant S58→ W in complex with nonhydrolyzable ATP analogues reveal a putative active conformation of the enzyme as a result of domain motion</text></passage><passage><infon key="fpage">1407</infon><infon key="issue">11</infon><infon key="lpage">18</infon><infon key="name_0">surname:Feese;given-names:MD</infon><infon key="name_1">surname:Faber;given-names:HR</infon><infon key="name_2">surname:Bystrom;given-names:CE</infon><infon key="name_3">surname:Pettigrew;given-names:DW</infon><infon key="name_4">surname:Remington;given-names:SJ</infon><infon key="pub-id_pmid">9817843</infon><infon key="section_type">REF</infon><infon key="source">Structure</infon><infon key="type">ref</infon><infon key="volume">6</infon><infon key="year">1998</infon><offset>35278</offset><text>Glycerol kinase from Escherichia coli and an Ala65→ Thr mutant: the crystal structures reveal conformational changes with implications for allosteric regulation</text></passage><passage><infon key="fpage">787</infon><infon key="issue">3</infon><infon key="lpage">97</infon><infon key="name_0">surname:Grueninger;given-names:D</infon><infon key="name_1">surname:Schulz;given-names:GE</infon><infon key="pub-id_pmid">16674975</infon><infon key="section_type">REF</infon><infon key="source">Journal of molecular biology</infon><infon key="type">ref</infon><infon key="volume">359</infon><infon key="year">2006</infon><offset>35441</offset><text>Structure and Reaction Mechanism of l-Rhamnulose Kinase from Escherichia coli</text></passage><passage><infon key="fpage">1469</infon><infon key="issue">7</infon><infon key="lpage">82</infon><infon key="name_0">surname:Higgins;given-names:MA</infon><infon key="name_1">surname:Suits;given-names:MD</infon><infon key="name_2">surname:Marsters;given-names:C</infon><infon key="name_3">surname:Boraston;given-names:AB</infon><infon key="pub-id_doi">10.1016/j.jmb.2013.12.006</infon><infon key="pub-id_pmid">24333485</infon><infon key="section_type">REF</infon><infon key="source">Journal of molecular biology</infon><infon key="type">ref</infon><infon key="volume">426</infon><infon key="year">2014</infon><offset>35519</offset><text>Structural and Functional Analysis of Fucose-Processing Enzymes from Streptococcus pneumoniae</text></passage><passage><infon key="fpage">236</infon><infon key="issue">2</infon><infon key="lpage">45</infon><infon key="name_0">surname:Pettigrew;given-names:DW</infon><infon key="name_1">surname:Smith;given-names:GB</infon><infon key="name_2">surname:Thomas;given-names:KP</infon><infon key="name_3">surname:D'Nette;given-names:CD</infon><infon key="pub-id_pmid">9448710</infon><infon key="section_type">REF</infon><infon key="source">Archives of biochemistry and biophysics</infon><infon key="type">ref</infon><infon key="volume">349</infon><infon key="year">1998</infon><offset>35613</offset><text>Conserved active site aspartates and domain–domain interactions in regulatory properties of the sugar kinase superfamily</text></passage><passage><infon key="fpage">136</infon><infon key="issue">1</infon><infon key="lpage">47</infon><infon key="name_0">surname:Karlsson;given-names:R</infon><infon key="name_1">surname:Katsamba;given-names:PS</infon><infon key="name_2">surname:Nordin;given-names:H</infon><infon key="name_3">surname:Pol;given-names:E</infon><infon key="name_4">surname:Myszka;given-names:DG</infon><infon key="pub-id_pmid">16337141</infon><infon key="section_type">REF</infon><infon key="source">Analytical biochemistry</infon><infon key="type">ref</infon><infon key="volume">349</infon><infon key="year">2006</infon><offset>35736</offset><text>Analyzing a kinetic titration series using affinity biosensors</text></passage><passage><infon key="fpage">362</infon><infon key="issue">2</infon><infon key="lpage">73</infon><infon key="name_0">surname:Yeh;given-names:JI</infon><infon key="name_1">surname:Charrier;given-names:V</infon><infon key="name_2">surname:Paulo;given-names:J</infon><infon key="name_3">surname:Hou;given-names:L</infon><infon key="name_4">surname:Darbon;given-names:E</infon><infon key="name_5">surname:Claiborne;given-names:A</infon><infon key="pub-id_pmid">14717590</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry</infon><infon key="type">ref</infon><infon key="volume">43</infon><infon key="year">2004</infon><offset>35799</offset><text>Structures of enterococcal glycerol kinase in the absence and presence of glycerol: correlation of conformation to substrate binding and a mechanism of activation by phosphorylation</text></passage><passage><infon key="fpage">137</infon><infon key="lpage">62</infon><infon key="name_0">surname:Hurley;given-names:JH</infon><infon key="pub-id_pmid">8800467</infon><infon key="section_type">REF</infon><infon key="source">Annual review of biophysics and biomolecular structure</infon><infon key="type">ref</infon><infon key="volume">25</infon><infon key="year">1996</infon><offset>35981</offset><text>The sugar kinase/heat shock protein 70/actin superfamily: implications of conserved structure for mechanism</text></passage><passage><infon key="fpage">1219</infon><infon key="issue">4</infon><infon key="lpage">27</infon><infon key="name_0">surname:Emekli;given-names:U</infon><infon key="name_1">surname:Schneidman-Duhovny;given-names:D</infon><infon key="name_2">surname:Wolfson;given-names:HJ</infon><infon key="name_3">surname:Nussinov;given-names:R</infon><infon key="name_4">surname:Haliloglu;given-names:T</infon><infon key="pub-id_pmid">17847101</infon><infon key="section_type">REF</infon><infon key="source">Proteins</infon><infon key="type">ref</infon><infon key="volume">70</infon><infon key="year">2008</infon><offset>36089</offset><text>HingeProt: automated prediction of hinges in protein structures</text></passage><passage><infon key="fpage">8</infon><infon key="issue">1</infon><infon key="lpage">15</infon><infon key="name_0">surname:Stols;given-names:L</infon><infon key="name_1">surname:Gu;given-names:M</infon><infon key="name_2">surname:Dieckman;given-names:L</infon><infon key="name_3">surname:Raffen;given-names:R</infon><infon key="name_4">surname:Collart;given-names:FR</infon><infon key="name_5">surname:Donnelly;given-names:MI</infon><infon key="pub-id_pmid">12071693</infon><infon key="section_type">REF</infon><infon key="source">Protein Expr Purif</infon><infon key="type">ref</infon><infon key="volume">25</infon><infon key="year">2002</infon><offset>36153</offset><text>A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site</text></passage><passage><infon key="fpage">307</infon><infon key="lpage">26</infon><infon key="name_0">surname:Otwinowski;given-names:Z</infon><infon key="name_1">surname:Minor;given-names:W</infon><infon key="section_type">REF</infon><infon key="source">Methods In Enzymology</infon><infon key="type">ref</infon><infon key="volume">276</infon><infon key="year">1997</infon><offset>36269</offset><text>Processing of X-ray diffraction data collected in oscillation mode</text></passage><passage><infon key="fpage">213</infon><infon key="issue">2</infon><infon key="lpage">21</infon><infon key="name_0">surname:Adams;given-names:PD</infon><infon key="name_1">surname:Afonine;given-names:PV</infon><infon key="name_2">surname:Bunkóczi;given-names:G</infon><infon key="name_3">surname:Chen;given-names:VB</infon><infon key="name_4">surname:Davis;given-names:IW</infon><infon key="name_5">surname:Echols;given-names:N</infon><infon key="pub-id_pmid">20124702</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallographica Section D: Biological Crystallography</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>36336</offset><text>PHENIX: a comprehensive Python-based system for macromolecular structure solution</text></passage><passage><infon key="fpage">2126</infon><infon key="issue">12</infon><infon key="lpage">32</infon><infon key="name_0">surname:Emsley;given-names:P</infon><infon key="name_1">surname:Cowtan;given-names:K</infon><infon key="pub-id_pmid">15572765</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallographica Section D: Biological Crystallography</infon><infon key="type">ref</infon><infon key="volume">60</infon><infon key="year">2004</infon><offset>36418</offset><text>Coot: model-building tools for molecular graphics</text></passage><passage><infon key="fpage">1194</infon><infon key="issue">11</infon><infon key="lpage">7</infon><infon key="name_0">surname:Afonine;given-names:PV</infon><infon key="name_1">surname:Grosse-Kunstleve;given-names:RW</infon><infon key="name_2">surname:Adams;given-names:PD</infon><infon key="name_3">surname:Lunin;given-names:VY</infon><infon key="name_4">surname:Urzhumtsev;given-names:A</infon><infon key="pub-id_pmid">18007035</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallographica Section D: Biological Crystallography</infon><infon key="type">ref</infon><infon key="volume">63</infon><infon key="year">2007</infon><offset>36468</offset><text>On macromolecular refinement at subatomic resolution with interatomic scatterers</text></passage><passage><infon key="fpage">283</infon><infon key="issue">2</infon><infon key="lpage">91</infon><infon key="name_0">surname:Laskowski;given-names:RA</infon><infon key="name_1">surname:MacArthur;given-names:MW</infon><infon key="name_2">surname:Moss;given-names:DS</infon><infon key="name_3">surname:Thornton;given-names:JM</infon><infon key="section_type">REF</infon><infon key="source">Journal of applied crystallography</infon><infon key="type">ref</infon><infon key="volume">26</infon><infon key="year">1993</infon><offset>36549</offset><text>PROCHECK: a program to check the stereochemical quality of protein structures</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>36627</offset><text>DeLano. W. The PyMOL Molecular Graphics System. Available: http://www.pymol.org.</text></passage><passage><infon key="fpage">615</infon><infon key="issue">6</infon><infon key="lpage">22</infon><infon key="name_0">surname:Sanghera;given-names:J</infon><infon key="name_1">surname:Li;given-names:R</infon><infon key="name_2">surname:Yan;given-names:J</infon><infon key="pub-id_doi">10.1089/adt.2009.0237</infon><infon key="pub-id_pmid">20105027</infon><infon key="section_type">REF</infon><infon key="source">Assay and drug development technologies</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2009</infon><offset>36708</offset><text>Comparison of the luminescent ADP-Glo assay to a standard radiometric assay for measurement of protein kinase activity</text></passage></document></collection>
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+<collection><source>PMC</source><date>20201219</date><key>pmc.key</key><document><id>4887326</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1007/s13238-016-0264-7</infon><infon key="article-id_pmc">4887326</infon><infon key="article-id_pmid">27113583</infon><infon key="article-id_publisher-id">264</infon><infon key="fpage">403</infon><infon key="issue">6</infon><infon key="kwd">the YfiBNR system c-di-GMP Vitamin B6 L-Trp peptidoglycan layer bioflim formation</infon><infon key="license">
+Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.</infon><infon key="lpage">416</infon><infon key="name_0">surname:Xu;given-names:Min</infon><infon key="name_1">surname:Yang;given-names:Xuan</infon><infon key="name_2">surname:Yang;given-names:Xiu-An</infon><infon key="name_3">surname:Zhou;given-names:Lei</infon><infon key="name_4">surname:Liu;given-names:Tie-Zheng</infon><infon key="name_5">surname:Fan;given-names:Zusen</infon><infon key="name_6">surname:Jiang;given-names:Tao</infon><infon key="section_type">TITLE</infon><infon key="title">KEYWORDS</infon><infon key="type">front</infon><infon key="volume">7</infon><infon key="year">2016</infon><offset>0</offset><text>Structural insights into the regulatory mechanism of the Pseudomonas aeruginosa YfiBNR system</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>94</offset><text>YfiBNR is a recently identified bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) signaling system in opportunistic pathogens. It is a key regulator of biofilm formation, which is correlated with prolonged persistence of infection and antibiotic drug resistance. In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. However, the detailed regulatory mechanism remains elusive. Here, we report the crystal structures of YfiB alone and of an active mutant YfiBL43P complexed with YfiR with 2:2 stoichiometry. Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation. Based on the structural and biochemical data, we propose an updated regulatory model of the YfiBNR system.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1498</offset><text>INTRODUCTION</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1511</offset><text>Bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) is a ubiquitous second messenger that bacteria use to facilitate behavioral adaptations to their ever-changing environment. An increase in c-di-GMP promotes biofilm formation, and a decrease results in biofilm degradation (Boehm et al.,; Duerig et al.,; Hickman et al.,; Jenal,; Romling et al.,). The c-di-GMP level is regulated by two reciprocal enzyme systems, namely, diguanylate cyclases (DGCs) that synthesize c-di-GMP and phosphodiesterases (PDEs) that hydrolyze c-di-GMP (Kulasakara et al.,; Ross et al.,; Ross et al.,). Many of these enzymes are multiple-domain proteins containing a variable N-terminal domain that commonly acts as a signal sensor or transduction module, followed by the relatively conserved GGDEF motif in DGCs or EAL/HD-GYP domains in PDEs (Hengge,; Navarro et al.,; Schirmer and Jenal,). Intriguingly, studies in diverse species have revealed that a single bacterium can have dozens of DGCs and PDEs (Hickman et al.,; Kirillina et al.,; Kulasakara et al.,; Tamayo et al.,). In Pseudomonas aeruginosa in particular, 42 genes containing putative DGCs and/or PDEs were identified (Kulasakara et al.,). The functional role of a number of downstream effectors of c-di-GMP has been characterized as affecting exopolysaccharide (EPS) production, transcription, motility, and surface attachment (Caly et al.,; Camilli and Bassler,; Ha and O’Toole,; Pesavento and Hengge,). However, due to the intricacy of c-di-GMP signaling networks and the diversity of experimental cues, the detailed mechanisms by which these signaling pathways specifically sense and integrate different inputs remain largely elusive.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3186</offset><text>Biofilm formation protects pathogenic bacteria from antibiotic treatment, and c-di-GMP-regulated biofilm formation has been extensively studied in P. aeruginosa (Evans,; Kirisits et al.,; Malone,; Reinhardt et al.,). In the lungs of cystic fibrosis (CF) patients, adherent biofilm formation and the appearance of small colony variant (SCV) morphologies of P. aeruginosa correlate with prolonged persistence of infection and poor lung function (Govan and Deretic,; Haussler et al.,; Haussler et al.,; Parsek and Singh,; Smith et al.,). Recently, Malone and coworkers identified the tripartite c-di-GMP signaling module system YfiBNR (also known as AwsXRO (Beaumont et al.,; Giddens et al.,) or Tbp (Ueda and Wood,)) by genetic screening for mutants that displayed SCV phenotypes in P. aeruginosa PAO1 (Malone et al.,; Malone et al.,). The YfiBNR system contains three protein members and modulates intracellular c-di-GMP levels in response to signals received in the periplasm (Malone et al.,). More recently, this system was also reported in other Gram-negative bacteria, such as Escherichia coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), Klebsiella pneumonia (Huertas et al.,) and Yersinia pestis (Ren et al.,). YfiN is an integral inner-membrane protein with two potential transmembrane helices, a periplasmic Per-Arnt-Sim (PAS) domain, and cytosolic domains containing a HAMP domain (mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and a C-terminal GGDEF domain indicating a DGC’s function (Giardina et al.,; Malone et al.,). YfiN is repressed by specific interaction between its periplasmic PAS domain and the periplasmic protein YfiR (Malone et al.,). YfiB is an OmpA/Pal-like outer-membrane lipoprotein (Parsons et al.,) that can activate YfiN by sequestering YfiR (Malone et al.,) in an unknown manner. Whether YfiB directly recruits YfiR or recruits YfiR via a third partner is an open question. After the sequestration of YfiR by YfiB, the c-di-GMP produced by activated YfiN increases the biosynthesis of the Pel and Psl EPSs, resulting in the appearance of the SCV phenotype, which indicates enhanced biofilm formation (Malone et al.,).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5437</offset><text>It has been reported that the activation of YfiN may be induced by redox-driven misfolding of YfiR (Giardina et al.,; Malone et al.,; Malone et al.,). It is also proposed that the sequestration of YfiR by YfiB can be induced by certain YfiB-mediated cell wall stress, and mutagenesis studies revealed a number of activation residues of YfiB that were located in close proximity to the predicted first helix of the periplasmic domain (Malone et al.,). In addition, quorum sensing-related dephosphorylation of the PAS domain of YfiN may also be involved in the regulation (Ueda and Wood,; Xu et al.,). Recently, we solved the crystal structure of YfiR in both the non-oxidized and the oxidized states, revealing breakage/formation of one disulfide bond (Cys71-Cys110) and local conformational change around the other one (Cys145-Cys152), indicating that Cys145-Cys152 plays an important role in maintaining the correct folding of YfiR (Yang et al.,).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6386</offset><text>In the present study, we solved the crystal structures of an N-terminal truncated form of YfiB (34–168) and YfiR in complex with an active mutant YfiBL43P. Most recently, Li and coworkers reported the crystal structures of YfiB (27–168) alone and YfiRC71S in complex with YfiB (59–168) (Li et al.,). Compared with the reported complex structure, YfiBL43P in our YfiB-YfiR complex structure has additional visible N-terminal residues 44–58 that are shown to play essential roles in YfiB activation and biofilm formation. Therefore, we are able to visualize the detailed allosteric arrangement of the N-terminal structure of YfiB and its important role in YfiB-YfiR interaction. In addition, we found that the YfiBL43P shows a much higher PG-binding affinity than wild-type YfiB, most likely due to its more compact PG-binding pocket. Moreover, we found that Vitamin B6 (VB6) or L-Trp can bind YfiR with an affinity in the ten millimolar range. Together with functional data, these results provide new mechanistic insights into how activated YfiB sequesters YfiR and releases the suppression of YfiN. These findings may facilitate the development and optimization of anti-biofilm drugs for the treatment of chronic infections.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>7619</offset><text>RESULTS</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>7627</offset><text>Overall structure of YfiB</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7653</offset><text>We obtained two crystal forms of YfiB (residues 34–168, lacking the signal peptide from residues 1–26 and periplasmic residues 27–33), crystal forms I and II, belonging to space groups P21 and P41, respectively.</text></passage><passage><infon key="file">13238_2016_264_Fig1_HTML.jpg</infon><infon key="id">Fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>7871</offset><text>Overall structure of YfiB. (A) The overall structure of the YfiB monomer. (B) A topology diagram of the YfiB monomer. (C and D) The analytical ultracentrifugation experiment results for the wild-type YfiB and YfiBL43P
+</text></passage><passage><infon key="file">13238_2016_264_Fig2_HTML.jpg</infon><infon key="id">Fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>8090</offset><text>Two dimeric types of YfiB dimer. (A–C) The “head to head” dimer. (D–F) The “back to back” dimer. (A) and (E) indicate the front views of the two dimers, (B) and (F) indicate the top views of the two dimers, and (C) and (D) indicate the details of the two dimeric interfaces</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8376</offset><text>The crystal structure of YfiB monomer consists of a five-stranded β-sheet (β1-2-5-3-4) flanked by five α-helices (α1–5) on one side. In addition, there is a short helix turn connecting the β4 strand and α4 helix (Fig. 1A and 1B). Each crystal form contains three different dimeric types of YfiB, two of which are present in both, suggesting that the rest of the dimeric types may result from crystal packing. Here, we refer to the two dimeric types as “head to head” and “back to back” according to the interacting mode (Fig. 2A and 2E), with the total buried surface areas being 316.8 Å2 and 554.3 Å2, respectively.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9014</offset><text>The “head to head” dimer exhibits a clamp shape. The dimerization occurs mainly via hydrophobic interactions formed by A37 and I40 on the α1 helices, L50 on the β1 strands, and W55 on the β2 strands of both molecules, making a hydrophobic interacting core (Fig. 2A–C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9293</offset><text>The “back to back” dimer presents a Y shape. The dimeric interaction is mainly hydrophilic, occurring among the main-chain and side-chain atoms of N68, L69, D70 and R71 on the α2-α3 loops and R116 and S120 on the α4 helices of both molecules, resulting in a complex hydrogen bond network (Fig. 2D–F).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>9606</offset><text>The YfiB-YfiR interaction</text></passage><passage><infon key="file">13238_2016_264_Fig3_HTML.jpg</infon><infon key="id">Fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>9632</offset><text>Overall structure of the YfiB-YfiR complex and the conserved surface in YfiR. (A) The overall structure of the YfiB-YfiR complex. The YfiBL43P molecules are shown in cyan and yellow. The YfiR molecules are shown in green and magenta. Two interacting regions are highlighted by red rectangles. (B) Structural superposition of apo YfiB and YfiR-bound YfiBL43P. To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34–70 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44–70 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P. The residues proposed to contribute to YfiB activation are illustrated in sticks. The key residues in apo YfiB are shown in red and those in YfiBL43P are shown in blue. (D) Close-up views showing interactions in regions I and II. YfiBL43P and YfiR are shown in cyan and green, respectively. (E and F) The conserved surface in YfiR contributes to the interaction with YfiB. (G) The residues of YfiR responsible for interacting with YfiB are shown in green sticks, and the proposed YfiN-interacting residues are shown in yellow sticks. The red sticks, which represent the YfiB-interacting residues, are also responsible for the proposed interactions with YfiN</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10966</offset><text>To gain structural insights into the YfiB-YfiR interaction, we co-expressed YfiB (residues 34–168) and YfiR (residues 35–190, lacking the signal peptide), but failed to obtain the complex, in accordance with a previous report in which no stable complex of YfiB-YfiR was observed (Malone et al.,). It has been reported that single mutants of Q39, L43, F48 and W55 contribute to YfiB activation leading to the induction of the SCV phenotype in P. aeruginosa PAO1 (Malone et al.,). It is likely that these residues may be involved in the conformational changes of YfiB that are related to YfiR sequestration (Fig. 3C). Therefore, we constructed two such single mutants of YfiB (YfiBL43P and YfiBF48S). As expected, both mutants form a stable complex with YfiR. Finally, we crystalized YfiR in complex with the YfiBL43P mutant and solved the structure at 1.78 Å resolution by molecular replacement using YfiR and YfiB as models.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11897</offset><text>The YfiB-YfiR complex is a 2:2 heterotetramer (Fig. 3A) in which the YfiR dimer is clamped by two separated YfiBL43P molecules with a total buried surface area of 3161.2 Å2. The YfiR dimer in the complex is identical to the non-oxidized YfiR dimer alone (Yang et al.,), with only Cys145-Cys152 of the two disulfide bonds well formed, suggesting Cys71-Cys110 disulfide bond formation is not essential for forming YfiB-YfiR complex. The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the α1 helix (residues 34–43) is invisible on the electron density map, and the α2 helix and β1 and β2 strands are rearranged to form a long loop containing two short α-helix turns (Fig. 3B and 3C), thus embracing the YfiR dimer. The observed changes in conformation of YfiB and the results of mutagenesis suggest a mechanism by which YfiB sequesters YfiR.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12877</offset><text>The YfiB-YfiR interface can be divided into two regions (Fig. 3A and 3D). Region I is formed by numerous main-chain and side-chain hydrophilic interactions between residues E45, G47 and E53 from the N-terminal extended loop of YfiB and residues S57, R60, A89 and H177 from YfiR (Fig. 3D-I(i)). Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). In region II, the side chains of R96, E98 and E157 from YfiB interact with the side chains of E163, S146 and R171 from YfiR, respectively. Additionally, the main chains of I163 and V165 from YfiB form hydrogen bonds with the main chains of L166 and A164 from YfiR, respectively, and the main chain of P166 from YfiB interacts with the side chain of R185 from YfiR (Fig. 3D-II). These two regions contribute a robust hydrogen-bonding network to the YfiB-YfiR interface, resulting in a tightly bound complex.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14067</offset><text>Based on the observations that two separated YfiBL43P molecules form a 2:2 complex structure with YfiR dimer, we performed an analytical ultracentrifugation experiment to check the oligomeric states of wild-type YfiB and YfiBL43P. The results showed that wild-type YfiB exists in both monomeric and dimeric states in solution, while YfiBL43P primarily adopts the monomer state in solution (Fig. 1C–D). This suggests that the N-terminus of YfiB plays an important role in forming the dimeric YfiB in solution and that the conformational change of residue L43 is associated with the stretch of the N-terminus and opening of the dimer. Therefore, it is possible that both dimeric types might exist in solution. For simplicity, we only discuss the “head to head” dimer in the following text.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>14862</offset><text>The PG-binding site of YfiB</text></passage><passage><infon key="file">13238_2016_264_Fig4_HTML.jpg</infon><infon key="id">Fig4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>14890</offset><text>The PG-binding site in YfiB. (A) Structural superposition of the PG-binding sites of the H. influenzae Pal/PG-P complex and YfiR-bound YfiBL43P complexed with sulfate ions. (B) Close-up view showing the key residues of Pal interacting with the m-Dap5 ε-carboxylate group of PG-P. Pal is shown in wheat and PG-P is in magenta. (C) Close-up view showing the key residues of YfiR-bound YfiBL43P interacting with a sulfate ion. YfiR-bound YfiBL43P is shown in cyan; the sulfate ion, in green; and the water molecule, in yellow. (D) Structural superposition of the PG-binding sites of apo YfiB and YfiR-bound YfiBL43P, the key residues are shown in stick. Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15823</offset><text>PG-associated lipoprotein (Pal) is highly conserved in Gram-negative bacteria and anchors to the outer membrane through an N-terminal lipid attachment and to PG layer through its periplasmic domain, which is implicated in maintaining outer membrane integrity. Previous homology modeling studies suggested that YfiB contains a Pal-like PG-binding site (Parsons et al.,), and the mutation of two residues at this site, D102 and G105, reduces the ability for biofilm formation and surface attachment (Malone et al.,). In the YfiB-YfiR complex, one sulfate ion is found at the bottom of each YfiBL43P molecule (Fig. 3A) and forms a strong hydrogen bond with D102 of YfiBL43P (Fig. 4A and 4C). Structural superposition between YfiBL43P and Haemophilus influenzae Pal complexed with biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala (m-Dap is meso-diaminopimelate) (PDB code: 2aiz) (Parsons et al.,), revealed that the sulfate ion is located at the position of the m-Dap5 ϵ-carboxylate group in the Pal/PG-P complex (Fig. 4A). In the Pal/PG-P complex structure, the m-Dap5 ϵ-carboxylate group interacts with the side-chain atoms of D71 and the main-chain amide of D37 (Fig. 4B). Similarly, in the YfiR-bound YfiBL43P structure, the sulfate ion interacts with the side-chain atoms of D102 (corresponding to D71 in Pal) and R117 (corresponding to R86 in Pal) and the main-chain amide of N68 (corresponding to D37 in Pal). Moreover, a water molecule was found to bridge the sulfate ion and the side chains of N67 and D102, strengthening the hydrogen bond network (Fig. 4C). In addition, sequence alignment of YfiB with Pal and the periplasmic domain of OmpA (proteins containing PG-binding site) showed that N68 and D102 are highly conserved (Fig. 4G, blue stars), suggesting that these residues contribute to the PG-binding ability of YfiB.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17719</offset><text>Interestingly, superposition of apo YfiB with YfiR-bound YfiBL43P revealed that the PG-binding region is largely altered mainly due to different conformation of the N68 containing loop. Compared to YfiBL43P, the N68-containing loop of the apo YfiB flips away about 7 Å, and D102 and R117 swing slightly outward; thus, the PG-binding pocket is enlarged with no sulfate ion or water bound (Fig. 4D). Therefore, we proposed that the PG-binding ability of inactive YfiB might be weaker than that of active YfiB. To validate this, we performed a microscale thermophoresis (MST) assay to measure the binding affinities of PG to wild-type YfiB and YfiBL43P, respectively. The results indicated that the PG-binding affinity of YfiBL43P is 65.5 μmol/L, which is about 16-fold stronger than that of wild-type YfiB (Kd = 1.1 mmol/L) (Fig. 4E–F). As the experiment is performed in the absence of YfiR, it suggests that an increase in the PG-binding affinity of YfiB is not a result of YfiB-YfiR interaction and is highly coupled to the activation of YfiB characterized by a stretched N-terminal conformation.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>18822</offset><text>The conserved surface in YfiR is functional for binding YfiB and YfiN</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18892</offset><text>Calculation using the ConSurf Server (http://consurf.tau.ac.il/), which estimates the evolutionary conservation of amino acid positions and visualizes information on the structure surface, revealed a conserved surface on YfiR that contributes to the interaction with YfiB (Fig. 3E and 3F). Interestingly, the majority of this conserved surface contributes to the interaction with YfiB (Fig. 3E and 3F). Malone JG et al. have reported that F151, E163, I169 and Q187, located near the C-terminus of YfiR, comprise a putative YfiN binding site (Malone et al.,). Interestingly, these residues are part of the conserved surface of YfiR (Fig. 3G). F151, E163 and I169 form a hydrophobic core while, Q187 is located at the end of the α6 helix. E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). Collectively, a part of the YfiB-YfiR interface overlaps with the proposed YfiR-YfiN interface, suggesting alteration in the association-disassociation equilibrium of YfiR-YfiN and hence the ability of YfiB to sequester YfiR.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>20101</offset><text>YfiR binds small molecules</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20128</offset><text>Previous studies indicated that YfiR constitutes a YfiB-independent sensing device that can activate YfiN in response to the redox status of the periplasm, and we have reported YfiR structures in both the non-oxidized and the oxidized states earlier, revealing that the Cys145-Cys152 disulfide bond plays an essential role in maintaining the correct folding of YfiR (Yang et al.,). However, whether YfiR is involved in other regulatory mechanisms is still an open question.</text></passage><passage><infon key="file">13238_2016_264_Fig5_HTML.jpg</infon><infon key="id">Fig5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>20602</offset><text>Overall Structures of VB6-bound and Trp-bound YfiR. (A) Superposition of the overall structures of VB6-bound and Trp-bound YfiR. (B) Close-up views showing the key residues of YfiR that bind VB6 and L-Trp. The electron densities of VB6 and Trp are countered at 3.0σ and 2.3σ, respectively, in |Fo|-|Fc| maps. (C) Superposition of the hydrophobic pocket of YfiR with VB6, L-Trp and F57 of YfiB</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21003</offset><text>Intriguingly, a Dali search (Holm and Rosenstrom,) indicated that the closest homologs of YfiR shared the characteristic of being able to bind several structurally similar small molecules, such as L-Trp, L-Phe, B-group vitamins and their analogs, encouraging us to test whether YfiR can recognize these molecules. For this purpose, we co-crystallized YfiR or soaked YfiR crystals with different small molecules, including L-Trp and B-group vitamins. Fortunately, we found obvious small-molecule density in the VB6-bound and Trp-bound YfiR crystal structures (Fig. 5A and 5B), and in both structures, the YfiR dimers resemble the oxidized YfiR structure in which both two disulfide bonds are well formed (Yang et al.,).</text></passage><passage><infon key="file">13238_2016_264_Fig6_HTML.jpg</infon><infon key="id">Fig6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>21723</offset><text>Functional analysis of VB6 and L-Trp. (A and B) The effect of increasing concentrations of VB6 or L-Trp on YfiBL43P-induced attachment (bars). The relative optical density is represented as curves. Wild-type YfiB is used as negative control. (C and D) BIAcore data and analysis for binding affinities of (C) VB6 and (D) L-Trp with YfiR. (E–G) ITC data and analysis for titration of (E) YfiB wild-type, (F) YfiBL43P, and (G) YfiBL43P/F57A into YfiR</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22173</offset><text>Structural analyses revealed that the VB6 and L-Trp molecules are bound at the periphery of the YfiR dimer, but not at the dimer interface. Interestingly, VB6 and L-Trp were found to occupy the same hydrophobic pocket, formed by L166/I169/V176/P178/L181 of YfiR, which is also a binding pocket for F57 of YfiB, as observed in the YfiB-YfiR complex (Fig. 5C). To evaluate the importance of F57 in YfiBL43P-YfiR interaction, the binding affinities of YfiBL43P and YfiBL43P/F57A for YfiR were measured by isothermal titration calorimetry (ITC). The results showed Kd values of 1.4 × 10−7 mol/L and 5.3 × 10−7 mol/L for YfiBL43P and YfiBL43P/F57A, respectively, revealing that the YfiBL43P/F57A mutant caused a 3.8-fold reduction in the binding affinity compared with the YfiBL43P mutant (Fig. 6F and 6G).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22983</offset><text>In parallel, to better understand the putative functional role of VB6 and L-Trp, yfiB was deleted in a PAO1 wild-type strain, and a construct expressing the YfiBL43P mutant was transformed into the PAO1 ΔyfiB strain to trigger YfiBL43P-induced biofilm formation. Growth and surface attachment assays were carried out for the yfiB-L43P strain in the presence of increasing concentrations of VB6 or L-Trp. As shown in Fig. 6A and 6B, the over-expression of YfiBL43P induced strong surface attachment and much slower growth of the yfiB-L43P strain, and as expected, a certain amount of VB6 or L-Trp (4–6 mmol/L for VB6 and 6–10 mmol/L for L-Trp) could reduce the surface attachment. Interestingly, at a concentration higher than 8 mmol/L, VB6 lost its ability to inhibit biofilm formation, implying that the VB6-involving regulatory mechanism is highly complicated and remains to be further investigated.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23891</offset><text>Of note, both VB6 and L-Trp have been reported to correlate with biofilm formation in certain Gram-negative bacteria (Grubman et al.,; Shimazaki et al.,). In Helicobacter pylori in particular, VB6 biosynthetic enzymes act as novel virulence factors, and VB6 is required for full motility and virulence (Grubman et al.,). In E. coli, mutants with decreased tryptophan synthesis show greater biofilm formation, and matured biofilm is degraded by L-tryptophan addition (Shimazaki et al.,). However, the detailed mechanism remains elusive.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24430</offset><text>To answer the question whether competition of VB6 or L-Trp for the YfiB F57-binding pocket of YfiR plays an essential role in inhibiting biofilm formation, we measured the binding affinities of VB6 and L-Trp for YfiR via BIAcore experiments. The results showed relatively weak Kd values of 35.2 mmol/L and 76.9 mmol/L for VB6 and L-Trp, respectively (Fig. 6C and 6D). Based on our results, we concluded that VB6 or L-Trp can bind to YfiR, however, VB6 or L-Trp alone may have little effects in interrupting the YfiB-YfiR interaction, the mechanism by which VB6 or L-Trp inhibits biofilm formation remains unclear and requires further investigation.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>25080</offset><text>DISCUSSION</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>25091</offset><text>Previous studies suggested that in response to cell stress, YfiB in the outer membrane sequesters the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus inducing the diguanylate cyclase activity of YfiN to allow c-di-GMP production (Giardina et al.,; Malone et al.,; Malone et al.,). However, the pattern of interaction between these proteins and the detailed regulatory mechanism remain unknown due to a lack of structural information.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>25567</offset><text>Here, we report the crystal structures of YfiB alone and an active mutant YfiBL43P in complex with YfiR, indicating that YfiR forms a 2:2 complex with YfiB via a region composed of conserved residues. Our structural data analysis shows that the activated YfiB has an N-terminal portion that is largely altered, adopting a stretched conformation compared with the compact conformation of the apo YfiB. The apo YfiB structure constructed beginning at residue 34 has a compact conformation of approximately 45 Å in length. In addition to the preceding 8 aa loop (from the lipid acceptor Cys26 to Gly34), the full length of the periplasmic portion of apo YfiB can reach approximately 60 Å. It was reported that the distance between the outer membrane and the cell wall is approximately 50 Å and that the thickness of the PG layer is approximately 70 Å (Matias et al.,). Thus, YfiB alone represents an inactive form that may only partially insert into the PG matrix. By contrast, YfiR-bound YfiBL43P (residues 44–168) has a stretched conformation of approximately 55 Å in length. In addition to the 17 preceding intracellular residues (from the lipid acceptor Cys26 to Leu43), the length of the intracellular portion of active YfiB may extend over 100 Å, assuming a fully stretched conformation. Provided that the diameter of the widest part of the YfiB dimer is approximately 64 Å, which is slightly smaller than the smallest diameter of the PG pore (70 Å) (Meroueh et al.,), the YfiB dimer should be able to penetrate the PG layer.</text></passage><passage><infon key="file">13238_2016_264_Fig7_HTML.jpg</infon><infon key="id">Fig7</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27106</offset><text>Regulatory model of the YfiBNR tripartite system. The periplasmic domain of YfiB and the YfiB-YfiR complex are depicted according to the crystal structures. The lipid acceptor Cys26 is indicated as blue ball. The loop connecting Cys26 and Gly34 of YfiB is modeled. The PAS domain of YfiN is shown as pink oval. Once activated by certain cell stress, the dimeric YfiB transforms from a compact conformation to a stretched conformation, allowing the periplasmic domain of the membrane-anchored YfiB to penetrate the cell wall and sequester the YfiR dimer, thus relieving the repression of YfiN</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>27698</offset><text>These results, together with our observation that activated YfiB has a much higher cell wall binding affinity, and previous mutagenesis data showing that (1) both PG binding and membrane anchoring are required for YfiB activity and (2) activating mutations possessing an altered N-terminal loop length are dominant over the loss of PG binding (Malone et al.,), suggest an updated regulatory model of the YfiBNR system (Fig. 7). In this model, in response to a particular cell stress that is yet to be identified, the dimeric YfiB is activated from a compact, inactive conformation to a stretched conformation, which possesses increased PG binding affinity. This allows the C-terminal portion of the membrane-anchored YfiB to reach, bind and penetrate the cell wall and sequester the YfiR dimer. The YfiBNR system provides a good example of a delicate homeostatic system that integrates multiple signals to regulate the c-di-GMP level. Homologs of the YfiBNR system are functionally conserved in P. aeruginosa (Malone et al.,; Malone et al.,), E. coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), K. pneumonia (Huertas et al.,) and Y. pestis (Ren et al.,), where they affect c-di-GMP production and biofilm formation. The mechanism by which activated YfiB relieves the repression of YfiN may be applicable to the YfiBNR system in other bacteria and to analogous outside-in signaling for c-di-GMP production, which in turn may be relevant to the development of drugs that can circumvent complicated antibiotic resistance.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>29236</offset><text>MATERIALS AND METHODS</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>29258</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>29294</offset><text>P. aeruginosa YfiR (residues 35–190, lacking the predicted N-terminal periplasmic localization signaling peptide) and YfiB (residues 34–168, lacking the signal peptide from residues 1–26 and periplasmic residues 27–33) were cloned into ORF1 of the pETDuet-1 (Merck Millipore, Darmstadt, Germany) vector via the BamHI and HindIII restriction sites, with a constructed N-terminal His6 and a TEV cleavage site, respectively. In addition, YfiB (residues 34–168) was ligated into the NdeI and XhoI restriction sites of ORF2 in the previously constructed YfiR expression vector. Site-directed mutagenesis was carried out using a QuikChange kit (Agilent Technologies, Santa Clara, CA), following the manufacturer’s instructions.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>30028</offset><text>The proteins were over-expressed in the E. coli BL21-CodonPlus(DE3)-RIPL strain. Protein expression was induced by adding 0.5–1 mmol/L IPTG at an OD600 of approximately 0.8. The cell cultures were then incubated for an additional 4.5 h at 37°C. The cells were subsequently harvested by centrifugation and stored at −80°C.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>30356</offset><text>Cell suspensions were thawed and homogenized using a high-pressure homogenizer (JNBIO, Beijing, China). YfiR was first purified by Ni affinity chromatography and then incubated with His6-tagged TEV protease overnight. The His6-TEV cleavage site was subsequently removed by incubation with Ni-NTA resin. Finally, YfiR was purified with a HiTrap STM column (GE Healthcare), followed by a Superdex 200 (GE Healthcare) column. YfiB was purified with Ni affinity chromatography, followed by a Superdex 200 (GE Healthcare) column. The YfiB-YfiR complex was first purified by Ni affinity chromatography, then by a Superdex 200 (GE Healthcare) column, and finally by a HiTrap STM column (GE Healthcare). All of the purified fractions were collected and concentrated to ~40 mg/mL in 20 mmol/L Tris-HCl (pH 8.0) and 200 mmol/L NaCl, frozen in liquid nitrogen and stored at −80°C.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>31229</offset><text>Crystallization and data collection</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31265</offset><text>Crystal screening was performed with commercial screening kits (Hampton Research, CA, USA) using the sitting-drop vapor diffusion method, and positive hits were optimized using the hanging-drop vapor diffusion method at 293 K. Crystals of the YfiB protein were obtained and optimized in buffer containing 0.2 mol/L lithium sulfate monohydrate, 0.1 mol/L Tris-HCl (pH 8.0) and 30% w/v polyethylene glycol 4000. After being soaked for a few seconds in cryoprotection solution (well solution complemented with 25% xylitol), the crystals were cooled by plunging them into liquid nitrogen. Diffraction-quality crystals of the YfiB-YfiR complex were grown in buffer containing 0.2 mol/L ammonium sulfate, 0.1 mol/L Tris-HCl (pH 8.0) and 12% w/v polyethylene glycol 8000. The crystals were cryoprotected with 8% (w/v) polyethylene glycol 8000 and 0.1 mol/L Tris-HCl (pH 7.5) supplemented with saturated sucrose prior to being flash frozen. Crystals of the native YfiR were obtained and optimized in 0.1 mol/L HEPES (pH 7.5) and 1.8 mol/L ammonium sulfate. VB6-bound YfiR crystals were obtained by soaking the native YfiR crystals in 2 mmol/L VB6 molecules. Trp-bound YfiR crystals were obtained by co-crystalizing the YfiR protein and 4 mmol/L L-Trp molecules in 0.2 mol/L NaCl, 0.1 mol/L BIS-TRIS (pH 5.5), and 25% w/v polyethylene glycol 3350. For cryoprotection, both the VB6-bound and the L-Trp-bound YfiR crystals were soaked in 2.5 mol/L lithium sulfate monohydrate for a few seconds before data collection. Diffraction data for the YfiB crystal belonging to space group P21 was collected in house, the data for the YfiB crystal belonging to space group P41 and for the Trp-bound YfiR crystal were collected on beamline BL17U at the Shanghai Synchrotron Radiation Facility (SSRF), and the data for the VB6-bound YfiR crystal were collected on beamline BL18U at SSRF. Finally, the data for the YfiB-YfiR complex crystal were collected on beamline BL-1A at the Photon Factory in Japan. The diffraction data were processed with the HKL2000 software program (Otwinowski and Minor,).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>33343</offset><text>Structure determination and refinement</text></passage><passage><infon key="file">Tab1.xml</infon><infon key="id">Tab1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>33382</offset><text>Data collection, phasing and refinement statistics</text></passage><passage><infon key="file">Tab1.xml</infon><infon key="id">Tab1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;thead&gt;&lt;tr&gt;&lt;th align=&quot;left&quot;&gt;
+&lt;bold&gt;Data collection&lt;/bold&gt;
+&lt;/th&gt;&lt;th align=&quot;left&quot;&gt;YfiB (crystal form I)&lt;/th&gt;&lt;th align=&quot;left&quot;&gt;YfiB (crystal form II)&lt;/th&gt;&lt;th align=&quot;left&quot;&gt;VB6-bound YfiR&lt;/th&gt;&lt;th align=&quot;left&quot;&gt;Trp-bound YfiR&lt;/th&gt;&lt;th align=&quot;left&quot;&gt;YfiBL43P-YfiR&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Space group&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;
+&lt;italic&gt;P&lt;/italic&gt;21&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;
+&lt;italic&gt;P&lt;/italic&gt;41&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;
+&lt;italic&gt;P&lt;/italic&gt;43212&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;
+&lt;italic&gt;P&lt;/italic&gt;43212&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;
+&lt;italic&gt;P&lt;/italic&gt;1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Wavelength (Å)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;1.54187&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0.9791&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0.97861&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0.9791&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;1.10000&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Resolution (Å)&lt;sup&gt;a&lt;/sup&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;50.0–2.15 (2.19–2.15)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;50.0–2.80 (2.85–2.8)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;50.0–2.4 (2.44–2.4)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;50.0–2.5 (2.54–2.5)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;50–1.78 (1.86–1.78)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; colspan=&quot;6&quot;&gt;Cell dimensions&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; a, b, c (Å)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;65.85, 90.45, 66.30&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;46.95, 46.95, 154.24&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;120.24, 120.24, 84.99&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;120.88, 120.88, 88.46&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;49.50, 58.57, 69.86&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; α, β, γ (°)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;90, 113.87, 90&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;72.93, 96.98, 90.19&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Unique reflections&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;37,625 (1866)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;8,105 (412)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;24,776 (1202)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;23170 (1132)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;67,774 (6615)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;
+&lt;italic&gt; I/&lt;/italic&gt;σ&lt;italic&gt;I&lt;/italic&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;19.59 (2.62)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;12.36 (4.15)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;20.17 (2.4)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;39.5 (4.68)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;17.75 (1.89)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Completeness (%)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;97.1 (95.4)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;97.8 (100)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;99.1 (98.8)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;99.9 (100)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;96.5 (94.6)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; &lt;italic&gt;R&lt;/italic&gt;
+&lt;sub&gt;merge&lt;/sub&gt; (%)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;6.5 (44.5)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;14.6 (49.7)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;8.9 (56.8)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;9.4 (89.2)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;5.6 (46.3)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;
+&lt;italic&gt; R&lt;/italic&gt;
+&lt;sub&gt;meas&lt;/sub&gt; (%)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;7.4 (51.6)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;15.4 (52.0)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;9.6 (61.7)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;9.6 (90.8)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;6.6 (55.1)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; CC1/2&lt;sup&gt;b&lt;/sup&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0.747&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0.952&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0.899&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0.974&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0.849&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; colspan=&quot;6&quot;&gt;
+&lt;bold&gt;Refinement&lt;/bold&gt;
+&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;
+&lt;italic&gt; R&lt;/italic&gt;
+&lt;sub&gt;work&lt;/sub&gt; (%)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;20.14&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;19.17&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;17.82&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;18.66&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;17.90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;
+&lt;italic&gt; R&lt;/italic&gt;
+&lt;sub&gt;free&lt;/sub&gt;(%)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;26.29&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;26.49&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;19.81&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;23.05&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;20.61&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; colspan=&quot;6&quot;&gt;Average B factors (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Protein&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;25.54&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;42.70&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;38.68&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;35.03&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;32.54&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; VB6&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;-&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;-&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;44.08&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;-&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;-&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Trp&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;-&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;-&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;-&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;87.51&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;-&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; SO&lt;sub&gt;4&lt;/sub&gt;
+&lt;sup&gt;2−&lt;/sup&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;37.16&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;66.52&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;51.55&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;41.93&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;45.51&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;32.91&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;36.09&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;40.58&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;34.75&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;43.52&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; colspan=&quot;6&quot;&gt;Root mean square deviations&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Bond lengths (Å)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0.009&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0.009&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0.007&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0.007&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0.007&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Bond angles (°)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;1.085&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;1.132&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;1.021&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0.977&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;1.110&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; colspan=&quot;6&quot;&gt;Ramachandran plot&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Most favored (%)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;92.6&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;87.7&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;96.5&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;98.1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;94.2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Additionally allowed (%)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;7.4&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;12.3&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;3.5&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;1.9&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;5.8&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Generously allowed (%)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Disallowed&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;0&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>33433</offset><text>Data collection	YfiB (crystal form I)	YfiB (crystal form II)	VB6-bound YfiR	Trp-bound YfiR	YfiBL43P-YfiR	 	Space group	P21	P41	P43212	P43212	P1	 	Wavelength (Å)	1.54187	0.9791	0.97861	0.9791	1.10000	 	Resolution (Å)a	50.0–2.15 (2.19–2.15)	50.0–2.80 (2.85–2.8)	50.0–2.4 (2.44–2.4)	50.0–2.5 (2.54–2.5)	50–1.78 (1.86–1.78)	 	Cell dimensions	 	 a, b, c (Å)	65.85, 90.45, 66.30	46.95, 46.95, 154.24	120.24, 120.24, 84.99	120.88, 120.88, 88.46	49.50, 58.57, 69.86	 	 α, β, γ (°)	90, 113.87, 90	90, 90, 90	90, 90, 90	90, 90, 90	72.93, 96.98, 90.19	 	 Unique reflections	37,625 (1866)	8,105 (412)	24,776 (1202)	23170 (1132)	67,774 (6615)	 	 I/σI	19.59 (2.62)	12.36 (4.15)	20.17 (2.4)	39.5 (4.68)	17.75 (1.89)	 	 Completeness (%)	97.1 (95.4)	97.8 (100)	99.1 (98.8)	99.9 (100)	96.5 (94.6)	 	 Rmerge (%)	6.5 (44.5)	14.6 (49.7)	8.9 (56.8)	9.4 (89.2)	5.6 (46.3)	 	 Rmeas (%)	7.4 (51.6)	15.4 (52.0)	9.6 (61.7)	9.6 (90.8)	6.6 (55.1)	 	 CC1/2b	0.747	0.952	0.899	0.974	0.849	 	Refinement	 	 Rwork (%)	20.14	19.17	17.82	18.66	17.90	 	 Rfree(%)	26.29	26.49	19.81	23.05	20.61	 	Average B factors (Å2)	 	 Protein	25.54	42.70	38.68	35.03	32.54	 	 VB6	-	-	44.08	-	-	 	 Trp	-	-	-	87.51	-	 	 SO42−	37.16	66.52	51.55	41.93	45.51	 	 H2O	32.91	36.09	40.58	34.75	43.52	 	Root mean square deviations	 	 Bond lengths (Å)	0.009	0.009	0.007	0.007	0.007	 	 Bond angles (°)	1.085	1.132	1.021	0.977	1.110	 	Ramachandran plot	 	 Most favored (%)	92.6	87.7	96.5	98.1	94.2	 	 Additionally allowed (%)	7.4	12.3	3.5	1.9	5.8	 	 Generously allowed (%)	0	0	0	0	0	 	 Disallowed	0	0	0	0	0	 	</text></passage><passage><infon key="file">Tab1.xml</infon><infon key="id">Tab1</infon><infon key="section_type">TABLE</infon><infon key="type">table_foot</infon><offset>35033</offset><text>
+a Numbers in parentheses are for the highest resolution shell</text></passage><passage><infon key="file">Tab1.xml</infon><infon key="id">Tab1</infon><infon key="section_type">TABLE</infon><infon key="type">table_foot</infon><offset>35096</offset><text>
+b The values of CC1/2 are for the highest resolution shell</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35156</offset><text>The two YfiB crystal structures respectively belonging to space groups P21 and P41 were both solved by molecular replacement (Lebedev et al.,) using the putative MotB-like protein DVU_2228 from D. vulgaris as a model (PDB code: 3khn) at 2.15 Å and 2.8 Å resolution, respectively. Both the VB6-bound and the Trp-bound YfiR crystals belonging to space group P43212, with a dimer in the asymmetric unit, were solved by molecular replacement (Lebedev et al.,) using native YfiR as a model (PDB code: 4YN7) at 2.4 Å and 2.5 Å resolution, respectively. The YfiB-YfiR crystal belonging to space group P1, with a 2:2 heterotetramer in the asymmetric unit, was solved by molecular replacement using YfiR and YfiB as models. Electron density maps were calculated using PHENIX (Adams et al.,). Model building was performed using COOT (Emsley et al.,) and refined with PHENIX (Adams et al.,; Afonine et al.,). The final structures were analyzed with PROCHECK (Laskowski et al.,). Data collection and refinement statistics are presented in Table 1. The figures depicting structures were prepared using PyMOL (http://www.pymol.org). Atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank (http://www.pdb.org) under accession codes 5EAZ, 5EB0, 5EB1, 5EB2 and 5EB3.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>36447</offset><text>Analytical ultracentrifugation</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>36478</offset><text>Sedimentation velocity measurements were performed on a Beckman ProteomeLab XL-I at 25°C. All protein samples were diluted to an OD280 of 0.7 in 20 mmol/L Tris (pH 8.0) and 200 mmol/L NaCl. Data were collected at 60,000 rpm. (262,000 ×g) every 3 min at a wavelength of 280 nm. Interference sedimentation coefficient distributions, or c(M), were calculated from the sedimentation velocity data using SEDFIT (Schuck,).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>36897</offset><text>PG preparation</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>36912</offset><text>PG was extracted from the E. coli DH5α strain by following a method described previously (Desmarais et al.,). Briefly, cells were cultured until they reached an OD600 of 0.7–0.8 and then collected at 5,000 ×g, 4°C. The collected bacteria were dripped into the boiling 6% (w/v) SDS and stirred at 500 rpm in a boiling water bath for 3 h before incubating overnight at room temperature. The large PG polymers were collected by ultracentrifugation at 130,000 ×g for 1 h at room temperature and washed repeatedly to remove SDS. The pellet was treated with Pronase E (200 μg/mL final concentration) for 3 h at 60°C followed by SDS to remove contaminating proteins and washed three times to remove the SDS by ultracentrifugation. Next, the samples were treated with lysozyme (200 μg/mL final concentration) for 16 h at 37°C. Finally, the purified PG is obtained by treating the samples in a boiling water bath for 10 min and centrifuging it at 13,000 ×g to remove the contaminating lysozyme.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>37909</offset><text>Microscale thermophoresis (MST)</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>37941</offset><text>Purified YfiB wild-type and it mutant YfiBL43P were fluorescently labeled using the NanoTemper blue protein-labeling kit according to the manufacturer’s protocol. This resulted in coupling of the fluorescent dye NT-495. PG was titrated in 1:1 dilutions starting at 1 mmol/L. To determine of the Kd values, 10 μL labeled protein was mixed with 10 μL PG at various concentrations in Hepes buffer (20 mmol/L Hepes, 200 mmol/L NaCl, 0.005% Tween-20, pH 7.5). After 10 min of incubation, all binding reaction mixtures were loaded into the MST-grade glass capillaries (NanoTemper Technologies), and thermophoresis was measured with a NanoTemper Monolith-NT115 system (20% light-emitting diode, 20% IR laser power).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>38654</offset><text>Deletion of the yfiB genes</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38681</offset><text>The yfiB deletion construct was produced by SOE-PCR (Hmelo et al.,) and contained homologous flanking regions to the target gene. This construct was ligated into the pEX18Gm vector between the HindIII and the KpnI sites. The resulting vector was then used to delete yfiB by two-step allelic exchange (Hmelo et al.,). After being introduced into PAO1 via biparental mating with E. coli SM10 (λpir), single crossovers were selected on Vogel-Bonner Minimal Medium (VBMM), which was used for counter-selection against E. coli (P. aeruginosa can utilize citrate as a sole carbon source and energy source, whereas E. coli cannot), containing 50 μg/mL gentamycin. Restreaking was then performed on no-salt Luria-Bertani (NSLB) agar that contained 15% sucrose to force the resolution of double crossovers. Deletion of yfiB in the strains was confirmed by colony PCR.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>39546</offset><text>For complementation experiments, yfiB wild-type and L43P mutant genes were cloned into the pJN105 vector via the EcoRI and XbaI restriction sites, respectively. The plasmids were then individually transformed into the PAO1 ΔyfiB strain using the rapid electroporation method described in Choi KH et al. (Choi et al.,). Transformants were selected on LB plates containing 50 μg/mL gentamycin. For induction, arabinose was added to a final concentration of 0.2%.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>40012</offset><text>Attachment assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>40030</offset><text>The attachment assays were carried out using the MBECTM (Minimum Biofilm Eradication Concentration, Innovotech, Inc.) biofilm inoculator, which consists of a plastic lid with 96 pegs and 96 individual wells. The MBEC plates containing 150 μL LB medium/well were inoculated with 1% overnight cultures of the yfiB-L43P strain and incubated overnight at 37°C without shaking. VB6, L-Trp and arabinose were added as appropriate. The peg lids were washed with distilled water, and the attached cell material was then stained with 0.1% crystal violet solution (5% methanol, 5% isopropanol) before further washing to remove excess dye. The crystal violet was re-dissolved in 20% acetic acid solution, and the absorbance was measured at 600 nm. Assays were performed with 12 wells/strain and repeated independently for each experiment.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>40860</offset><text>BIAcore analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>40877</offset><text>The interaction kinetics of YfiR with VB6 and L-Trp were examined on a SPR machine Biacore 3000 (GE Healthcare) at 25°C. The running buffer (20 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 0.005% (v/v) Tween-20) was vacuum filtered, and degassed immediately prior to use. YfiR at 10 μg/mL in 10 mmol/L sodium acetate (pH 5.5) was immobilized to 3000 response units on the carboxymethylated dextran surface-modified chip (CM5 chip). The binding affinities were evaluated over a range of 2.5–40 mmol/L concentrations. Meanwhile, for both binding assays, the concentration of 10 mmol/L was repeated as an internal control. All of the data collected were analyzed using BIAevaluation software version 4.1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>41577</offset><text>ITC assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>41588</offset><text>ITC experiments were performed in a buffer composed of 20 mmol/L Tris (pH 8.0) and 150 mmol/L NaCl at 25°C using an iTC200 calorimeter (GE Healthcare). YfiB wild-type or its mutants (YfiBL43P, YfiBL43P/F57A) (0.4 mmol/L, in the syringe) was titrated into YfiR (0.04 mmol/L, in the cell), respectively. The titration sequence included a single 0.5 µL injection, followed by 19 injections of 2 µL each, with a 2-min interval between injections and a stirring rate of 1000 rpm. The calorimetric data were then analyzed with OriginLab software (GE Healthcare).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">footnote</infon><offset>42153</offset><text>Min Xu, Xuan Yang and Xiu-An Yang have contributed equally to this work.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>42226</offset><text>References</text></passage><passage><infon key="fpage">213</infon><infon key="lpage">221</infon><infon key="name_0">surname:Adams;given-names:PD</infon><infon key="name_1">surname:Afonine;given-names:PV</infon><infon key="name_2">surname:Bunkoczi;given-names:G</infon><infon key="name_3">surname:Chen;given-names:VB</infon><infon key="name_4">surname:Davis;given-names:IW</infon><infon key="name_5">surname:Echols;given-names:N</infon><infon key="name_6">surname:Headd;given-names:JJ</infon><infon key="name_7">surname:Hung;given-names:LW</infon><infon key="name_8">surname:Kapral;given-names:GJ</infon><infon key="name_9">surname:Grosse-Kunstleve;given-names:RW</infon><infon key="pub-id_doi">10.1107/S0907444909052925</infon><infon key="pub-id_pmid">20124702</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>42237</offset><text>PHENIX: a comprehensive Python-based system for macromolecular structure solution</text></passage><passage><infon key="fpage">352</infon><infon key="lpage">367</infon><infon key="name_0">surname:Afonine;given-names:PV</infon><infon key="name_1">surname:Grosse-Kunstleve;given-names:RW</infon><infon key="name_2">surname:Echols;given-names:N</infon><infon key="name_3">surname:Headd;given-names:JJ</infon><infon key="name_4">surname:Moriarty;given-names:NW</infon><infon key="name_5">surname:Mustyakimov;given-names:M</infon><infon key="name_6">surname:Terwilliger;given-names:TC</infon><infon key="name_7">surname:Urzhumtsev;given-names:A</infon><infon key="name_8">surname:Zwart;given-names:PH</infon><infon key="name_9">surname:Adams;given-names:PD</infon><infon key="pub-id_doi">10.1107/S0907444912001308</infon><infon key="pub-id_pmid">22505256</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">68</infon><infon key="year">2012</infon><offset>42319</offset><text>Towards automated crystallographic structure refinement with phenix.refine</text></passage><passage><infon key="fpage">90</infon><infon key="lpage">93</infon><infon key="name_0">surname:Beaumont;given-names:HJ</infon><infon key="name_1">surname:Gallie;given-names:J</infon><infon key="name_2">surname:Kost;given-names:C</infon><infon key="name_3">surname:Ferguson;given-names:GC</infon><infon key="name_4">surname:Rainey;given-names:PB</infon><infon key="pub-id_doi">10.1038/nature08504</infon><infon key="pub-id_pmid">19890329</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">462</infon><infon key="year">2009</infon><offset>42394</offset><text>Experimental evolution of bet hedging</text></passage><passage><infon key="fpage">107</infon><infon key="lpage">116</infon><infon key="name_0">surname:Boehm;given-names:A</infon><infon key="name_1">surname:Kaiser;given-names:M</infon><infon key="name_2">surname:Li;given-names:H</infon><infon key="name_3">surname:Spangler;given-names:C</infon><infon key="name_4">surname:Kasper;given-names:CA</infon><infon key="name_5">surname:Ackermann;given-names:M</infon><infon key="name_6">surname:Kaever;given-names:V</infon><infon key="name_7">surname:Sourjik;given-names:V</infon><infon key="name_8">surname:Roth;given-names:V</infon><infon key="name_9">surname:Jenal;given-names:U</infon><infon key="pub-id_doi">10.1016/j.cell.2010.01.018</infon><infon key="pub-id_pmid">20303158</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">141</infon><infon key="year">2010</infon><offset>42432</offset><text>Second messenger-mediated adjustment of bacterial swimming velocity</text></passage><passage><infon key="fpage">12</infon><infon key="lpage">24</infon><infon key="name_0">surname:Caly;given-names:DL</infon><infon key="name_1">surname:Bellini;given-names:D</infon><infon key="name_2">surname:Walsh;given-names:MA</infon><infon key="name_3">surname:Dow;given-names:JM</infon><infon key="name_4">surname:Ryan;given-names:RP</infon><infon key="pub-id_doi">10.2174/1381612820666140905124701</infon><infon key="pub-id_pmid">25189859</infon><infon key="section_type">REF</infon><infon key="source">Curr Pharm Des</infon><infon key="type">ref</infon><infon key="volume">21</infon><infon key="year">2015</infon><offset>42500</offset><text>Targeting cyclic di-GMP signalling: a strategy to control biofilm formation?</text></passage><passage><infon key="fpage">1113</infon><infon key="lpage">1116</infon><infon key="name_0">surname:Camilli;given-names:A</infon><infon key="name_1">surname:Bassler;given-names:BL</infon><infon key="pub-id_doi">10.1126/science.1121357</infon><infon key="pub-id_pmid">16497924</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">311</infon><infon key="year">2006</infon><offset>42577</offset><text>Bacterial small-molecule signaling pathways</text></passage><passage><infon key="fpage">391</infon><infon key="lpage">397</infon><infon key="name_0">surname:Choi;given-names:KH</infon><infon key="name_1">surname:Kumar;given-names:A</infon><infon key="name_2">surname:Schweizer;given-names:HP</infon><infon key="pub-id_doi">10.1016/j.mimet.2005.06.001</infon><infon key="pub-id_pmid">15987659</infon><infon key="section_type">REF</infon><infon key="source">J Microbiol Methods</infon><infon key="type">ref</infon><infon key="volume">64</infon><infon key="year">2006</infon><offset>42621</offset><text>A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>42795</offset><text>Desmarais SM, Cava F, de Pedro MA, Huang KC (2014) Isolation and preparation of bacterial cell walls for compositional analysis by ultra performance liquid chromatography. J Vis Exp 83:e51183</text></passage><passage><infon key="fpage">93</infon><infon key="lpage">104</infon><infon key="name_0">surname:Duerig;given-names:A</infon><infon key="name_1">surname:Abel;given-names:S</infon><infon key="name_2">surname:Folcher;given-names:M</infon><infon key="name_3">surname:Nicollier;given-names:M</infon><infon key="name_4">surname:Schwede;given-names:T</infon><infon key="name_5">surname:Amiot;given-names:N</infon><infon key="name_6">surname:Giese;given-names:B</infon><infon key="name_7">surname:Jenal;given-names:U</infon><infon key="pub-id_doi">10.1101/gad.502409</infon><infon key="pub-id_pmid">19136627</infon><infon key="section_type">REF</infon><infon key="source">Genes Dev</infon><infon key="type">ref</infon><infon key="volume">23</infon><infon key="year">2009</infon><offset>42987</offset><text>Second messenger-mediated spatiotemporal control of protein degradation regulates bacterial cell cycle progression</text></passage><passage><infon key="fpage">486</infon><infon key="lpage">501</infon><infon key="name_0">surname:Emsley;given-names:P</infon><infon key="name_1">surname:Lohkamp;given-names:B</infon><infon key="name_2">surname:Scott;given-names:WG</infon><infon key="name_3">surname:Cowtan;given-names:K</infon><infon key="pub-id_doi">10.1107/S0907444910007493</infon><infon key="pub-id_pmid">20383002</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>43102</offset><text>Features and development of Coot</text></passage><passage><infon key="fpage">231</infon><infon key="lpage">239</infon><infon key="name_0">surname:Evans;given-names:TJ</infon><infon key="pub-id_doi">10.2217/fmb.14.107</infon><infon key="pub-id_pmid">25689535</infon><infon key="section_type">REF</infon><infon key="source">Future Microbiol</infon><infon key="type">ref</infon><infon key="volume">10</infon><infon key="year">2015</infon><offset>43135</offset><text>Small colony variants of Pseudomonas aeruginosa in chronic bacterial infection of the lung in cystic fibrosis</text></passage><passage><infon key="fpage">e81324</infon><infon key="name_0">surname:Giardina;given-names:G</infon><infon key="name_1">surname:Paiardini;given-names:A</infon><infon key="name_2">surname:Fernicola;given-names:S</infon><infon key="name_3">surname:Franceschini;given-names:S</infon><infon key="name_4">surname:Rinaldo;given-names:S</infon><infon key="name_5">surname:Stelitano;given-names:V</infon><infon key="name_6">surname:Cutruzzola;given-names:F</infon><infon key="pub-id_doi">10.1371/journal.pone.0081324</infon><infon key="pub-id_pmid">24278422</infon><infon key="section_type">REF</infon><infon key="source">PLoS One</infon><infon key="type">ref</infon><infon key="volume">8</infon><infon key="year">2013</infon><offset>43245</offset><text>Investigating the allosteric regulation of YfiN from Pseudomonas aeruginosa: clues from the structure of the catalytic domain</text></passage><passage><infon key="fpage">18247</infon><infon key="lpage">18252</infon><infon key="name_0">surname:Giddens;given-names:SR</infon><infon key="name_1">surname:Jackson;given-names:RW</infon><infon key="name_2">surname:Moon;given-names:CD</infon><infon key="name_3">surname:Jacobs;given-names:MA</infon><infon key="name_4">surname:Zhang;given-names:XX</infon><infon key="name_5">surname:Gehrig;given-names:SM</infon><infon key="name_6">surname:Rainey;given-names:PB</infon><infon key="pub-id_doi">10.1073/pnas.0706739104</infon><infon key="pub-id_pmid">17989226</infon><infon key="section_type">REF</infon><infon key="source">Proc Natl Acad Sci USA</infon><infon key="type">ref</infon><infon key="volume">104</infon><infon key="year">2007</infon><offset>43371</offset><text>Mutational activation of niche-specific genes provides insight into regulatory networks and bacterial function in a complex environment</text></passage><passage><infon key="fpage">539</infon><infon key="lpage">574</infon><infon key="name_0">surname:Govan;given-names:JR</infon><infon key="name_1">surname:Deretic;given-names:V</infon><infon key="pub-id_pmid">8840786</infon><infon key="section_type">REF</infon><infon key="source">Microbiol Rev</infon><infon key="type">ref</infon><infon key="volume">60</infon><infon key="year">1996</infon><offset>43507</offset><text>Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>43605</offset><text>Grubman A, Phillips A, Thibonnier M, Kaparakis-Liaskos M, Johnson C, Thiberge JM, Radcliff FJ, Ecobichon C, Labigne A, de Reuse H. et al (2010) Vitamin B6 is required for full motility and virulence in Helicobacter pylori. MBio 1</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>43835</offset><text>Ha DG, O’Toole GA (2015) c-di-GMP and its effects on biofilm formation and dispersion: a Pseudomonas aeruginosa review. Microbiol Spectr 3, MB-0003-2014</text></passage><passage><infon key="fpage">621</infon><infon key="lpage">625</infon><infon key="name_0">surname:Haussler;given-names:S</infon><infon key="name_1">surname:Tummler;given-names:B</infon><infon key="name_2">surname:Weissbrodt;given-names:H</infon><infon key="name_3">surname:Rohde;given-names:M</infon><infon key="name_4">surname:Steinmetz;given-names:I</infon><infon key="pub-id_doi">10.1086/598644</infon><infon key="pub-id_pmid">10530458</infon><infon key="section_type">REF</infon><infon key="source">Clin Infect Dis</infon><infon key="type">ref</infon><infon key="volume">29</infon><infon key="year">1999</infon><offset>43990</offset><text>Small-colony variants of Pseudomonas aeruginosa in cystic fibrosis</text></passage><passage><infon key="fpage">295</infon><infon key="lpage">301</infon><infon key="name_0">surname:Haussler;given-names:S</infon><infon key="name_1">surname:Ziegler;given-names:I</infon><infon key="name_2">surname:Lottel;given-names:A</infon><infon key="name_3">surname:von Gotz;given-names:F</infon><infon key="name_4">surname:Rohde;given-names:M</infon><infon key="name_5">surname:Wehmhohner;given-names:D</infon><infon key="name_6">surname:Saravanamuthu;given-names:S</infon><infon key="name_7">surname:Tummler;given-names:B</infon><infon key="name_8">surname:Steinmetz;given-names:I</infon><infon key="pub-id_doi">10.1099/jmm.0.05069-0</infon><infon key="pub-id_pmid">12676867</infon><infon key="section_type">REF</infon><infon key="source">J Med Microbiol</infon><infon key="type">ref</infon><infon key="volume">52</infon><infon key="year">2003</infon><offset>44057</offset><text>Highly adherent small-colony variants of Pseudomonas aeruginosa in cystic fibrosis lung infection</text></passage><passage><infon key="fpage">263</infon><infon key="lpage">273</infon><infon key="name_0">surname:Hengge;given-names:R</infon><infon key="pub-id_doi">10.1038/nrmicro2109</infon><infon key="pub-id_pmid">19287449</infon><infon key="section_type">REF</infon><infon key="source">Nat Rev Microbiol</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2009</infon><offset>44155</offset><text>Principles of c-di-GMP signalling in bacteria</text></passage><passage><infon key="fpage">14422</infon><infon key="lpage">14427</infon><infon key="name_0">surname:Hickman;given-names:JW</infon><infon key="name_1">surname:Tifrea;given-names:DF</infon><infon key="name_2">surname:Harwood;given-names:CS</infon><infon key="pub-id_doi">10.1073/pnas.0507170102</infon><infon key="pub-id_pmid">16186483</infon><infon key="section_type">REF</infon><infon key="source">Proc Natl Acad Sci USA</infon><infon key="type">ref</infon><infon key="volume">102</infon><infon key="year">2005</infon><offset>44201</offset><text>A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels</text></passage><passage><infon key="fpage">1820</infon><infon key="lpage">1841</infon><infon key="name_0">surname:Hmelo;given-names:LR</infon><infon key="name_1">surname:Borlee;given-names:BR</infon><infon key="name_2">surname:Almblad;given-names:H</infon><infon key="name_3">surname:Love;given-names:ME</infon><infon key="name_4">surname:Randall;given-names:TE</infon><infon key="name_5">surname:Tseng;given-names:BS</infon><infon key="name_6">surname:Lin;given-names:C</infon><infon key="name_7">surname:Irie;given-names:Y</infon><infon key="name_8">surname:Storek;given-names:KM</infon><infon key="name_9">surname:Yang;given-names:JJ</infon><infon key="pub-id_doi">10.1038/nprot.2015.115</infon><infon key="pub-id_pmid">26492139</infon><infon key="section_type">REF</infon><infon key="source">Nat Protoc</infon><infon key="type">ref</infon><infon key="volume">10</infon><infon key="year">2015</infon><offset>44304</offset><text>Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange</text></passage><passage><infon key="fpage">W545</infon><infon key="lpage">W549</infon><infon key="name_0">surname:Holm;given-names:L</infon><infon key="name_1">surname:Rosenstrom;given-names:P</infon><infon key="pub-id_doi">10.1093/nar/gkq366</infon><infon key="pub-id_pmid">20457744</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res</infon><infon key="type">ref</infon><infon key="volume">38</infon><infon key="year">2010</infon><offset>44391</offset><text>Dali server: conservation mapping in 3D</text></passage><passage><infon key="fpage">2595</infon><infon key="lpage">2606</infon><infon key="name_0">surname:Huertas;given-names:MG</infon><infon key="name_1">surname:Zarate;given-names:L</infon><infon key="name_2">surname:Acosta;given-names:IC</infon><infon key="name_3">surname:Posada;given-names:L</infon><infon key="name_4">surname:Cruz;given-names:DP</infon><infon key="name_5">surname:Lozano;given-names:M</infon><infon key="name_6">surname:Zambrano;given-names:MM</infon><infon key="pub-id_doi">10.1099/mic.0.081992-0</infon><infon key="pub-id_pmid">25261190</infon><infon key="section_type">REF</infon><infon key="source">Microbiology</infon><infon key="type">ref</infon><infon key="volume">160</infon><infon key="year">2014</infon><offset>44431</offset><text>Klebsiella pneumoniae yfiRNB operon affects biofilm formation, polysaccharide production and drug susceptibility</text></passage><passage><infon key="fpage">3690</infon><infon key="lpage">3699</infon><infon key="name_0">surname:Hufnagel;given-names:DA</infon><infon key="name_1">surname:DePas;given-names:WH</infon><infon key="name_2">surname:Chapman;given-names:MR</infon><infon key="pub-id_doi">10.1128/JB.02019-14</infon><infon key="pub-id_pmid">25112475</infon><infon key="section_type">REF</infon><infon key="source">J Bacteriol</infon><infon key="type">ref</infon><infon key="volume">196</infon><infon key="year">2014</infon><offset>44544</offset><text>The disulfide bonding system suppresses CsgD-independent cellulose production in Escherichia coli</text></passage><passage><infon key="fpage">185</infon><infon key="lpage">191</infon><infon key="name_0">surname:Jenal;given-names:U</infon><infon key="pub-id_doi">10.1016/j.mib.2004.02.007</infon><infon key="pub-id_pmid">15063857</infon><infon key="section_type">REF</infon><infon key="source">Curr Opin Microbiol</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2004</infon><offset>44642</offset><text>Cyclic di-guanosine-monophosphate comes of age: a novel secondary messenger involved in modulating cell surface structures in bacteria?</text></passage><passage><infon key="fpage">75</infon><infon key="lpage">88</infon><infon key="name_0">surname:Kirillina;given-names:O</infon><infon key="name_1">surname:Fetherston;given-names:JD</infon><infon key="name_2">surname:Bobrov;given-names:AG</infon><infon key="name_3">surname:Abney;given-names:J</infon><infon key="name_4">surname:Perry;given-names:RD</infon><infon key="pub-id_doi">10.1111/j.1365-2958.2004.04253.x</infon><infon key="pub-id_pmid">15458406</infon><infon key="section_type">REF</infon><infon key="source">Mol Microbiol</infon><infon key="type">ref</infon><infon key="volume">54</infon><infon key="year">2004</infon><offset>44778</offset><text>HmsP, a putative phosphodiesterase, and HmsT, a putative diguanylate cyclase, control Hms-dependent biofilm formation in Yersinia pestis</text></passage><passage><infon key="fpage">4809</infon><infon key="lpage">4821</infon><infon key="name_0">surname:Kirisits;given-names:MJ</infon><infon key="name_1">surname:Prost;given-names:L</infon><infon key="name_2">surname:Starkey;given-names:M</infon><infon key="name_3">surname:Parsek;given-names:MR</infon><infon key="pub-id_doi">10.1128/AEM.71.8.4809-4821.2005</infon><infon key="pub-id_pmid">16085879</infon><infon key="section_type">REF</infon><infon key="source">Appl Environ Microbiol</infon><infon key="type">ref</infon><infon key="volume">71</infon><infon key="year">2005</infon><offset>44915</offset><text>Characterization of colony morphology variants isolated from Pseudomonas aeruginosa biofilms</text></passage><passage><infon key="fpage">2839</infon><infon key="lpage">2844</infon><infon key="name_0">surname:Kulasakara;given-names:H</infon><infon key="name_1">surname:Lee;given-names:V</infon><infon key="name_2">surname:Brencic;given-names:A</infon><infon key="name_3">surname:Liberati;given-names:N</infon><infon key="name_4">surname:Urbach;given-names:J</infon><infon key="name_5">surname:Miyata;given-names:S</infon><infon key="name_6">surname:Lee;given-names:DG</infon><infon key="name_7">surname:Neely;given-names:AN</infon><infon key="name_8">surname:Hyodo;given-names:M</infon><infon key="name_9">surname:Hayakawa;given-names:Y</infon><infon key="pub-id_doi">10.1073/pnas.0511090103</infon><infon key="pub-id_pmid">16477007</infon><infon key="section_type">REF</infon><infon key="source">Proc Natl Acad Sci USA</infon><infon key="type">ref</infon><infon key="volume">103</infon><infon key="year">2006</infon><offset>45008</offset><text>Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3′-5′)-cyclic-GMP in virulence</text></passage><passage><infon key="fpage">283</infon><infon key="lpage">291</infon><infon key="name_0">surname:Laskowski;given-names:RA</infon><infon key="name_1">surname:MacArthur;given-names:MW</infon><infon key="name_2">surname:Moss;given-names:DS</infon><infon key="name_3">surname:Thornton;given-names:JM</infon><infon key="pub-id_doi">10.1107/S0021889892009944</infon><infon key="section_type">REF</infon><infon key="source">J Appl Crystallogr</infon><infon key="type">ref</infon><infon key="volume">26</infon><infon key="year">1993</infon><offset>45146</offset><text>PROCHECK: a program to check the stereochemical quality of protein structures</text></passage><passage><infon key="fpage">33</infon><infon key="lpage">39</infon><infon key="name_0">surname:Lebedev;given-names:AA</infon><infon key="name_1">surname:Vagin;given-names:AA</infon><infon key="name_2">surname:Murshudov;given-names:GN</infon><infon key="pub-id_doi">10.1107/S0907444907049839</infon><infon key="pub-id_pmid">18094465</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">64</infon><infon key="year">2008</infon><offset>45224</offset><text>Model preparation in MOLREP and examples of model improvement using X-ray data</text></passage><passage><infon key="fpage">16915</infon><infon key="name_0">surname:Li;given-names:S</infon><infon key="name_1">surname:Li;given-names:T</infon><infon key="name_2">surname:Xu;given-names:Y</infon><infon key="name_3">surname:Zhang;given-names:Q</infon><infon key="name_4">surname:Zhang;given-names:W</infon><infon key="name_5">surname:Che;given-names:S</infon><infon key="name_6">surname:Liu;given-names:R</infon><infon key="name_7">surname:Wang;given-names:Y</infon><infon key="name_8">surname:Bartlam;given-names:M</infon><infon key="pub-id_doi">10.1038/srep16915</infon><infon key="pub-id_pmid">26593397</infon><infon key="section_type">REF</infon><infon key="source">Sci Rep</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2015</infon><offset>45303</offset><text>Structural insights into YfiR sequestering by YfiB in Pseudomonas aeruginosa PAO1</text></passage><passage><infon key="fpage">237</infon><infon key="lpage">247</infon><infon key="name_0">surname:Malone;given-names:JG</infon><infon key="pub-id_doi">10.2147/IDR.S68214</infon><infon key="pub-id_pmid">26251621</infon><infon key="section_type">REF</infon><infon key="source">Infect Drug Resist</infon><infon key="type">ref</infon><infon key="volume">8</infon><infon key="year">2015</infon><offset>45385</offset><text>Role of small colony variants in persistence of Pseudomonas aeruginosa infections in cystic fibrosis lungs</text></passage><passage><infon key="fpage">e1000804</infon><infon key="name_0">surname:Malone;given-names:JG</infon><infon key="name_1">surname:Jaeger;given-names:T</infon><infon key="name_2">surname:Spangler;given-names:C</infon><infon key="name_3">surname:Ritz;given-names:D</infon><infon key="name_4">surname:Spang;given-names:A</infon><infon key="name_5">surname:Arrieumerlou;given-names:C</infon><infon key="name_6">surname:Kaever;given-names:V</infon><infon key="name_7">surname:Landmann;given-names:R</infon><infon key="name_8">surname:Jenal;given-names:U</infon><infon key="pub-id_doi">10.1371/journal.ppat.1000804</infon><infon key="pub-id_pmid">20300602</infon><infon key="section_type">REF</infon><infon key="source">PLoS Pathog</infon><infon key="type">ref</infon><infon key="volume">6</infon><infon key="year">2010</infon><offset>45492</offset><text>YfiBNR mediates cyclic di-GMP dependent small colony variant formation and persistence in Pseudomonas aeruginosa</text></passage><passage><infon key="fpage">e1002760</infon><infon key="name_0">surname:Malone;given-names:JG</infon><infon key="name_1">surname:Jaeger;given-names:T</infon><infon key="name_2">surname:Manfredi;given-names:P</infon><infon key="name_3">surname:Dotsch;given-names:A</infon><infon key="name_4">surname:Blanka;given-names:A</infon><infon key="name_5">surname:Bos;given-names:R</infon><infon key="name_6">surname:Cornelis;given-names:GR</infon><infon key="name_7">surname:Haussler;given-names:S</infon><infon key="name_8">surname:Jenal;given-names:U</infon><infon key="pub-id_doi">10.1371/journal.ppat.1002760</infon><infon key="pub-id_pmid">22719254</infon><infon key="section_type">REF</infon><infon key="source">PLoS Pathog</infon><infon key="type">ref</infon><infon key="volume">8</infon><infon key="year">2012</infon><offset>45605</offset><text>The YfiBNR signal transduction mechanism reveals novel targets for the evolution of persistent Pseudomonas aeruginosa in cystic fibrosis airways</text></passage><passage><infon key="fpage">6112</infon><infon key="lpage">6118</infon><infon key="name_0">surname:Matias;given-names:VR</infon><infon key="name_1">surname:Al-Amoudi;given-names:A</infon><infon key="name_2">surname:Dubochet;given-names:J</infon><infon key="name_3">surname:Beveridge;given-names:TJ</infon><infon key="pub-id_doi">10.1128/JB.185.20.6112-6118.2003</infon><infon key="pub-id_pmid">14526023</infon><infon key="section_type">REF</infon><infon key="source">J Bacteriol</infon><infon key="type">ref</infon><infon key="volume">185</infon><infon key="year">2003</infon><offset>45750</offset><text>Cryo-transmission electron microscopy of frozen-hydrated sections of Escherichia coli and Pseudomonas aeruginosa</text></passage><passage><infon key="fpage">4404</infon><infon key="lpage">4409</infon><infon key="name_0">surname:Meroueh;given-names:SO</infon><infon key="name_1">surname:Bencze;given-names:KZ</infon><infon key="name_2">surname:Hesek;given-names:D</infon><infon key="name_3">surname:Lee;given-names:M</infon><infon key="name_4">surname:Fisher;given-names:JF</infon><infon key="name_5">surname:Stemmler;given-names:TL</infon><infon key="name_6">surname:Mobashery;given-names:S</infon><infon key="pub-id_doi">10.1073/pnas.0510182103</infon><infon key="pub-id_pmid">16537437</infon><infon key="section_type">REF</infon><infon key="source">Proc Natl Acad Sci USA</infon><infon key="type">ref</infon><infon key="volume">103</infon><infon key="year">2006</infon><offset>45863</offset><text>Three-dimensional structure of the bacterial cell wall peptidoglycan</text></passage><passage><infon key="fpage">e1000588</infon><infon key="name_0">surname:Navarro;given-names:MV</infon><infon key="name_1">surname:Newell;given-names:PD</infon><infon key="name_2">surname:Krasteva;given-names:PV</infon><infon key="name_3">surname:Chatterjee;given-names:D</infon><infon key="name_4">surname:Madden;given-names:DR</infon><infon key="name_5">surname:O’Toole;given-names:GA</infon><infon key="name_6">surname:Sondermann;given-names:H</infon><infon key="pub-id_doi">10.1371/journal.pbio.1000588</infon><infon key="pub-id_pmid">21304926</infon><infon key="section_type">REF</infon><infon key="source">PLoS Biol</infon><infon key="type">ref</infon><infon key="volume">9</infon><infon key="year">2011</infon><offset>45932</offset><text>Structural basis for c-di-GMP-mediated inside-out signaling controlling periplasmic proteolysis</text></passage><passage><infon key="fpage">307</infon><infon key="lpage">326</infon><infon key="name_0">surname:Otwinowski;given-names:Z</infon><infon key="name_1">surname:Minor;given-names:W</infon><infon key="pub-id_doi">10.1016/S0076-6879(97)76066-X</infon><infon key="section_type">REF</infon><infon key="source">Methods Enzymol</infon><infon key="type">ref</infon><infon key="volume">276</infon><infon key="year">1997</infon><offset>46028</offset><text>Processing of X-ray diffraction data collected in oscillation mode</text></passage><passage><infon key="fpage">677</infon><infon key="lpage">701</infon><infon key="name_0">surname:Parsek;given-names:MR</infon><infon key="name_1">surname:Singh;given-names:PK</infon><infon key="pub-id_doi">10.1146/annurev.micro.57.030502.090720</infon><infon key="pub-id_pmid">14527295</infon><infon key="section_type">REF</infon><infon key="source">Annu Rev Microbiol</infon><infon key="type">ref</infon><infon key="volume">57</infon><infon key="year">2003</infon><offset>46095</offset><text>Bacterial biofilms: an emerging link to disease pathogenesis</text></passage><passage><infon key="fpage">2122</infon><infon key="lpage">2128</infon><infon key="name_0">surname:Parsons;given-names:LM</infon><infon key="name_1">surname:Lin;given-names:F</infon><infon key="name_2">surname:Orban;given-names:J</infon><infon key="pub-id_doi">10.1021/bi052227i</infon><infon key="pub-id_pmid">16475801</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry</infon><infon key="type">ref</infon><infon key="volume">45</infon><infon key="year">2006</infon><offset>46156</offset><text>Peptidoglycan recognition by Pal, an outer membrane lipoprotein</text></passage><passage><infon key="fpage">170</infon><infon key="lpage">176</infon><infon key="name_0">surname:Pesavento;given-names:C</infon><infon key="name_1">surname:Hengge;given-names:R</infon><infon key="pub-id_doi">10.1016/j.mib.2009.01.007</infon><infon key="pub-id_pmid">19318291</infon><infon key="section_type">REF</infon><infon key="source">Curr Opin Microbiol</infon><infon key="type">ref</infon><infon key="volume">12</infon><infon key="year">2009</infon><offset>46220</offset><text>Bacterial nucleotide-based second messengers</text></passage><passage><infon key="fpage">3089</infon><infon key="lpage">3098</infon><infon key="name_0">surname:Raterman;given-names:EL</infon><infon key="name_1">surname:Shapiro;given-names:DD</infon><infon key="name_2">surname:Stevens;given-names:DJ</infon><infon key="name_3">surname:Schwartz;given-names:KJ</infon><infon key="name_4">surname:Welch;given-names:RA</infon><infon key="pub-id_doi">10.1128/IAI.01396-12</infon><infon key="pub-id_pmid">23774594</infon><infon key="section_type">REF</infon><infon key="source">Infect Immun</infon><infon key="type">ref</infon><infon key="volume">81</infon><infon key="year">2013</infon><offset>46265</offset><text>Genetic analysis of the role of yfiR in the ability of Escherichia coli CFT073 to control cellular cyclic dimeric GMP levels and to persist in the urinary tract</text></passage><passage><infon key="fpage">1341</infon><infon key="lpage">1350</infon><infon key="name_0">surname:Reinhardt;given-names:A</infon><infon key="name_1">surname:Kohler;given-names:T</infon><infon key="name_2">surname:Wood;given-names:P</infon><infon key="name_3">surname:Rohner;given-names:P</infon><infon key="name_4">surname:Dumas;given-names:JL</infon><infon key="name_5">surname:Ricou;given-names:B</infon><infon key="name_6">surname:van Delden;given-names:C</infon><infon key="pub-id_doi">10.1128/AAC.01278-06</infon><infon key="pub-id_pmid">17261619</infon><infon key="section_type">REF</infon><infon key="source">Antimicrob Agents Chemother</infon><infon key="type">ref</infon><infon key="volume">51</infon><infon key="year">2007</infon><offset>46426</offset><text>Development and persistence of antimicrobial resistance in Pseudomonas aeruginosa: a longitudinal observation in mechanically ventilated patients</text></passage><passage><infon key="fpage">1202</infon><infon key="lpage">1216</infon><infon key="name_0">surname:Ren;given-names:GX</infon><infon key="name_1">surname:Yan;given-names:HQ</infon><infon key="name_2">surname:Zhu;given-names:H</infon><infon key="name_3">surname:Guo;given-names:XP</infon><infon key="name_4">surname:Sun;given-names:YC</infon><infon key="pub-id_doi">10.1111/1462-2920.12323</infon><infon key="pub-id_pmid">24192006</infon><infon key="section_type">REF</infon><infon key="source">Environ Microbiol</infon><infon key="type">ref</infon><infon key="volume">16</infon><infon key="year">2014</infon><offset>46572</offset><text>HmsC, a periplasmic protein, controls biofilm formation via repression of HmsD, a diguanylate cyclase in Yersinia pestis</text></passage><passage><infon key="fpage">1</infon><infon key="lpage">52</infon><infon key="name_0">surname:Romling;given-names:U</infon><infon key="name_1">surname:Galperin;given-names:MY</infon><infon key="name_2">surname:Gomelsky;given-names:M</infon><infon key="pub-id_doi">10.1128/MMBR.00043-12</infon><infon key="pub-id_pmid">23471616</infon><infon key="section_type">REF</infon><infon key="source">Microbiol Mol Biol Rev</infon><infon key="type">ref</infon><infon key="volume">77</infon><infon key="year">2013</infon><offset>46693</offset><text>Cyclic di-GMP: the first 25 years of a universal bacterial second messenger</text></passage><passage><infon key="fpage">279</infon><infon key="lpage">281</infon><infon key="name_0">surname:Ross;given-names:P</infon><infon key="name_1">surname:Weinhouse;given-names:H</infon><infon key="name_2">surname:Aloni;given-names:Y</infon><infon key="name_3">surname:Michaeli;given-names:D</infon><infon key="name_4">surname:Weinberger-Ohana;given-names:P</infon><infon key="name_5">surname:Mayer;given-names:R</infon><infon key="name_6">surname:Braun;given-names:S</infon><infon key="name_7">surname:de Vroom;given-names:E</infon><infon key="name_8">surname:van der Marel;given-names:GA</infon><infon key="name_9">surname:van Boom;given-names:JH</infon><infon key="pub-id_doi">10.1038/325279a0</infon><infon key="pub-id_pmid">18990795</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">325</infon><infon key="year">1987</infon><offset>46769</offset><text>Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid</text></passage><passage><infon key="fpage">35</infon><infon key="lpage">58</infon><infon key="name_0">surname:Ross;given-names:P</infon><infon key="name_1">surname:Mayer;given-names:R</infon><infon key="name_2">surname:Benziman;given-names:M</infon><infon key="pub-id_pmid">2030672</infon><infon key="section_type">REF</infon><infon key="source">Microbiol Rev</infon><infon key="type">ref</infon><infon key="volume">55</infon><infon key="year">1991</infon><offset>46852</offset><text>Cellulose biosynthesis and function in bacteria</text></passage><passage><infon key="fpage">651</infon><infon key="lpage">658</infon><infon key="name_0">surname:Sanchez-Torres;given-names:V</infon><infon key="name_1">surname:Hu;given-names:H</infon><infon key="name_2">surname:Wood;given-names:TK</infon><infon key="pub-id_doi">10.1007/s00253-010-3074-5</infon><infon key="pub-id_pmid">21181144</infon><infon key="section_type">REF</infon><infon key="source">Appl Microbiol Biotechnol</infon><infon key="type">ref</infon><infon key="volume">90</infon><infon key="year">2011</infon><offset>46900</offset><text>GGDEF proteins YeaI, YedQ, and YfiN reduce early biofilm formation and swimming motility in Escherichia coli</text></passage><passage><infon key="fpage">724</infon><infon key="lpage">735</infon><infon key="name_0">surname:Schirmer;given-names:T</infon><infon key="name_1">surname:Jenal;given-names:U</infon><infon key="pub-id_doi">10.1038/nrmicro2203</infon><infon key="pub-id_pmid">19756011</infon><infon key="section_type">REF</infon><infon key="source">Nat Rev Microbiol</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">2009</infon><offset>47009</offset><text>Structural and mechanistic determinants of c-di-GMP signalling</text></passage><passage><infon key="fpage">1606</infon><infon key="lpage">1619</infon><infon key="name_0">surname:Schuck;given-names:P</infon><infon key="pub-id_doi">10.1016/S0006-3495(00)76713-0</infon><infon key="pub-id_pmid">10692345</infon><infon key="section_type">REF</infon><infon key="source">Biophys J</infon><infon key="type">ref</infon><infon key="volume">78</infon><infon key="year">2000</infon><offset>47072</offset><text>Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling</text></passage><passage><infon key="fpage">715</infon><infon key="lpage">718</infon><infon key="name_0">surname:Shimazaki;given-names:J</infon><infon key="name_1">surname:Furukawa;given-names:S</infon><infon key="name_2">surname:Ogihara;given-names:H</infon><infon key="name_3">surname:Morinaga;given-names:Y</infon><infon key="pub-id_doi">10.1016/j.bbrc.2012.02.085</infon><infon key="pub-id_pmid">22386992</infon><infon key="section_type">REF</infon><infon key="source">Biochem Biophys Res Commun</infon><infon key="type">ref</infon><infon key="volume">419</infon><infon key="year">2012</infon><offset>47190</offset><text>L-Tryptophan prevents Escherichia coli biofilm formation and triggers biofilm degradation</text></passage><passage><infon key="fpage">8487</infon><infon key="lpage">8492</infon><infon key="name_0">surname:Smith;given-names:EE</infon><infon key="name_1">surname:Buckley;given-names:DG</infon><infon key="name_2">surname:Wu;given-names:Z</infon><infon key="name_3">surname:Saenphimmachak;given-names:C</infon><infon key="name_4">surname:Hoffman;given-names:LR</infon><infon key="name_5">surname:D’Argenio;given-names:DA</infon><infon key="name_6">surname:Miller;given-names:SI</infon><infon key="name_7">surname:Ramsey;given-names:BW</infon><infon key="name_8">surname:Speert;given-names:DP</infon><infon key="name_9">surname:Moskowitz;given-names:SM</infon><infon key="pub-id_doi">10.1073/pnas.0602138103</infon><infon key="pub-id_pmid">16687478</infon><infon key="section_type">REF</infon><infon key="source">Proc Natl Acad Sci USA</infon><infon key="type">ref</infon><infon key="volume">103</infon><infon key="year">2006</infon><offset>47280</offset><text>Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients</text></passage><passage><infon key="fpage">33324</infon><infon key="lpage">33330</infon><infon key="name_0">surname:Tamayo;given-names:R</infon><infon key="name_1">surname:Tischler;given-names:AD</infon><infon key="name_2">surname:Camilli;given-names:A</infon><infon key="pub-id_doi">10.1074/jbc.M506500200</infon><infon key="pub-id_pmid">16081414</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">280</infon><infon key="year">2005</infon><offset>47368</offset><text>The EAL domain protein VieA is a cyclic diguanylate phosphodiesterase</text></passage><passage><infon key="fpage">e1000483</infon><infon key="name_0">surname:Ueda;given-names:A</infon><infon key="name_1">surname:Wood;given-names:TK</infon><infon key="pub-id_doi">10.1371/journal.ppat.1000483</infon><infon key="pub-id_pmid">19543378</infon><infon key="section_type">REF</infon><infon key="source">PLoS Pathog</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2009</infon><offset>47438</offset><text>Connecting quorum sensing, c-di-GMP, pel polysaccharide, and biofilm formation in Pseudomonas aeruginosa through tyrosine phosphatase TpbA (PA3885)</text></passage><passage><infon key="fpage">e0124330</infon><infon key="name_0">surname:Xu;given-names:K</infon><infon key="name_1">surname:Li;given-names:S</infon><infon key="name_2">surname:Yang;given-names:W</infon><infon key="name_3">surname:Li;given-names:K</infon><infon key="name_4">surname:Bai;given-names:Y</infon><infon key="name_5">surname:Xu;given-names:Y</infon><infon key="name_6">surname:Jin;given-names:J</infon><infon key="name_7">surname:Wang;given-names:Y</infon><infon key="name_8">surname:Bartlam;given-names:M</infon><infon key="pub-id_doi">10.1371/journal.pone.0124330</infon><infon key="pub-id_pmid">25909591</infon><infon key="section_type">REF</infon><infon key="source">PLoS One</infon><infon key="type">ref</infon><infon key="volume">10</infon><infon key="year">2015</infon><offset>47586</offset><text>Structural and biochemical analysis of tyrosine phosphatase related to biofilm formation A (TpbA) from the opportunistic pathogen Pseudomonas aeruginosa PAO1</text></passage><passage><infon key="fpage">14</infon><infon key="lpage">20</infon><infon key="name_0">surname:Yang;given-names:X</infon><infon key="name_1">surname:Yang;given-names:XA</infon><infon key="name_2">surname:Xu;given-names:M</infon><infon key="name_3">surname:Zhou;given-names:L</infon><infon key="name_4">surname:Fan;given-names:Z</infon><infon key="name_5">surname:Jiang;given-names:T</infon><infon key="pub-id_doi">10.1016/j.bbrc.2015.03.160</infon><infon key="pub-id_pmid">25849887</infon><infon key="section_type">REF</infon><infon key="source">Biochem Biophys Res Commun</infon><infon key="type">ref</infon><infon key="volume">461</infon><infon key="year">2015</infon><offset>47744</offset><text>Crystal structures of YfiR from Pseudomonas aeruginosa in two redox states</text></passage></document></collection>
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+<collection><source>PMC</source><date>20201223</date><key>pmc.key</key><document><id>4919469</id><infon key="license">CC BY</infon><passage><infon key="alt-title">Investigation of the TOCA1-Cdc42 Interaction</infon><infon key="article-id_doi">10.1074/jbc.M116.724294</infon><infon key="article-id_pmc">4919469</infon><infon key="article-id_pmid">27129201</infon><infon key="article-id_publisher-id">M116.724294</infon><infon key="fpage">13875</infon><infon key="issue">26</infon><infon key="kwd">actin CDC42 endocytosis nuclear magnetic resonance (NMR) protein-protein interaction BAR domain CIP4 FBP17 TOCA1 WASP</infon><infon key="license">Author's Choice—Final version free via Creative Commons CC-BY license.</infon><infon key="lpage">13890</infon><infon key="name_0">surname:Watson;given-names:Joanna R.</infon><infon key="name_1">surname:Fox;given-names:Helen M.</infon><infon key="name_2">surname:Nietlispach;given-names:Daniel</infon><infon key="name_3">surname:Gallop;given-names:Jennifer L.</infon><infon key="name_4">surname:Owen;given-names:Darerca</infon><infon key="name_5">surname:Mott;given-names:Helen R.</infon><infon key="section_type">TITLE</infon><infon key="subtitle">HANDOVER OF Cdc42 TO THE ACTIN REGULATOR N-WASP IS FACILITATED BY DIFFERENTIAL BINDING AFFINITIES*</infon><infon key="type">front</infon><infon key="volume">291</infon><infon key="year">2016</infon><offset>0</offset><text>Investigation of the Interaction between Cdc42 and Its Effector TOCA1</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>70</offset><text>Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42. It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain. TOCA1 binding to Cdc42 leads to actin rearrangements, which are thought to be involved in processes such as endocytosis, filopodia formation, and cell migration. We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein. We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains. We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42. NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1594</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1607</offset><text>The Ras superfamily of small GTPases comprises over 150 members that regulate a multitude of cellular processes in eukaryotes. The superfamily can be divided into five families based on structural and functional similarities: Ras, Rho, Rab, Arf, and Ran. All members share a well defined core structure of ∼20 kDa known as the G domain, which is responsible for guanine nucleotide binding. It is this guanine nucleotide binding that underlies their function as molecular switches, controlling a vast array of signaling pathways. These molecular switches cycle between active, GTP-bound, and inactive, GDP-bound, states with the help of auxiliary proteins. The guanine nucleotide exchange factors mediate formation of the active state by promoting the dissociation of GDP, allowing GTP to bind. The GTPase-activating proteins stimulate the rate of intrinsic GTP hydrolysis, mediating the return to the inactive state (reviewed in Ref.).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2545</offset><text>The overall conformation of small G proteins in the active and inactive states is similar, but they differ significantly in two main regions known as switch I and switch II. These regions are responsible for “sensing” the nucleotide state, with the GTP-bound state showing greater rigidity and the GDP-bound state adopting a more relaxed conformation (reviewed in Ref.). In the active state, G proteins bind to an array of downstream effectors, through which they exert their extensive roles within the cell. The structures of more than 60 small G protein·effector complexes have been solved, and, not surprisingly, the switch regions have been implicated in a large proportion of the G protein-effector interactions (reviewed in Ref.). However, because each of the 150 members of the superfamily interacts with multiple effectors, there are still a huge number of known G protein-effector interactions that have not yet been studied structurally.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3498</offset><text>The Rho family comprises 20 members, of which three, RhoA, Rac1, and Cdc42, have been relatively well studied. The role of these three proteins in the coordination of the actin cytoskeleton has been examined extensively. RhoA acts to rearrange existing actin structures to form stress fibers, whereas Rac1 and Cdc42 promote de novo actin polymerization to form lamellipodia and filopodia, respectively. A number of RhoA and Rac1 effector proteins, including the formins and members of the protein kinase C-related kinase (PRK)6 family, along with Cdc42 effectors, including the Wiskott-Aldrich syndrome (WASP) family and the transducer of Cdc42-dependent actin assembly (TOCA) family, have also been linked to the pathways that govern cytoskeletal dynamics.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4256</offset><text>Cdc42 effectors, TOCA1 and the ubiquitously expressed member of the WASP family, N-WASP, have been implicated in the regulation of actin polymerization downstream of Cdc42 and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). N-WASP exists in an autoinhibited conformation, which is released upon PI(4,5)P2 and Cdc42 binding or by other factors, such as phosphorylation. Following their release, the C-terminal regions of N-WASP are free to interact with G-actin and a known nucleator of actin assembly, the Arp2/3 complex. The importance of TOCA1 in actin polymerization has been demonstrated in a range of in vitro and in vivo studies, but the exact role of TOCA1 in the many pathways involving actin assembly remains unclear. The most widely studied role of TOCA1 is in membrane invagination and endocytosis, although it has also been implicated in filopodia formation, neurite elongation, transcriptional reprogramming via nuclear actin, and interaction with ZO-1 at tight junctions. A role in cell motility and invasion has also been established.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5309</offset><text>TOCA1 comprises an N-terminal F-BAR domain, a central homology region 1 (HR1) domain, and a C-terminal SH3 domain. The F-BAR domain is a known dimerization, membrane-binding, and membrane-deforming module found in a number of cell signaling proteins. The TOCA1 SH3 domain has many known binding partners, including N-WASP and dynamin. The HR1 domain has been directly implicated in the interaction between TOCA1 and Cdc42, representing the first Cdc42-HR1 domain interaction to be identified.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5802</offset><text>Other HR1 domains studied so far, including those from the PRK family, have been found to bind their cognate Rho family G protein-binding partner with high specificity and affinities in the nanomolar range. The structures of the PRK1 HR1a domain in complex with RhoA and the HR1b domain in complex with Rac1 show that the HR1 domain comprises an anti-parallel coiled-coil that interacts with its G protein binding partner via both helices. Both of the G protein switch regions are involved in the interaction. The coiled-coil fold is shared by the HR1 domain of the TOCA family protein, CIP4, and, based on sequence homology, by TOCA1 itself. These HR1 domains, however, show specificity for Cdc42, rather than RhoA or Rac1. How different HR1 domain proteins distinguish their specific G protein partners remains only partially understood, and structural characterization of a novel G protein-HR1 domain interaction would add to the growing body of information pertaining to these protein complexes. Furthermore, the biological function of the interaction between TOCA1 and Cdc42 remains poorly understood, and so far there has been no biophysical or structural insight.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6973</offset><text>The interactions of TOCA1 and N-WASP with Cdc42 as well as with each other have raised questions as to whether the two Cdc42 effectors can interact with a single molecule of Cdc42 simultaneously. There is some evidence for a ternary complex between Cdc42, N-WASP, and TOCA1, but there was no direct demonstration of simultaneous contacts between the two effectors and a single molecule of Cdc42. Nonetheless, the substantial difference between the structures of the G protein-binding regions of the two effectors is intriguing and implies that they bind to Cdc42 quite differently, providing motivation for investigating the possibility that Cdc42 can bind both effectors concurrently. WASP interacts with Cdc42 via a conserved, unstructured binding motif known as the Cdc42- and Rac-interactive binding region (CRIB), which forms an intermolecular β-sheet, expanding the anti-parallel β2 and β3 strands of Cdc42. In contrast, the TOCA family proteins are thought to interact via the HR1 domain, which may form a triple coiled-coil with switch II of Rac1, like the HR1b domain of PRK1.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>8068</offset><text>Here, we present the solution NMR structure of the HR1 domain of TOCA1, providing the first structural data for this protein. We also present data pertaining to binding of the TOCA HR1 domain to Cdc42, which is the first biophysical description of an HR1 domain binding this particular Rho family small G protein. Finally, we investigate the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and N-WASP, contributing to our understanding of G protein-effector interactions as well as the roles of Cdc42, N-WASP, and TOCA1 in the pathways that govern actin dynamics.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>8667</offset><text>Experimental Procedures</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>8691</offset><text>Expression Constructs</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>8713</offset><text>The Xenopus tropicalis TOCA1 HR1 domain (residues 330–426 and N-terminally extended constructs as indicated) were amplified from cDNA (TOCA1 accession number NM_001005148) and cloned into pGEX-6P-1 (GE Healthcare) or pGEX-HisP. The HR1 domain of human CIP4 (residues 388–481) was amplified from IMAGE clone 3532036, the Xenopus laevis FBP17 HR1 domain (residues 385–486) from IMAGE clone 5514481, and the X. tropicalis N-WASP G protein-binding domain (GBD) (residues 197–255) from IMAGE clone 5379332, and all were cloned into pGEX-6P-1. The resulting constructs express the proteins as N-terminal GST fusions with a 3C protease-cleavable tag, with pGEX-HisP expressing an additional C-terminal His6 tag. Human Cdc42Δ7Q61L and full-length Cdc42 were cloned into pGEX-2T (GE Healthcare) and pGEX-6P-1, respectively. A C-terminally extended construct of TOCA1 comprising residues 330–545 was cloned into pMAT10-P.7 The resulting construct expresses TOCA1 330–545 as an N-terminal His-MBP fusion protein with a 3C protease-cleavable tag. Full-length X. tropicalis TOCA1, TOCA1 F-BAR (residues 1–287), and TOCA1 ΔSH3 (residues 1–480) were PCR-amplified from a cDNA clone (IMAGE 5157175) and cloned into pET-His6-SNAP using FseI and AscI sites that had been incorporated into the primers to create His-SNAP-TOCA1 proteins.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>10049</offset><text>Protein Expression</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>10068</offset><text>GST fusion proteins (HR1 domains and Cdc42) were expressed in E. coli BL21 cells (Invitrogen). Stationary cultures were diluted 1:10 and grown at 37 °C until an A600 of ∼0.8 was reached and then induced with 0.1 mm isopropyl-β-d-thiogalactopyranoside for 20 h at 20 °C. The GST-N-WASP GBD construct was expressed in E. coli BL21-CodonPlus®-RIL (Agilent Technologies). The proteins were purified using glutathione-agarose beads (Sigma) and eluted from the beads by cleavage of the GST tag with 3C protease (HR1 domains, N-WASP GBD, and full-length Cdc42Q61L) or thrombin (Novagen, Cdc42Δ7Q61L) prior to gel filtration on a 16/60 S75 column (GE Healthcare). His-MBP-HR1-SH3 was purified using nickel-nitrilotriacetic acid-agarose beads (Life Technologies) prior to cleavage with 3C protease and gel filtration. Full-length TOCA1, TOCA1 F-BAR, and TOCA1 ΔSH3 were expressed from pET-His6-SNAP in BL21 pLysS, grown at 37 °C until an A600 of ∼0.6 was reached, and induced with 0.3 mm isopropyl-β-d-thiogalactopyranoside overnight at 19 °C. Proteins were coupled to nickel-nitrilotriacetic acid-agarose (Qiagen), eluted using increasing concentrations of imidazole, and further purified by gel filtration using a 16/60 S200 column (GE Healthcare). All protein concentrations were determined by amino acid analysis (Protein and Nucleic Acid Chemistry Facility, Department of Biochemistry, University of Cambridge).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>11490</offset><text>Nucleotide Exchange</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>11510</offset><text>For NMR experiments, Cdc42 was nucleotide-exchanged for the non-hydrolyzable GTP analogue GMPPNP (Sigma) as described previously. For scintillation proximity assays (SPAs), Cdc42 was loaded with [3H]GTP using [8-3H]GTP (PerkinElmer Life Sciences), as described previously. The protein was confirmed as full-length using mass spectrometry (PNAC facility, Department of Biochemistry, University of Cambridge).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>11918</offset><text>SPAs</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>11923</offset><text>For direct assays, GST-PAK, GST-ACK, or His-tagged TOCA1 constructs were attached to a fluoromicrosphere via an anti-GST or anti-His antibody in the presence of Cdc42Δ7Q61L·[3H]GTP. Binding curves were fitted using a direct binding isotherm to obtain Kd values and their curve-fitting errors for the G protein-effector interactions. For competition assays, free ACK GBD, TOCA1 HR1, TOCA1 HR1SH3, or N-WASP GBD was titrated into a mixture of 30 nm Cdc42Δ7Q61L·[3H]GTP and 30 nm GST-ACK immobilized on a fluoromicrosphere as above. Data were fitted to competition binding isotherms to obtain Kd values and curve-fitting errors, as described previously.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>12582</offset><text>NMR Spectroscopy</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>12599</offset><text>The NMR experiments and resonance assignments of the HR1 domain are described. The NMR experiments were carried out with 0.9 mm 13C/15N-labeled HR1 domain in 20 mm sodium phosphate, pH 7.5, 150 mm NaCl, 5 mm MgCl2, 5 mm DTT, 10% D2O. Distance restraints were derived from a 15N-separated NOESY (100-ms mixing time) recorded on a Bruker DRX500 and a 13C-separated NOESY (100-ms mixing time) recorded on an Avance AV600. NMR data were processed using AZARA (W. Boucher, University of Cambridge) and analyzed using ANALYSIS.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>13121</offset><text>Structure Calculation</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>13143</offset><text>Structures were calculated iteratively using CNS version 1.0 interfaced to Aria version 2.3.1. The PROSLQ force field was used for non-bonded parameters. Backbone torsion angles were estimated from CA, CO, CB, N, and HA chemical shifts using TALOS-N. The “strong” φ and ψ restraints were included with an error of ±2 S.D. values of the averaged TALOS-N predictions. Dihedral angle predictions for residues 323–340 were weak, so no restraints were included for this region.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>13625</offset><text>NMR Titrations</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>13640</offset><text>All of the 15N and 13C HSQCs were recorded at 25 °C in 50 mm sodium phosphate, pH 5.5, 25 mm NaCl, 5 mm MgCl2, 5 mm DTT, 10% D2O on a Bruker DRX500. 15N-HR1 HSQC experiments were recorded on 0.2 mm 15N-HR1 domain with HR1/Cdc42·GMPPNP ratios of 1:0, 1:0.25, 1:0.5, 1:1, and 1:4. Experiments were recorded on 0.27 mm 15N-Cdc42·GMPPNP at Cdc42/HR1 ratios of 1:0, 1:0.25, 1:0.5, and 1:2.2. The 15N HSQC titrations with N-WASP were recorded on 0.6 mm 15N-HR1 domain or 0.15 mm 15N-Cdc42 at the ratios indicated in the figures.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>14166</offset><text>Chemical Shift Mapping</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>14189</offset><text>The chemical shift changes, δ, were calculated using the equation,  where δ(1H) and δ(15N) are the chemical shift changes for the 1H and 15N dimensions, respectively. Residues that had disappeared were assigned a δ value larger than the maximum calculated δ for the data set, and residues that were too overlapped to be reliably assigned in the complex spectra were assigned δ = 0. The residues that had shifted more than the mean chemical shift change across the spectrum were classed as significant and were filtered for solvent accessibility using NACCESS. Residues with &lt;50% solvent accessibility were considered to be buried and unavailable for binding.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>14872</offset><text>Pyrene Actin Assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>14892</offset><text>Pyrene actin assays were carried out as described previously. Xenopus high speed supernatant was used at 5 mg/ml and supplemented with 0.12 mg/ml pyrene actin as described previously. TOCA1 HR1 domain or N-WASP CRIB domain was added at the concentrations indicated. Liposomes were made, using methods described previously, from 60% phosphatidylcholine, 30% phosphatidylserine, and 10% PI(4,5)P2 to 2 mm final lipid concentration. All of the lipids used were natural brain or liver lipids from Avanti Polar Lipids. The assays were initiated by the addition of 5 μl of liposomes per 200 μl of reaction mix.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>15499</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>15507</offset><text>Cdc42-TOCA1 Binding</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15527</offset><text>TOCA1 was identified in Xenopus extracts as a protein necessary for Cdc42-dependent actin assembly and was shown to bind to Cdc42·GTPγS but not to Cdc42·GDP or to Rac1 and RhoA. Given its homology to other Rho family binding modules, it is likely that the HR1 domain of TOCA1 is sufficient to bind Cdc42. The C. elegans TOCA1 orthologues also bind to Cdc42 via their consensus HR1 domain. The HR1 domains from the PRK family bind their G protein partners with a high affinity, exhibiting a range of submicromolar dissociation constants (Kd) as low as 26 nm. A Kd in the nanomolar range was therefore expected for the interaction of the TOCA1 HR1 domain with Cdc42.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16196</offset><text>We generated an X. tropicalis TOCA1 HR1 domain construct encompassing residues 330–426. This region comprises the complete HR1 domain based on secondary structure predictions and sequence alignments with another TOCA family member, CIP4, whose structure has been determined. The interaction between [3H]GTP·Cdc42 and a C-terminally His-tagged TOCA1 HR1 domain construct was investigated using SPA. The binding isotherm for the interaction is shown in Fig. 1A, together with the Cdc42-PAK interaction as a positive control. The binding of TOCA1 HR1 to Cdc42 was unexpectedly weak, with a Kd of &gt;1 μm. It was not possible to estimate the Kd more accurately using direct SPA experiments, because saturation could not be reached due to nonspecific signal at higher protein concentrations.</text></passage><passage><infon key="file">zbc0281646060001.jpg</infon><infon key="id">F1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>16985</offset><text>The TOCA1 HR1-Cdc42 interaction is low affinity.
+A, curves derived from direct binding assays in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-PAK or HR1-His6 in SPAs. The SPA signal was corrected by subtraction of control data with no GST-PAK or HR1-His6. The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal; B and C, competition SPA experiments were carried out with the indicated concentrations of ACK GBD (B) or HR1 domain (C) titrated into 30 nm GST-ACK and either 30 nm Cdc42Δ7Q61L·[3H]GTP or full-length Cdc42Q61L·[3H]GTP. The Kd values derived for the ACK GBD with Cdc42Δ7 and full-length Cdc42 were 0.032 ± 0.01 and 0.011 ± 0.01 μm, respectively. The Kd values derived for the TOCA1 HR1 with Cdc42Δ7 and full-length Cdc42 were 6.05 ± 1.96 and 5.39 ± 1.69 μm, respectively.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17901</offset><text>It was possible that the low affinity observed was due to negative effects of immobilization of the HR1 domain, so other methods were employed, which utilized untagged proteins. Isothermal titration calorimetry was carried out, but no heat changes were observed at a range of concentrations and temperatures (data not shown), suggesting that the interaction is predominantly entropically driven. Other G protein-HR1 domain interactions have also failed to show heat changes in our hands.7 Infrared interferometry with immobilized Cdc42 was also attempted but was unsuccessful for both TOCA1 HR1 and for the positive control, ACK.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18531</offset><text>The affinity was therefore determined using competition SPAs. A complex of a GST fusion of the GBD of ACK, which binds with a high affinity to Cdc42, with radiolabeled [3H]GTP·Cdc42 was preformed, and the effect of increasing concentrations of untagged TOCA1 HR1 domain was examined. Competition of GST-ACK GBD bound to [3H]GTP·Cdc42 by free ACK GBD was used as a control and to establish the value of background counts when Cdc42 is fully displaced. The data were fitted to a binding isotherm describing competition. Free ACK competed with itself with an affinity of 32 nm, similar to the value obtained by direct binding of 23 nm. The TOCA1 HR1 domain also fully competed with the GST-ACK but bound with an affinity of 6 μm (Fig. 1, B and C), in agreement with the low affinity observed in the direct binding experiments.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19358</offset><text>The Cdc42 construct used in the binding assays has seven residues deleted from the C terminus to facilitate purification. These residues are not generally required for G protein-effector interactions, including the interaction between RhoA and the PRK1 HR1a domain. In contrast, the C terminus of Rac1 contains a polybasic sequence, which is crucial for Rac1 binding to the HR1b domain from PRK1. As the observed affinity between TOCA1 HR1 and Cdc42 was much lower than expected, we reasoned that the C terminus of Cdc42 might be necessary for a high affinity interaction. The binding experiments were repeated with full-length [3H]GTP·Cdc42, but the affinity of the HR1 domain for full-length Cdc42 was similar to its affinity for truncated Cdc42 (Kd ≈ 5 μm; Fig. 1C). Thus, the C-terminal region of Cdc42 is not required for maximal binding of TOCA1 HR1.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20219</offset><text>Another possible explanation for the low affinities observed was that the HR1 domain alone is not sufficient for maximal binding of the TOCA proteins to Cdc42 and that the other domains are required. Indeed, GST pull-downs performed with in vitro translated human TOCA1 fragments had suggested that residues N-terminal to the HR1 domain may be required to stabilize the HR1 domain structure. Furthermore, both BAR and SH3 domains have been implicated in interactions with small G proteins (e.g. the BAR domain of Arfaptin2 binds to Rac1 and Arl1), while an SH3 domain mediates the interaction between Rac1 and the guanine nucleotide exchange factor, β-PIX. TOCA1 dimerizes via its F-BAR domain, which could also affect Cdc42 binding, for example by presenting two HR1 domains for Cdc42 interactions. Various TOCA1 fragments (Fig. 2A) were therefore assessed for binding to full-length Cdc42 by direct SPA. The isolated F-BAR domain showed no binding to full-length Cdc42 (Fig. 2B). Full-length TOCA1 and ΔSH3 TOCA1 bound with micromolar affinity (Fig. 2B), in a similar manner to the isolated HR1 domain (Fig. 1A). The HR1-SH3 protein could not be purified to homogeneity as a fusion protein, so it was assayed in competition assays after cleavage of the His tag. This construct competed with GST-ACK GBD to give a similar affinity to the HR1 domain alone (Kd = 4.6 ± 4 μm; Fig. 2C). Taken together, these data suggest that the TOCA1 HR1 domain is sufficient for maximal binding and that this binding is of a relatively low affinity compared with many other Cdc42·effector complexes.</text></passage><passage><infon key="file">zbc0281646060002.jpg</infon><infon key="id">F2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>21812</offset><text>The Cdc42-HR1 interaction is of low affinity in the context of full-length protein and in TOCA1 paralogues.
+A, diagram illustrating the TOCA1 constructs assayed for Cdc42 binding. Domain boundaries are derived from secondary structure predictions; B, binding curves derived from direct binding assays, in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-ACK or His-tagged TOCA1 constructs, as indicated, in SPAs. The SPA signal was corrected by subtraction of control data with no fusion protein. The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal. C–E, representative examples of competition SPA experiments carried out with the indicated concentrations of the TOCA1 HR1-SH3 construct titrated into 30 nm GST-ACK and 30 nm Cdc42Δ7Q61L·[3H]GTP (C) or HR1CIP4 (D) or HR1FBP17 (E) titrated into 30 nm GST-ACK and 30 nm Cdc42FLQ61L·[3H]GTP.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22768</offset><text>The low affinity of the TOCA1 HR1-Cdc42 interaction raised the question of whether the other known Cdc42-binding TOCA family proteins, FBP17 and CIP4, also bind weakly. The HR1 domains from FBP17 and CIP4 were purified and assayed for Cdc42 binding in competition SPAs, analogous to those carried out with the TOCA1 HR1 domain. The affinities of both the FBP17 and CIP4 HR1 domains were also in the low micromolar range (10 and 5 μm, respectively) (Fig. 2, D and E), suggesting that low affinity interactions with Cdc42 are a common feature within the TOCA family.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>23334</offset><text>Structure of the TOCA1 HR1 Domain</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23368</offset><text>Because the TOCA1 HR1 domain was sufficient for maximal Cdc42-binding, we used this construct for structural studies. Initial experiments were performed with TOCA1 residues 324–426, but we observed that the N terminus was cleaved during purification to yield a new N terminus at residue 330 (data not shown). We therefore engineered a construct comprising residues 330–426 to produce the minimal, stable HR1 domain. Backbone and side chain resonances were assigned as described. 2,778 non-degenerate NOE restraints were used in initial structure calculations (1,791 unambiguous and 987 ambiguous), derived from three-dimensional 15N-separated NOESY and 13C-separated NOESY experiments. There were 1,845 unambiguous NOEs and 757 ambiguous NOEs after eight iterations. 100 structures were calculated in the final iteration; the 50 lowest energy structures were water-refined; and of these, the 35 lowest energy structures were analyzed. Table 1 indicates that the HR1 domain structure is well defined by the NMR data.</text></passage><passage><infon key="file"></infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>24388</offset><text>Experimental restraints and structural statistics</text></passage><passage><infon key="file"></infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>24438</offset><text>a &lt;SA&gt;, the average root mean square deviations for the ensemble ± S.D.</text></passage><passage><infon key="file"></infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>24511</offset><text>b &lt;SA&gt;c, values for the structure that is closest to the mean.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24574</offset><text>The structure closest to the mean is shown in Fig. 3A. The two α-helices of the HR1 domain interact to form an anti-parallel coiled-coil with a slight left-handed twist, reminiscent of the HR1 domains of CIP4 (PDB code 2KE4) and PRK1 (PDB codes 1CXZ and 1URF). A sequence alignment illustrating the secondary structure elements of the TOCA1 and CIP4 HR1 domains and the HR1a and HR1b domains from PRK1 is shown in Fig. 3B.</text></passage><passage><infon key="file">zbc0281646060003.jpg</infon><infon key="id">F3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>25001</offset><text>The structure of the TOCA1 HR1 domain.
+A, the backbone trace of the 35 lowest energy structures of the HR1 domain overlaid with the structure closest to the mean is shown alongside a schematic representation of the structure closest to the mean. Flexible regions at the N and C termini (residues 330–333 and 421–426) are omitted for clarity. B, a sequence alignment of the HR1 domains from TOCA1, CIP4, and PRK1. The secondary structure was deduced using Stride, based on the Ramachandran angles, and is indicated as follows: gray, turn; yellow, α-helix; blue, 310 helix; white, coil. C, a close-up of the N-terminal region of TOCA1 HR1, indicating some of the NOEs defining its position with respect to the two α-helices. Dotted lines, NOE restraints. D, a close-up of the interhelix loop region showing some of the contacts between the loop and helix 1. NOEs are indicated with dotted lines. All structural figures were generated using PyMOL.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>25954</offset><text>In the HR1a domain of PRK1, a region N-terminal to helix 1 forms a short α-helix, which packs against both helices of the HR1 domain. This region of TOCA1 HR1 (residues 334–340) is well defined in the family of structures (Fig. 3A) but does not form an α-helix. It instead forms a series of turns, defined by NOE restraints observed between residues separated by one (residues 332–334, 333–335, etc.) or two (residues 337–340) residues in the sequence and the φ and ψ angles, assessed using Stride. These turns cause the chain to reverse direction, allowing the N-terminal segment (residues 334–340) to contact both helices of the HR1 domain. Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1. These contacts are summarized in Fig. 3C.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>26850</offset><text>The two α-helices of TOCA1 HR1 are separated by a long loop of 10 residues (residues 380–389) that contains two short 310 helices (residues 381–383 and 386–389). Interestingly, side chains of residues within the loop region point back toward helix 1; for example, there are numerous distinct NOEs between the side chains of Asn-380 and Met-383 of the loop region and Tyr-377 and Val-376 of helix 1 (Fig. 3D). The backbone NH and CHα groups of Gly-384 and Asp-385 also show NOEs with the side chain of Tyr-377.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>27368</offset><text>Mapping the TOCA1 and Cdc42 Binding Interfaces</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>27415</offset><text>The HR1TOCA1-Cdc42 interface was investigated using NMR spectroscopy. A series of 15N HSQC experiments was recorded on 15N-labeled TOCA1 HR1 domain in the presence of increasing concentrations of unlabeled Cdc42Δ7Q61L·GMPPNP to map the Cdc42-binding surface. A comparison of the 15N HSQC spectra of free HR1 and HR1 in the presence of excess Cdc42 shows that although some peaks were shifted, several were much broader in the complex, and a considerable subset had disappeared (Fig. 4A). This behavior cannot be explained by the increase in molecular mass (from 12 to 33 kDa) when Cdc42 binds and is more likely to be due to conformational exchange. This leads to broadening of the peaks so that they are not detectable. Overall chemical shift perturbations (CSPs) were calculated for each residue, whereas those that had disappeared were assigned a shift change of 0.2 (Fig. 4B). A peak that disappeared or had a CSP above the mean CSP for the spectrum was considered to be significantly affected.</text></passage><passage><infon key="file">zbc0281646060004.jpg</infon><infon key="id">F4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28418</offset><text>Mapping the binding surface of Cdc42 onto the TOCA1 HR1 domain.
+A, the 15N HSQC of 200 μm TOCA1 HR1 domain is shown in the free form (black) and in the presence of a 4-fold molar excess of Cdc42Δ7Q61L·GMPPNP (red). Expansions of two regions are shown with peak assignments, showing backbone amides in fast or intermediate exchange. B, CSPs were calculated as described under “Experimental Procedures” and are shown for backbone and side chain NH groups. The mean CSP is marked with a red line. Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.2 but were excluded when calculating the mean CSP and are indicated with open bars. Those that were not traceable due to spectral overlap were assigned a CSP of zero and are marked with an asterisk below the bar. Residues with affected side chain CSPs derived from 13C HSQCs are marked with green asterisks above the bars. Secondary structure elements are shown below the graph. C, a schematic representation of the HR1 domain. Residues with significantly affected backbone or side chain chemical shifts when Cdc42 bound and that are buried are colored dark blue, whereas those that are solvent-accessible are colored yellow. Residues with significantly affected backbone and side chain groups that are solvent-accessible are colored red. A close-up of the binding region is shown, with affected side chain heavy atoms shown as sticks. D, the G protein-binding region is marked in red onto structures of the HR1 domains as indicated.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>29929</offset><text>15N HSQC shift mapping experiments report on changes to amide groups, which are mainly inaccessible because they are buried inside the helices and are involved in hydrogen bonds. Therefore, 13C HSQC and methyl-selective SOFAST-HMQC experiments were also recorded on 15N,13C-labeled TOCA1 HR1 to yield more information on side chain involvement. The affected CH groups underwent significant line broadening, similarly to the NH peaks. Side chains whose CH groups disappeared in the presence of Cdc42 are marked on the graph in Fig. 4B with green asterisks.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>30485</offset><text>TOCA1 residues whose signals were affected by Cdc42 binding were mapped onto the structure of TOCA1 HR1 (Fig. 4C). The changes were localized to one end of the coiled-coil, and the binding site appeared to include residues from both α-helices and the loop region that joins them. Residues outside of this region were not significantly affected, indicating that there was no widespread conformational change. The residues in the interhelical loop and helix 1 that contact each other (Fig. 3D) show shift changes in their backbone NH and side chains in the presence of Cdc42. For example, the side chain of Asn-380 and the backbones of Val-376 and Tyr-377 were significantly affected but are all buried in the free TOCA1 HR1 structure, indicating that local conformational changes in the loop may facilitate complex formation. The chemical shift mapping data indicate that the G protein-binding region of the TOCA1 HR1 domain is broadly similar to that of the CIP4 and PRK1 HR1 domains (Figs. 3B and 4D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>31492</offset><text>The corresponding 15N and 13C NMR experiments were also recorded on 15N-Cdc42Δ7Q61L·GMPPNP or 15N/13C -Cdc42Δ7Q61L·GMPPNP in the presence of unlabeled HR1 domain. The overall CSP was calculated for each residue. As was the case when labeled HR1 was observed, several peaks were shifted in the complex, but many disappeared, indicating exchange on an unfavorable, millisecond time scale (Fig. 5A). Detailed side chain data could not be obtained for all residues due to spectral overlap, but constant time 13C HSQC and methyl-selective SOFAST-HMQC experiments provided further information on certain well resolved side chains (marked with green asterisks in Fig. 5B).</text></passage><passage><infon key="file">zbc0281646060005.jpg</infon><infon key="id">F5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>32166</offset><text>Mapping the binding surface of the HR1 domain onto Cdc42.
+A, the 15N HSQC of Cdc42Δ7Q61L·GMPPNP is shown in its free form (black) and in the presence of excess TOCA1 HR1 domain (1:2.2, red). Expansions of two regions are shown, with most peaks in fast or intermediate exchange. B, CSPs are shown for backbone NH groups. The red line indicates the mean CSP, plus one S.D. Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.1 and are indicated with open bars. Those that were not traceable due to overlap are marked with an asterisk. Residues with disappeared peaks in 13C HSQC experiments are marked on the chart with green asterisks. Secondary structure elements are indicated below the graph. C, the residues with significantly affected backbone and side chain groups are highlighted on an NMR structure of free Cdc42Δ7Q61L·GMPPNP; those that are buried are colored dark blue, whereas those that are solvent-accessible are colored red. Residues with either side chain or backbone groups affected are colored blue if buried and yellow if solvent-accessible. Residues without information from shift mapping are colored gray. The flexible switch regions are circled.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>33364</offset><text>As many of the peaks disappeared, the mean chemical shift change was relatively low, so a threshold of the mean plus one S.D. value was used to define a significant CSP. Residues that disappeared were also classed as significantly affected. Parts of the switch regions (Fig. 5, B and C) are invisible in NMR spectra recorded on free Cdc42 due to conformational exchange. These switch regions become visible in Cdc42 and other small G protein·effector complexes due to a decrease in conformational freedom upon complex formation. The switch regions of Cdc42 did not, however, become visible in the presence of the TOCA1 HR1 domain. Indeed, Ser-30 of switch I and Arg-66, Arg-68, Leu-70, and Ser-71 of switch II are visible in free Cdc42 but disappear in the presence of the HR1 domain. This suggests that the switch regions are not rigidified in the HR1 complex and are still in conformational exchange. Nevertheless, mapping of the affected residues onto the NMR structure of free Cdc42Δ7Q61L·GMPPNP (Fig. 5C)8 shows that, although they are relatively widespread compared with changes in the HR1 domain, in general, they are on the face of the protein that includes the switches. Although the binding interface may be overestimated, this suggests that the switch regions are involved in binding to TOCA1.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>34673</offset><text>Modeling the Cdc42·TOCA1 HR1 Complex</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>34711</offset><text>The Cdc42·HR1TOCA1 complex was not amenable to full structural analysis due to the weak interaction and the extensive exchange broadening seen in the NMR experiments. HADDOCK was therefore used to perform rigid body docking based on the structures of free HR1 domain and Cdc42 and ambiguous interaction restraints derived from the titration experiments described above. Residues with significantly affected resonances and more than 50% solvent accessibility were defined as active. Passive residues were defined automatically as those neighboring active residues.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>35276</offset><text>The orientation of the HR1 domain with respect to Cdc42 cannot be definitively concluded in the absence of unambiguous distance restraints; hence, HADDOCK produced a set of models in which the HR1 domain contacts the same surface on Cdc42 but is in various orientations with respect to Cdc42. The cluster with the lowest root mean square deviation from the lowest energy structure is assumed to be the best model. By these criteria, in the best model, the HR1 domain is in a similar orientation to the HR1a domain of PRK1 bound to RhoA and the HR1b domain bound to Rac1. A representative model from this cluster is shown in Fig. 6A alongside the Rac1-HR1b structure (PDB code 2RMK) in Fig. 6B.</text></passage><passage><infon key="file">zbc0281646060006.jpg</infon><infon key="id">F6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>35970</offset><text>Model of Cdc42·HR1 complex.
+A, a representative model of the Cdc42·HR1 complex from the cluster closest to the lowest energy model produced using HADDOCK. Residues of Cdc42 that are affected in the presence of the HR1 domain but are not in close proximity to it are colored in red and labeled. B, structure of Rac1 in complex with the HR1b domain of PRK1 (PDB code 2RMK). C, sequence alignment of RhoA, Cdc42 and Rac1. Contact residues of RhoA and Rac1 to PRK1 HR1a and HR1b, respectively, are colored cyan. Residues of Cdc42 that disappear or show chemical shift changes in the presence of TOCA1 are colored cyan if also identified as contacts in RhoA and Rac1 and yellow if they are not. Residues equivalent to Rac1 and RhoA contact sites but that are invisible in free Cdc42 are gray. D, regions of interest of the Cdc42·HR1 domain model. The four lowest energy structures in the chosen HADDOCK cluster are shown overlaid, with the residues of interest shown as sticks and labeled. Cdc42 is shown in cyan, and TOCA1 is shown in purple.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>37012</offset><text>A sequence alignment of RhoA, Cdc42, and Rac1 is shown in Fig. 6C. The RhoA and Rac1 contact residues in the switch regions are invisible in the spectra of Cdc42, but they are generally conserved between all three G proteins. Several Cdc42 residues identified by chemical shift mapping are not in close contact in the Cdc42·TOCA1 model (Fig. 6A). Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II. Other residues that are affected in the Cdc42·TOCA1 complex but that do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) include Gln-2Cdc42, Lys-16Cdc42, Thr-52Cdc42, and Arg-68Cdc42. Lys-16Cdc42 is unlikely to be a contact residue because it is involved in nucleotide binding, but the others may represent specific Cdc42-TOCA1 contacts. In the model, these side chains are involved in direct contacts (Fig. 6D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>38047</offset><text>Competition between N-WASP and TOCA1</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>38084</offset><text>From the known interactions and effects of the proteins in biological systems, it has been suggested that TOCA1 and N-WASP could bind Cdc42 simultaneously. Studies in CHO cells indicated that a Cdc42·N-WASP·TOCA1 complex existed because FRET was observed between RFP-TOCA1 and GFP-N-WASP, and the efficiency was decreased when an N-WASP mutant was used that no longer binds Cdc42. An overlay of the HADDOCK model of the Cdc42·HR1TOCA1 complex and the structure of Cdc42 in complex with the GBD of the N-WASP homologue, WASP (PDB code 1CEE), shows that the HR1 and GBD binding sites only partly overlap, and, therefore, a ternary complex remained possible (Fig. 7A). Interestingly, the presence of the TOCA1 HR1 would not prevent the core CRIB of WASP from binding to Cdc42, although the regions C-terminal to the CRIB that are required for high affinity binding of WASP would interfere sterically with the TOCA1 HR1. A basic region in WASP including three lysines (residues 230–232), N-terminal to the core CRIB, has been implicated in an electrostatic steering mechanism, and these residues would be free to bind in the presence of TOCA1 HR1 (Fig. 7A).</text></passage><passage><infon key="file">zbc0281646060007.jpg</infon><infon key="id">F7</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>39243</offset><text>The N-WASP GBD displaces the TOCA1 HR1 domain.
+A, the model of the Cdc42·TOCA1 HR1 domain complex overlaid with the Cdc42-WASP structure. Cdc42 is shown in green, and TOCA1 is shown in purple. The core CRIB region of WASP is shown in red, whereas its basic region is shown in orange and the C-terminal region required for maximal affinity is shown in cyan. A semitransparent surface representation of Cdc42 and WASP is shown overlaid with the schematic. B, competition SPA experiments carried out with indicated concentrations of the N-WASP GBD construct titrated into 30 nm GST-ACK or GST-WASP GBD and 30 nm Cdc42Δ7Q61L·[3H]GTP. C, Selected regions of the 15N HSQC of 145 μm Cdc42Δ7Q61L·GMPPNP with the indicated ratios of the TOCA1 HR1 domain, the N-WASP GBD, or both, showing that the TOCA HR1 domain does not displace the N-WASP GBD. D, selected regions of the 15N HSQC of 600 μm TOCA1 HR1 domain in complex with Cdc42 in the absence and presence of the N-WASP GBD, showing displacement of Cdc42 from the HR1 domain by N-WASP.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>40284</offset><text>An N-WASP GBD construct was produced, and its affinity for Cdc42 was measured by competition SPA (Fig. 7B). The Kd that was determined (37 nm) is consistent with the previously reported affinity. Unlabeled N-WASP GBD was titrated into 15N-Cdc42Δ7Q61L·GMPPNP, and the backbone NH groups were monitored using HSQCs (Fig. 7C). Unlabeled HR1TOCA1 was then added to the Cdc42·N-WASP complex, and no changes were seen, suggesting that the N-WASP GBD was not displaced even in the presence of a 5-fold excess of HR1TOCA1. These experiments were recorded at sufficiently high protein concentrations (145 μm Cdc42, 145 μm N-WASP GBD, 725 μm TOCA1 HR1 domain) to be far in excess of the Kd values of the individual interactions (TOCA1 Kd ≈ 5 μm, N-WASP Kd = 37 nm). A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>41289</offset><text>Furthermore, 15N-TOCA1 HR1 was monitored in the presence of unlabeled Cdc42Δ7Q61L·GMPPNP (1:1) before and after the addition of 0.25 and 1.0 eq of unlabeled N-WASP GBD. The spectrum when N-WASP and TOCA1 were equimolar was identical to that of the free HR1 domain, whereas the spectrum in the presence of 0.25 eq of N-WASP was intermediate between the TOCA1 HR1 free and complex spectra (Fig. 7D). When in fast exchange, the NMR signal represents a population-weighted average between free and bound states, so the intermediate spectrum indicates that the population comprises a mixture of free and bound HR1 domain. Hence, a third, intermediate state that includes all three proteins is unlikely. Again, the experiments were recorded on protein samples far in excess of the individual Kd values (600 μm each protein). These data indicate that the HR1 domain is displaced from Cdc42 by N-WASP and that a ternary complex comprising TOCA1 HR1, N-WASP GBD, and Cdc42 is not formed. Taken together, the data in Fig. 7, C and D, indicate unidirectional competition for Cdc42 binding in which the N-WASP GBD displaces TOCA1 HR1 but not vice versa.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>42436</offset><text>To extend these studies to a more complex system and to assess the ability of TOCA1 HR1 to compete with full-length N-WASP, pyrene actin assays were employed. These assays, described in detail elsewhere, were carried out using pyrene actin-supplemented Xenopus extracts into which exogenous TOCA1 HR1 domain or N-WASP GBD was added, to assess their effects on actin polymerization. Actin polymerization in all cases was initiated by the addition of PI(4,5)P2-containing liposomes. Actin polymerization triggered by the addition of PI(4,5)P2-containing liposomes has previously been shown to depend on TOCA1 and N-WASP. Endogenous N-WASP is present at ∼100 nm in Xenopus extracts, whereas TOCA1 is present at a 10-fold lower concentration than N-WASP.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>43189</offset><text>The addition of the isolated N-WASP GBD significantly inhibited the polymerization of actin at concentrations as low as 100 nm and completely abolished polymerization at higher concentrations (Fig. 8). The GBD presumably acts as a dominant negative, sequestering endogenous Cdc42 and preventing endogenous full-length N-WASP from binding and becoming activated. The addition of the TOCA1 HR1 domain to 100 μm had no significant effect on the rate of actin polymerization or maximum fluorescence. This is consistent with endogenous N-WASP, activated by other components of the assay, outcompeting the TOCA1 HR1 domain for Cdc42 binding.</text></passage><passage><infon key="file">zbc0281646060008.jpg</infon><infon key="id">F8</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>43826</offset><text>Actin polymerization downstream of Cdc42·N-WASP·TOCA1 is inhibited by excess N-WASP GBD but not by the TOCA1 HR1 domain. Fluorescence curves show actin polymerization in the presence of increasing concentrations of N-WASP GBD or TOCA1 HR1 domain as indicated. Maximal rates of actin polymerization derived from the linear region of the curves are represented in bar charts below. Error bars, S.E.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>44225</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_4</infon><offset>44236</offset><text>The Cdc42-TOCA1 Interaction</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>44264</offset><text>The TOCA1 HR1 domain alone is sufficient for Cdc42 binding in vitro, yet the affinity of the TOCA1 HR1 domain for Cdc42 is remarkably low (Kd ≈ 5 μm). This is over 100 times lower than that of the N-WASP GBD (Kd = 37 nm) and considerably lower than other known G protein-HR1 domain interactions. The polybasic tract within the C-terminal region of Cdc42 does not appear to be required for binding to TOCA1, which is in contrast to the interaction between Rac1 and the HR1b domain of PRK1 but more similar to the PRK1 HR1a-RhoA interaction. A single binding interface on both the HR1 domain and Cdc42 can be concluded from the data presented here. Furthermore, the interfaces are comparable with those of other G protein-HR1 interactions (Fig. 4), and the lowest energy model produced in rigid body docking resembles previously studied G protein·HR1 complexes (Fig. 6). It seems, therefore, that the interaction, despite its relatively low affinity, is specific and sterically similar to other HR1 domain-G protein interactions.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>45296</offset><text>The TOCA1 HR1 domain is a left-handed coiled-coil comparable with other known HR1 domains. A short region N-terminal to the coiled-coil exhibits a series of turns and contacts residues of both helices of the coiled-coil (Fig. 3). The corresponding sequence in CIP4 also includes a series of turns but is flexible, whereas in the HR1a domain of PRK1, the equivalent region adopts an α-helical structure that packs against the coiled-coil. The contacts between the N-terminal region and the coiled-coil are predominantly hydrophobic in both cases, but sequence-specific contacts do not appear to be conserved. This region is distant from the G protein-binding interface of the HR1 domains, so the structural differences may relate to the structure and regulation of these domains rather than their G protein interactions.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>46120</offset><text>The interhelical loops of TOCA1 and CIP4 differ from the same region in the HR1 domains of PRK1 in that they are longer and contain two short stretches of 310-helix. This region lies within the G protein-binding surface of all of the HR1 domains (Fig. 4D). TOCA1 and CIP4 both bind weakly to Cdc42, whereas the HR1a domain of PRK1 binds tightly to RhoA and Rac1, and the HR1b domain binds to Rac1. The structural features shared by TOCA1 and CIP4 may therefore be related to Cdc42 binding specificity and the low affinities. In free TOCA1, the side chains of the interhelical region make extensive contacts with residues in helix 1. Many of these residues are significantly affected in the presence of Cdc42, so it is likely that the conformation of this loop is altered in the Cdc42 complex. These observations therefore provide a molecular mechanism whereby mutation of Met383-Gly384-Asp385 to Ile383-Ser384-Thr385 abolishes TOCA1 binding to Cdc42.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>47071</offset><text>The lowest energy model produced by HADDOCK using ambiguous interaction restraints from the titration data resembled the NMR structures of RhoA and Rac1 in complex with their HR1 domain partners. Some speculative conclusions can be made based on this model. For example, Phe-56Cdc42, which is not visible in free Cdc42 or Cdc42·HR1TOCA1, is close to the TOCA1 HR1 (Fig. 6A). Phe-56Cdc42, which is a Trp in both Rac1 and RhoA (Fig. 6C), is thought to pack behind switch I when Cdc42 interacts with ACK, maintaining the switch in a binding-competent orientation. This residue has also been identified as important for Cdc42-WASP binding. Phe-56Cdc42 is therefore likely to be involved in the Cdc42-TOCA1 interaction, probably by stabilizing the position of switch I.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>47837</offset><text>Some residues that are affected in the Cdc42·HR1TOCA1 complex but do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) may contact HR1TOCA1 directly (Fig. 6D). Gln-2Cdc42, which has also been identified as a contact residue in the Cdc42·ACK complex, contacts Val-376TOCA1 and Asn-380TOCA1 in the model and disrupts the contacts between the interhelical loop and the first helix of the TOCA1 coiled-coil. Thr-52Cdc42, which has also been identified as making minor contacts with ACK, falls near the side chains of HR1TOCA1 helix 1, particularly Lys-372TOCA1, whereas the equivalent position in Rac1 is Asn-52Rac1. N52T is one of a combination of seven residues found to confer ACK binding on Rac1 and so may represent a specific Cdc42-effector contact residue. The position equivalent to Lys-372TOCA1 in PRK1 is Glu-58HR1a or Gln-151HR1b. Thr-52Cdc42-Lys-372TOCA1 may therefore represent a specific Cdc42-HR1TOCA1 contact. Arg-68Cdc42 of switch II is positioned close to Glu-395TOCA1 (Fig. 6D), suggesting a direct electrostatic contact between switch II of Cdc42 and helix 2 of the HR1 domain. The equivalent Arg in Rac1 and RhoA is pointing away from the HR1 domains of PRK1. The importance of this residue in the Cdc42-TOCA1 interaction remains unclear, although its mutation reduces binding to RhoGAP, suggesting that it can be involved in Cdc42 interactions.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>49213</offset><text>The solution structure of the TOCA1 HR1 domain presented here, along with the model of the HR1TOCA1·Cdc42 complex is consistent with a conserved mode of binding across the known HR1 domain-Rho family interactions, despite their differing affinities. The weak binding prevented detailed structural and thermodynamic studies of the complex. Nonetheless, structural studies of the TOCA1 HR1 domain, combined with chemical shift mapping, have highlighted some potentially interesting differences between Cdc42-HR1TOCA1 and RhoA/Rac1-HR1PRK1 binding.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>49760</offset><text>We have previously postulated that the inherent flexibility of HR1 domains contributes to their ability to bind to different Rho family G proteins, with Rho-binding HR1 domains displaying increased flexibility, reflected in their lower melting temperatures (Tm) and Rac binders being more rigid. The Tm of the TOCA1 HR1 domain is 61.9 °C (data not shown), which is the highest Tm that we have measured for an HR1 domain thus far. As such, the ability of the TOCA1 HR1 domain to bind to Cdc42 (a close relative of Rac1 rather than RhoA) fits this trend. An investigation into the local motions, particularly in the G protein-binding regions, may offer further insight into the differential specificities and affinities of G protein-HR1 domain interactions.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_4</infon><offset>50517</offset><text>Significance of a Weak, Transient Interaction</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>50563</offset><text>The low affinity of the Cdc42-HR1TOCA1 interaction is consistent with a tightly spatially and temporally regulated pathway, requiring combinatorial signals leading to a series of coincident weak interactions that elicit full activation. The HR1 domains from other TOCA family members, CIP4 and FBP17, also bind at low micromolar affinities to Cdc42, so the low affinity interaction appears to be commonplace among this family of HR1 domain proteins, in contrast to the PRK family. Weak, transient protein-protein interactions are functionally significant in several systems; for example, the binding of adaptor proteins to protein cargo during the formation of clathrin-coated vesicles in endocytosis involves multiple interactions of micromolar affinity.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>51319</offset><text>The low affinity of the HR1TOCA1-Cdc42 interaction in the context of the physiological concentration of TOCA1 in Xenopus extracts (∼10 nm) suggests that binding between TOCA1 and Cdc42 is likely to occur in vivo only when TOCA1 is at high local concentrations and membrane-localized and therefore in close proximity to activated Cdc42. Evidence suggests that the TOCA family of proteins are recruited to the membrane via an interaction between their F-BAR domain and specific signaling lipids. For example, electrostatic interactions between the F-BAR domain and the membrane are required for TOCA1 recruitment to membrane vesicles and tubules, and TOCA1-dependent actin polymerization is known to depend specifically on PI(4,5)P2. Furthermore, the isolated F-BAR domain of FBP17 has been shown to induce membrane tubulation of brain liposomes and BAR domain proteins that promote tubulation cluster on membranes at high densities. Once at the membrane, high local concentrations of TOCA1 could exceed the Kd of F-BAR dimerization (likely to be comparable with that of the FCHo2 F-BAR domain (2.5 μm)) and that of the Cdc42-HR1TOCA1 interaction. Cdc42-HR1TOCA1 binding would then be favorable, as long as coincident activation of Cdc42 had occurred, leading to stabilization of TOCA1 at the membrane and downstream activation of N-WASP.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>52659</offset><text>It has been postulated that WASP and N-WASP exist in equilibrium between folded (inactive) and unfolded (active) forms, and the affinity of Cdc42 for the unfolded WASP proteins is significantly enhanced. The unfolded, high affinity state of WASP is represented by a short peptide, the GBD, which binds with a low nanomolar affinity to Cdc42. In contrast, the best estimate of the affinity of full-length WASP for Cdc42 is low micromolar. In the inactive state of WASP, the actin- and Arp2/3-binding VCA domain contacts the GBD, competing for Cdc42 binding. The high affinity of Cdc42 for the unfolded, active form pushes the equilibrium in favor of (N-)WASP activation. Binding of PI(4,5)P2 to the basic region just N-terminal to the GBD further favors the active conformation. A substantial body of data has illuminated the complex regulation of WASP/N-WASP proteins, and current evidence suggests that these allosteric activation mechanisms and oligomerization combine to regulate WASP activity, allowing the synchronization and integration of multiple potential activation signals (reviewed in Ref.). Our data are easily reconciled with this model.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>53811</offset><text>We envisage that TOCA1 is first recruited to the appropriate membrane in response to PI(4,5)P2 via its F-BAR domain, where the local increase in concentration favors F-BAR-mediated dimerization of TOCA1. Cdc42 is activated in response to co-incident signals and can then bind to TOCA1, further stabilizing TOCA1 at the membrane. TOCA1 can then recruit N-WASP via an interaction between its SH3 domain and the N-WASP proline-rich region. The recruitment of N-WASP alone and of the N-WASP·WIP complex by TOCA1 and FBP17 has been demonstrated. WIP inhibits the activation of N-WASP by Cdc42, an effect that is reversed by TOCA1. It may therefore be envisaged that WIP and TOCA1 exert opposing allosteric effects on N-WASP, with TOCA1 favoring the unfolded, active conformation of N-WASP and increasing its affinity for Cdc42. TOCA1 may also activate N-WASP by effective oligomerization because clustering of TOCA1 at the membrane following coincident interactions with PI(4,5)P2 and Cdc42 would in turn lead to clustering of N-WASP, in addition to pushing the equilibrium toward the unfolded, active state.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>54916</offset><text>In a cellular context, full-length TOCA1 and N-WASP are likely to have similar affinities for active Cdc42, but in the unfolded, active conformation, the affinity of N-WASP for Cdc42 dramatically increases. Our binding data suggest that TOCA1 HR1 binding is not allosterically regulated, and our NMR data, along with the high stability of TOCA1 HR1, suggest that there is no widespread conformational change in the presence of Cdc42. As full-length TOCA1 and the isolated HR1 domain bind Cdc42 with similar affinities, the N-WASP-Cdc42 interaction will be favored because the N-WASP GBD can easily outcompete the TOCA1 HR1 for Cdc42. A combination of allosteric activation by PI(4,5)P2, activated Cdc42 and TOCA1, and oligomeric activation implemented by TOCA1 would lead to full activation of N-WASP and downstream actin polymerization.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>55754</offset><text>In such an array of molecules localized to a discrete region of the membrane, it is plausible that WASP could bind to a second Cdc42 molecule rather than displacing TOCA1 from its cognate Cdc42. Our NMR and affinity data, however, are consistent with displacement of the TOCA1 HR1 by the N-WASP GBD. Furthermore, TOCA1 is required for Cdc42-mediated activation of N-WASP·WIP, implying that it may not be possible for Cdc42 to bind and activate N-WASP prior to TOCA1-Cdc42 binding. The commonly used MGD → IST (Cdc42-binding deficient) mutant of TOCA1 has a reduced ability to activate the N-WASP·WIP complex, further indicating the importance of the Cdc42-HR1TOCA1 interaction prior to downstream activation of N-WASP.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>56477</offset><text>In light of this, we favor an “effector handover” scheme whereby TOCA1 interacts with Cdc42 prior to N-WASP activation, after which N-WASP displaces TOCA1 from its bound Cdc42 in order to be fully activated rather than binding a second Cdc42 molecule. Potentially, the TOCA1-Cdc42 interaction functions to position N-WASP and Cdc42 such that they are poised to interact with high affinity. The concomitant release of TOCA1 from Cdc42 while still bound to N-WASP presumably enhances the ability of TOCA1 to further activate N-WASP·WIP-induced actin polymerization. There is an advantage to such an effector handover, in that N-WASP would only be robustly recruited when F-BAR domains are already present. Hence, actin polymerization cannot occur until F-BAR domains are poised for membrane distortion.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>57283</offset><text>Our model of the Cdc42·HR1TOCA1 complex indicates a mechanism by which such a handover could take place (Fig. 9) because it shows that the effector binding sites only partially overlap on Cdc42. The lysine residues thought to be involved in an electrostatic steering mechanism in WASP-Cdc42 binding are conserved in N-WASP and would be able to interact with Cdc42 even when the TOCA1 HR1 domain is already bound. It has been postulated that the initial interactions between this basic region and Cdc42 could stabilize the active conformation of WASP, leading to high affinity binding between the core CRIB and Cdc42. The region C-terminal to the core CRIB, required for maximal affinity binding, would then fully displace the TOCA1 HR1.</text></passage><passage><infon key="file">zbc0281646060009.jpg</infon><infon key="id">F9</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>58021</offset><text>A simplified model of the early stages of Cdc42·N-WASP·TOCA1-dependent actin polymerization.
+Step 1, TOCA1 is recruited to the membrane via its F-BAR domain and/or Cdc42 interactions. F-BAR oligomerization is expected to occur following membrane binding, but a single monomer is shown for clarity. Step 2, N-WASP exists in an inactive, folded conformation. The TOCA1 SH3 domain interacts with N-WASP, causing an activatory allosteric effect. The HR1TOCA1-Cdc42 and SH3TOCA1-N-WASP interactions position Cdc42 and N-WASP for binding. Step 3, electrostatic interactions between Cdc42 and the basic region upstream of the CRIB initiate Cdc42·N-WASP binding. Step 4, the core CRIB binds with high affinity while the region C-terminal to the CRIB displaces the TOCA1 HR1 domain and increases the affinity of the N-WASP-Cdc42 interaction further. The VCA domain is released for downstream interactions, and actin polymerization proceeds. WH1, WASP homology 1 domain; PP, proline-rich region; VCA, verprolin homology, cofilin homology, acidic region.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>59068</offset><text>In conclusion, the data presented here show that the TOCA1 HR1 domain is sufficient for Cdc42 binding in vitro and that the interaction is of micromolar affinity, lower than that of other G protein-HR1 domain interactions. The analogous HR1 domains from other TOCA1 family members, FBP17 and CIP4, also exhibit micromolar affinity for Cdc42. A role for the TOCA1-, FBP17-, and CIP4-Cdc42 interactions in the recruitment of these proteins to the membrane therefore appears unlikely. Instead, our findings agree with earlier suggestions that the F-BAR domain is responsible for membrane recruitment. The role of the Cdc42-TOCA1 interaction remains somewhat elusive, but it may serve to position activated Cdc42 and N-WASP to allow full activation of N-WASP and as such serve to couple F-BAR-mediated membrane deformation with N-WASP activation. We envisage a complex interplay of equilibria between free and bound, active and inactive Cdc42, TOCA family, and WASP family proteins, facilitating a tightly spatially and temporally regulated pathway requiring numerous simultaneous events in order to achieve appropriate and robust activation of the downstream pathway. Our data are therefore easily reconciled with the dynamic instability models described in relation to the formation of endocytic vesicles and with the current data pertaining to the complex activation of WASP/N-WASP pathways by allosteric and oligomeric effects.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>60496</offset><text>It is clear from the data presented here that TOCA1 and N-WASP do not bind Cdc42 simultaneously and that N-WASP is likely to outcompete TOCA1 for Cdc42 binding. We therefore postulate an effector handover mechanism based on current evidence surrounding WASP/N-WASP activation and our model of the Cdc42·HR1TOCA1 complex. The displacement of the TOCA1 HR1 domain from Cdc42 by N-WASP may represent a unidirectional step in the pathway of Cdc42·N-WASP·TOCA1-dependent actin assembly.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">title_1</infon><offset>60981</offset><text>Author Contributions</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">paragraph</infon><offset>61002</offset><text>J. R. W. generated constructs and proteins, set up NMR experiments, analyzed NMR data, and performed binding experiments; D. N. set up NMR experiments; H. M. F. generated longer TOCA clones and proteins; J. L. G. supervised the pyrene actin assays; D. O. supervised the protein binding assays; and H. R. M. performed NMR experiments and analyzed NMR data. J. R. W., D. O., and H. R. M. wrote the paper with input from all authors.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>61433</offset><text>The authors declare that they have no conflicts of interest with the contents of this article.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>61528</offset><text>The atomic coordinates and structure factors (code 5FRG) have been deposited in the Protein Data Bank (http://wwpdb.org/).</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>61651</offset><text>D. Owen, unpublished data.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>61678</offset><text>H. R. Mott and D. Owen, unpublished data.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>61720</offset><text>PRK</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>61724</offset><text>protein kinase C related kinase</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>61756</offset><text>WASP</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>61761</offset><text>Wiskott-Aldrich syndrome protein</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>61794</offset><text>TOCA</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>61799</offset><text>transducer of Cdc42-dependent actin assembly protein</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>61852</offset><text>N-WASP</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>61859</offset><text>neural Wiskott-Aldrich syndrome protein</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>61899</offset><text>PI(4,5)P2</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>61909</offset><text>phosphatidylinositol 4,5-bisphosphate</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>61947</offset><text>HR1</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>61951</offset><text>homology region 1</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>61969</offset><text>F-BAR</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>61975</offset><text>Fes/CIP4 homology BAR</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>61997</offset><text>SH3</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62001</offset><text>Src homology 3</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62016</offset><text>CRIB</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62021</offset><text>Cdc42- and Rac-interactive binding</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62056</offset><text>CIP4</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62061</offset><text>Cdc42-interacting protein 4</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62089</offset><text>MBP</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62093</offset><text>maltose-binding protein</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62117</offset><text>GBD</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62121</offset><text>G protein binding domain</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62146</offset><text>SPA</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62150</offset><text>scintillation proximity assay</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62180</offset><text>PAK</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62184</offset><text>p21-activated kinase</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62205</offset><text>ACK</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62209</offset><text>activated Cdc42-associated kinase</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62243</offset><text>HSQC</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62248</offset><text>heteronuclear single quantum correlation</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62289</offset><text>GMPPNP</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62296</offset><text>guanosine 5′-[β,γ-imido] triphosphate</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62341</offset><text>GTPγS</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62351</offset><text>guanosine 5′-3-O-(thio)triphosphate</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62389</offset><text>GBD</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62393</offset><text>G protein-binding domain</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62418</offset><text>CSP</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62422</offset><text>chemical shift perturbation</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62450</offset><text>PDB</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62454</offset><text>Protein Data Bank.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>62473</offset><text>The abbreviations used are: </text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>62502</offset><text>References</text></passage><passage><infon key="fpage">117</infon><infon key="lpage">127</infon><infon key="name_0">surname:Bourne;given-names:H. R.</infon><infon key="name_1">surname:Sanders;given-names:D. A.</infon><infon key="name_2">surname:McCormick;given-names:F.</infon><infon key="pub-id_pmid">1898771</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">349</infon><infon key="year">1991</infon><offset>62513</offset><text>The GTPase superfamily: conserved structure and molecular mechanism</text></passage><passage><infon key="fpage">269</infon><infon key="lpage">309</infon><infon key="name_0">surname:Cherfils;given-names:J.</infon><infon key="name_1">surname:Zeghouf;given-names:M.</infon><infon key="pub-id_pmid">23303910</infon><infon key="section_type">REF</infon><infon key="source">Physiol. Rev</infon><infon key="type">ref</infon><infon key="volume">93</infon><infon key="year">2013</infon><offset>62581</offset><text>Regulation of small GTPases by GEFs, GAPs, and GDIs</text></passage><passage><infon key="fpage">1299</infon><infon key="lpage">1304</infon><infon key="name_0">surname:Vetter;given-names:I. R.</infon><infon key="name_1">surname:Wittinghofer;given-names:A.</infon><infon key="pub-id_pmid">11701921</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">294</infon><infon key="year">2001</infon><offset>62633</offset><text>The guanine nucleotide-binding switch in three dimensions</text></passage><passage><infon key="fpage">85</infon><infon key="lpage">133</infon><infon key="name_0">surname:Mott;given-names:H. R.</infon><infon key="name_1">surname:Owen;given-names:D.</infon><infon key="pub-id_pmid">25830673</infon><infon key="section_type">REF</infon><infon key="source">Crit. Rev. Biochem. Mol. Biol</infon><infon key="type">ref</infon><infon key="volume">50</infon><infon key="year">2015</infon><offset>62691</offset><text>Structures of Ras superfamily effector complexes: what have we learnt in two decades?</text></passage><passage><infon key="fpage">304</infon><infon key="lpage">310</infon><infon key="name_0">surname:Machesky;given-names:L. M.</infon><infon key="name_1">surname:Hall;given-names:A.</infon><infon key="pub-id_pmid">15157438</infon><infon key="section_type">REF</infon><infon key="source">Trends Cell Biol</infon><infon key="type">ref</infon><infon key="volume">6</infon><infon key="year">1996</infon><offset>62777</offset><text>Rho: a connection between membrane receptor signalling and the cytoskeleton</text></passage><passage><infon key="fpage">389</infon><infon key="lpage">399</infon><infon key="name_0">surname:Ridley;given-names:A. J.</infon><infon key="name_1">surname:Hall;given-names:A.</infon><infon key="pub-id_pmid">1643657</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">70</infon><infon key="year">1992</infon><offset>62853</offset><text>The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors</text></passage><passage><infon key="fpage">401</infon><infon key="lpage">410</infon><infon key="name_0">surname:Ridley;given-names:A. J.</infon><infon key="name_1">surname:Paterson;given-names:H. F.</infon><infon key="name_2">surname:Johnston;given-names:C. L.</infon><infon key="name_3">surname:Diekmann;given-names:D.</infon><infon key="name_4">surname:Hall;given-names:A.</infon><infon key="pub-id_pmid">1643658</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">70</infon><infon key="year">1992</infon><offset>62983</offset><text>The small GTP-binding protein rac regulates growth factor-induced membrane ruffling</text></passage><passage><infon key="fpage">1256</infon><infon key="lpage">1264</infon><infon key="name_0">surname:Ridley;given-names:A. J.</infon><infon key="pub-id_pmid">8939567</infon><infon key="section_type">REF</infon><infon key="source">Curr. Biol</infon><infon key="type">ref</infon><infon key="volume">6</infon><infon key="year">1996</infon><offset>63067</offset><text>Rho: theme and variations</text></passage><passage><infon key="fpage">53</infon><infon key="lpage">62</infon><infon key="name_0">surname:Nobes;given-names:C. D.</infon><infon key="name_1">surname:Hall;given-names:A.</infon><infon key="pub-id_pmid">7536630</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">81</infon><infon key="year">1995</infon><offset>63093</offset><text>Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia</text></passage><passage><infon key="fpage">509</infon><infon key="lpage">514</infon><infon key="name_0">surname:Hall;given-names:A.</infon><infon key="pub-id_pmid">9438836</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">279</infon><infon key="year">1998</infon><offset>63242</offset><text>Rho GTPases and the actin cytoskeleton</text></passage><passage><infon key="fpage">267</infon><infon key="lpage">272</infon><infon key="name_0">surname:Machesky;given-names:L. M.</infon><infon key="name_1">surname:Insall;given-names:R. H.</infon><infon key="pub-id_pmid">10427083</infon><infon key="section_type">REF</infon><infon key="source">J. Cell Biol</infon><infon key="type">ref</infon><infon key="volume">146</infon><infon key="year">1999</infon><offset>63281</offset><text>Signaling to actin dynamics</text></passage><passage><infon key="fpage">1942</infon><infon key="lpage">1952</infon><infon key="name_0">surname:Kozma;given-names:R.</infon><infon key="name_1">surname:Ahmed;given-names:S.</infon><infon key="name_2">surname:Best;given-names:A.</infon><infon key="name_3">surname:Lim;given-names:L.</infon><infon key="pub-id_pmid">7891688</infon><infon key="section_type">REF</infon><infon key="source">Mol. Cell. Biol</infon><infon key="type">ref</infon><infon key="volume">15</infon><infon key="year">1995</infon><offset>63309</offset><text>The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts</text></passage><passage><infon key="fpage">e29513</infon><infon key="name_0">surname:Kühn;given-names:S.</infon><infon key="name_1">surname:Geyer;given-names:M.</infon><infon key="pub-id_pmid">24914801</infon><infon key="section_type">REF</infon><infon key="source">Small GTPases</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2014</infon><offset>63445</offset><text>Formins as effector proteins of Rho GTPases</text></passage><passage><infon key="fpage">645</infon><infon key="lpage">648</infon><infon key="name_0">surname:Watanabe;given-names:G.</infon><infon key="name_1">surname:Saito;given-names:Y.</infon><infon key="name_2">surname:Madaule;given-names:P.</infon><infon key="name_3">surname:Ishizaki;given-names:T.</infon><infon key="name_4">surname:Fujisawa;given-names:K.</infon><infon key="name_5">surname:Morii;given-names:N.</infon><infon key="name_6">surname:Mukai;given-names:H.</infon><infon key="name_7">surname:Ono;given-names:Y.</infon><infon key="name_8">surname:Kakizuka;given-names:A.</infon><infon key="name_9">surname:Narumiya;given-names:S.</infon><infon key="pub-id_pmid">8571126</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">271</infon><infon key="year">1996</infon><offset>63489</offset><text>Protein kinase N (PKN) and PKN-related protein rhophilin as targets of small GTPase Rho</text></passage><passage><infon key="fpage">723</infon><infon key="lpage">734</infon><infon key="name_0">surname:Symons;given-names:M.</infon><infon key="name_1">surname:Derry;given-names:J. M. .</infon><infon key="name_2">surname:Karlak;given-names:B.</infon><infon key="name_3">surname:Jiang;given-names:S.</infon><infon key="name_4">surname:Lemahieu;given-names:V.</infon><infon key="name_5">surname:Mccormick;given-names:F.</infon><infon key="name_6">surname:Francke;given-names:U.</infon><infon key="name_7">surname:Abo;given-names:A.</infon><infon key="pub-id_pmid">8625410</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">84</infon><infon key="year">1996</infon><offset>63577</offset><text>Wiskott-Aldrich syndrome protein, a novel effector for the GTPase CDC42Hs, is implicated in actin polymerization</text></passage><passage><infon key="fpage">203</infon><infon key="lpage">216</infon><infon key="name_0">surname:Ho;given-names:H. Y.</infon><infon key="name_1">surname:Rohatgi;given-names:R.</infon><infon key="name_2">surname:Lebensohn;given-names:A. M.</infon><infon key="name_3">surname:Le;given-names:Ma</infon><infon key="name_4">surname:Li;given-names:J.</infon><infon key="name_5">surname:Gygi;given-names:S. P.</infon><infon key="name_6">surname:Kirschner;given-names:M. W.</infon><infon key="pub-id_pmid">15260990</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">118</infon><infon key="year">2004</infon><offset>63690</offset><text>Toca-1 mediates Cdc42-dependent actin nucleation by activating the N-WASP-WIP complex</text></passage><passage><infon key="fpage">479</infon><infon key="lpage">487</infon><infon key="name_0">surname:Aspenström;given-names:P.</infon><infon key="pub-id_pmid">9210375</infon><infon key="section_type">REF</infon><infon key="source">Curr. Biol</infon><infon key="type">ref</infon><infon key="volume">7</infon><infon key="year">1997</infon><offset>63776</offset><text>A Cdc42 target protein with homology to the non-kinase domain of FER has a potential role in regulating the actin cytoskeleton</text></passage><passage><infon key="fpage">1339</infon><infon key="lpage">1344</infon><infon key="name_0">surname:Wu;given-names:M.</infon><infon key="name_1">surname:Wu;given-names:X.</infon><infon key="name_2">surname:De Camilli;given-names:P.</infon><infon key="pub-id_pmid">23297209</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl. Acad. Sci. U.S.A</infon><infon key="type">ref</infon><infon key="volume">110</infon><infon key="year">2013</infon><offset>63903</offset><text>Calcium oscillations-coupled conversion of actin travelling waves to standing oscillations</text></passage><passage><infon key="fpage">6932</infon><infon key="lpage">6941</infon><infon key="name_0">surname:Miki;given-names:H.</infon><infon key="name_1">surname:Suetsugu;given-names:S.</infon><infon key="name_2">surname:Takenawa;given-names:T.</infon><infon key="pub-id_pmid">9843499</infon><infon key="section_type">REF</infon><infon key="source">EMBO J</infon><infon key="type">ref</infon><infon key="volume">17</infon><infon key="year">1998</infon><offset>63994</offset><text>WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac</text></passage><passage><infon key="fpage">221</infon><infon key="lpage">231</infon><infon key="name_0">surname:Rohatgi;given-names:R.</infon><infon key="name_1">surname:Ma;given-names:L.</infon><infon key="name_2">surname:Miki;given-names:H.</infon><infon key="name_3">surname:Lopez;given-names:M.</infon><infon key="name_4">surname:Kirchhausen;given-names:T.</infon><infon key="name_5">surname:Takenawa;given-names:T.</infon><infon key="name_6">surname:Kirschner;given-names:M. W.</infon><infon key="pub-id_pmid">10219243</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">97</infon><infon key="year">1999</infon><offset>64076</offset><text>The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly</text></passage><passage><infon key="fpage">1299</infon><infon key="lpage">1310</infon><infon key="name_0">surname:Rohatgi;given-names:R.</infon><infon key="name_1">surname:Ho;given-names:H. Y.</infon><infon key="name_2">surname:Kirschner;given-names:M. W.</infon><infon key="pub-id_pmid">10995436</infon><infon key="section_type">REF</infon><infon key="source">J. Cell Biol</infon><infon key="type">ref</infon><infon key="volume">150</infon><infon key="year">2000</infon><offset>64178</offset><text>Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4,5-bisphosphate</text></passage><passage><infon key="fpage">1125</infon><infon key="lpage">1136</infon><infon key="name_0">surname:Ma;given-names:L.</infon><infon key="name_1">surname:Cantley;given-names:L. C.</infon><infon key="name_2">surname:Janmey;given-names:P. A.</infon><infon key="name_3">surname:Kirschner;given-names:M. W.</infon><infon key="pub-id_pmid">9490725</infon><infon key="section_type">REF</infon><infon key="source">J. Cell Biol</infon><infon key="type">ref</infon><infon key="volume">140</infon><infon key="year">1998</infon><offset>64260</offset><text>Corequirement of specific phosphoinositides and small GTP-binding protein Cdc42 in inducing actin assembly in Xenopus egg extracts</text></passage><passage><infon key="fpage">151</infon><infon key="lpage">158</infon><infon key="name_0">surname:Kim;given-names:A. S.</infon><infon key="name_1">surname:Kakalis;given-names:L. T.</infon><infon key="name_2">surname:Abdul-Manan;given-names:N.</infon><infon key="name_3">surname:Liu;given-names:G. A.</infon><infon key="name_4">surname:Rosen;given-names:M. K.</infon><infon key="pub-id_pmid">10724160</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">404</infon><infon key="year">2000</infon><offset>64391</offset><text>Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein</text></passage><passage><infon key="fpage">707</infon><infon key="lpage">735</infon><infon key="name_0">surname:Padrick;given-names:S. B.</infon><infon key="name_1">surname:Rosen;given-names:M. K.</infon><infon key="pub-id_pmid">20533885</infon><infon key="section_type">REF</infon><infon key="source">Annu. Rev. Biochem</infon><infon key="type">ref</infon><infon key="volume">79</infon><infon key="year">2010</infon><offset>64472</offset><text>Physical mechanisms of signal integration by WASP family proteins</text></passage><passage><infon key="fpage">15362</infon><infon key="lpage">15367</infon><infon key="name_0">surname:Ma;given-names:L.</infon><infon key="name_1">surname:Rohatgi;given-names:R.</infon><infon key="name_2">surname:Kirschner;given-names:M. W.</infon><infon key="pub-id_pmid">9860974</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl. Acad. Sci. U.S.A</infon><infon key="type">ref</infon><infon key="volume">95</infon><infon key="year">1998</infon><offset>64538</offset><text>The Arp2/3 complex mediates actin polymerization induced by the small GTP-binding protein Cdc42</text></passage><passage><infon key="fpage">2817</infon><infon key="lpage">2828</infon><infon key="name_0">surname:Takano;given-names:K.</infon><infon key="name_1">surname:Takano;given-names:K.</infon><infon key="name_2">surname:Toyooka;given-names:K.</infon><infon key="name_3">surname:Suetsugu;given-names:S.</infon><infon key="pub-id_pmid">18923421</infon><infon key="section_type">REF</infon><infon key="source">EMBO J</infon><infon key="type">ref</infon><infon key="volume">27</infon><infon key="year">2008</infon><offset>64634</offset><text>EFC/F-BAR proteins and the N-WASP-WIP complex induce membrane curvature-dependent actin polymerization</text></passage><passage><infon key="fpage">11622</infon><infon key="lpage">11636</infon><infon key="name_0">surname:Bu;given-names:W.</infon><infon key="name_1">surname:Chou;given-names:A. M.</infon><infon key="name_2">surname:Lim;given-names:K. B.</infon><infon key="name_3">surname:Sudhaharan;given-names:T.</infon><infon key="name_4">surname:Ahmed;given-names:S.</infon><infon key="pub-id_pmid">19213734</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem</infon><infon key="type">ref</infon><infon key="volume">284</infon><infon key="year">2009</infon><offset>64737</offset><text>The Toca-1-N-WASP complex links filopodial formation to endocytosis</text></passage><passage><infon key="fpage">1429</infon><infon key="lpage">1437</infon><infon key="name_0">surname:Fricke;given-names:R.</infon><infon key="name_1">surname:Gohl;given-names:C.</infon><infon key="name_2">surname:Dharmalingam;given-names:E.</infon><infon key="name_3">surname:Grevelhörster;given-names:A.</infon><infon key="name_4">surname:Zahedi;given-names:B.</infon><infon key="name_5">surname:Harden;given-names:N.</infon><infon key="name_6">surname:Kessels;given-names:M.</infon><infon key="name_7">surname:Qualmann;given-names:B.</infon><infon key="name_8">surname:Bogdan;given-names:S.</infon><infon key="pub-id_pmid">19716703</infon><infon key="section_type">REF</infon><infon key="source">Curr. Biol</infon><infon key="type">ref</infon><infon key="volume">19</infon><infon key="year">2009</infon><offset>64805</offset><text>Drosophila Cip4/Toca-1 integrates membrane trafficking and actin dynamics through WASP and SCAR/WAVE</text></passage><passage><infon key="fpage">e1000675</infon><infon key="name_0">surname:Giuliani;given-names:C.</infon><infon key="name_1">surname:Troglio;given-names:F.</infon><infon key="name_10">surname:Hardin;given-names:J. D.</infon><infon key="name_11">surname:Soto;given-names:M. C.</infon><infon key="name_12">surname:Grant;given-names:B. D.</infon><infon key="name_13">surname:Scita;given-names:G.</infon><infon key="name_2">surname:Bai;given-names:Z.</infon><infon key="name_3">surname:Patel;given-names:F. B.</infon><infon key="name_4">surname:Zucconi;given-names:A.</infon><infon key="name_5">surname:Malabarba;given-names:M. G.</infon><infon key="name_6">surname:Disanza;given-names:A.</infon><infon key="name_7">surname:Stradal;given-names:T. B.</infon><infon key="name_8">surname:Cassata;given-names:G.</infon><infon key="name_9">surname:Confalonieri;given-names:S.</infon><infon key="pub-id_pmid">19798448</infon><infon key="section_type">REF</infon><infon key="source">PLoS Genet</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2009</infon><offset>64906</offset><text>Requirements for F-BAR proteins TOCA-1 and TOCA-2 in actin dynamics and membrane trafficking during Caenorhabditis elegans oocyte growth and embryonic epidermal morphogenesis</text></passage><passage><infon key="fpage">1</infon><infon key="lpage">16</infon><infon key="name_0">surname:Bu;given-names:W.</infon><infon key="name_1">surname:Lim;given-names:K. B.</infon><infon key="name_2">surname:Yu;given-names:Y. H.</infon><infon key="name_3">surname:Chou;given-names:A. M.</infon><infon key="name_4">surname:Sudhaharan;given-names:T.</infon><infon key="name_5">surname:Ahmed;given-names:S.</infon><infon key="section_type">REF</infon><infon key="source">PLoS ONE</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2010</infon><offset>65081</offset><text>Cdc42 interaction with N-WASP and Toca-1 regulates membrane tubulation, vesicle formation and vesicle motility: implications for endocytosis</text></passage><passage><infon key="fpage">1341</infon><infon key="lpage">1345</infon><infon key="name_0">surname:Lee;given-names:K.</infon><infon key="name_1">surname:Gallop;given-names:J. L.</infon><infon key="name_2">surname:Rambani;given-names:K.</infon><infon key="name_3">surname:Kirschner;given-names:M. W.</infon><infon key="pub-id_pmid">20829485</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">329</infon><infon key="year">2010</infon><offset>65222</offset><text>Self-assembly of filopodia-like structures on supported lipid bilayers</text></passage><passage><infon key="fpage">7193</infon><infon key="lpage">7198</infon><infon key="name_0">surname:Gallop;given-names:J. L.</infon><infon key="name_1">surname:Walrant;given-names:A.</infon><infon key="name_2">surname:Cantley;given-names:L. C.</infon><infon key="name_3">surname:Kirschner;given-names:M. W.</infon><infon key="pub-id_pmid">23589871</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl. Acad. Sci. U.S.A</infon><infon key="type">ref</infon><infon key="volume">110</infon><infon key="year">2013</infon><offset>65293</offset><text>Phosphoinositides and membrane curvature switch the mode of actin polymerization via selective recruitment of toca-1 and Snx9</text></passage><passage><infon key="fpage">269</infon><infon key="lpage">279</infon><infon key="name_0">surname:Tsujita;given-names:K.</infon><infon key="name_1">surname:Suetsugu;given-names:S.</infon><infon key="name_2">surname:Sasaki;given-names:N.</infon><infon key="name_3">surname:Furutani;given-names:M.</infon><infon key="name_4">surname:Oikawa;given-names:T.</infon><infon key="name_5">surname:Takenawa;given-names:T.</infon><infon key="pub-id_pmid">16418535</infon><infon key="section_type">REF</infon><infon key="source">J. Cell Biol</infon><infon key="type">ref</infon><infon key="volume">172</infon><infon key="year">2006</infon><offset>65419</offset><text>Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis</text></passage><passage><infon key="fpage">E1443</infon><infon key="lpage">E1452</infon><infon key="name_0">surname:Bai;given-names:Z.</infon><infon key="name_1">surname:Grant;given-names:B. D.</infon><infon key="pub-id_pmid">25775511</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl. Acad. Sci. U.S.A</infon><infon key="type">ref</infon><infon key="volume">112</infon><infon key="year">2015</infon><offset>65569</offset><text>A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling</text></passage><passage><infon key="fpage">29042</infon><infon key="lpage">29053</infon><infon key="name_0">surname:Kakimoto;given-names:T.</infon><infon key="name_1">surname:Katoh;given-names:H.</infon><infon key="name_2">surname:Negishi;given-names:M.</infon><infon key="pub-id_pmid">16885158</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem</infon><infon key="type">ref</infon><infon key="volume">281</infon><infon key="year">2006</infon><offset>65654</offset><text>Regulation of neuronal morphology by Toca-1, an F-BAR/EFC protein that induces plasma membrane invagination</text></passage><passage><infon key="fpage">946</infon><infon key="lpage">958</infon><infon key="name_0">surname:Miyamoto;given-names:K.</infon><infon key="name_1">surname:Pasque;given-names:V.</infon><infon key="name_2">surname:Jullien;given-names:J.</infon><infon key="name_3">surname:Gurdon;given-names:J. B.</infon><infon key="pub-id_pmid">21536734</infon><infon key="section_type">REF</infon><infon key="source">Genes Dev</infon><infon key="type">ref</infon><infon key="volume">25</infon><infon key="year">2011</infon><offset>65762</offset><text>Nuclear actin polymerization is required for transcriptional reprogramming of Oct4 by oocytes</text></passage><passage><infon key="fpage">2769</infon><infon key="lpage">2787</infon><infon key="name_0">surname:Van Itallie;given-names:C. M.</infon><infon key="name_1">surname:Tietgens;given-names:A. J.</infon><infon key="name_2">surname:Krystofiak;given-names:E.</infon><infon key="name_3">surname:Kachar;given-names:B.</infon><infon key="name_4">surname:Anderson;given-names:J. M.</infon><infon key="pub-id_pmid">26063734</infon><infon key="section_type">REF</infon><infon key="source">Mol. Biol. Cell</infon><infon key="type">ref</infon><infon key="volume">26</infon><infon key="year">2015</infon><offset>65856</offset><text>A complex of ZO-1 and the BAR-domain protein TOCA-1 regulates actin assembly at the tight junction</text></passage><passage><infon key="fpage">2261</infon><infon key="lpage">2272</infon><infon key="name_0">surname:Hu;given-names:J.</infon><infon key="name_1">surname:Mukhopadhyay;given-names:A.</infon><infon key="name_2">surname:Craig;given-names:A. W. B.</infon><infon key="pub-id_pmid">21062739</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem</infon><infon key="type">ref</infon><infon key="volume">286</infon><infon key="year">2011</infon><offset>65955</offset><text>Transducer of Cdc42-dependent actin assembly promotes epidermal growth factor-induced cell motility and invasiveness</text></passage><passage><infon key="fpage">3080</infon><infon key="lpage">3090</infon><infon key="name_0">surname:Chander;given-names:H.</infon><infon key="name_1">surname:Truesdell;given-names:P.</infon><infon key="name_2">surname:Meens;given-names:J.</infon><infon key="name_3">surname:Craig;given-names:A. W. B.</infon><infon key="pub-id_pmid">22824798</infon><infon key="section_type">REF</infon><infon key="source">Oncogene</infon><infon key="type">ref</infon><infon key="volume">32</infon><infon key="year">2013</infon><offset>66072</offset><text>Transducer of Cdc42-dependent actin assembly promotes breast cancer invasion and metastasis</text></passage><passage><infon key="fpage">791</infon><infon key="lpage">804</infon><infon key="name_0">surname:Itoh;given-names:T.</infon><infon key="name_1">surname:Erdmann;given-names:K. S.</infon><infon key="name_2">surname:Roux;given-names:A.</infon><infon key="name_3">surname:Habermann;given-names:B.</infon><infon key="name_4">surname:Werner;given-names:H.</infon><infon key="name_5">surname:De Camilli;given-names:P.</infon><infon key="pub-id_pmid">16326391</infon><infon key="section_type">REF</infon><infon key="source">Dev. Cell</infon><infon key="type">ref</infon><infon key="volume">9</infon><infon key="year">2005</infon><offset>66164</offset><text>Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins</text></passage><passage><infon key="fpage">839</infon><infon key="lpage">852</infon><infon key="name_0">surname:Henne;given-names:W. M.</infon><infon key="name_1">surname:Kent;given-names:H. M.</infon><infon key="name_2">surname:Ford;given-names:M. G. J.</infon><infon key="name_3">surname:Hegde;given-names:B. G.</infon><infon key="name_4">surname:Daumke;given-names:O.</infon><infon key="name_5">surname:Butler;given-names:P. J. G.</infon><infon key="name_6">surname:Mittal;given-names:R.</infon><infon key="name_7">surname:Langen;given-names:R.</infon><infon key="name_8">surname:Evans;given-names:P. R.</infon><infon key="name_9">surname:McMahon;given-names:H. T.</infon><infon key="pub-id_pmid">17540576</infon><infon key="section_type">REF</infon><infon key="source">Structure</infon><infon key="type">ref</infon><infon key="volume">15</infon><infon key="year">2007</infon><offset>66277</offset><text>Structure and analysis of FCHo2 F-BAR domain: a dimerizing and membrane recruitment module that effects membrane curvature</text></passage><passage><infon key="fpage">793</infon><infon key="lpage">803</infon><infon key="name_0">surname:Maesaki;given-names:R.</infon><infon key="name_1">surname:Ihara;given-names:K.</infon><infon key="name_2">surname:Shimizu;given-names:T.</infon><infon key="name_3">surname:Kuroda;given-names:S.</infon><infon key="name_4">surname:Kaibuchi;given-names:K.</infon><infon key="name_5">surname:Hakoshima;given-names:T.</infon><infon key="pub-id_pmid">10619026</infon><infon key="section_type">REF</infon><infon key="source">Mol. Cell</infon><infon key="type">ref</infon><infon key="volume">4</infon><infon key="year">1999</infon><offset>66400</offset><text>The structural basis of Rho effector recognition revealed by the crystal structure of human RhoA complexed with the effector domain of PKN/PRK1</text></passage><passage><infon key="fpage">50578</infon><infon key="lpage">50587</infon><infon key="name_0">surname:Owen;given-names:D.</infon><infon key="name_1">surname:Lowe;given-names:P. N.</infon><infon key="name_2">surname:Nietlispach;given-names:D.</infon><infon key="name_3">surname:Brosnan;given-names:C. E.</infon><infon key="name_4">surname:Chirgadze;given-names:D. Y.</infon><infon key="name_5">surname:Parker;given-names:P. J.</infon><infon key="name_6">surname:Blundell;given-names:T. L.</infon><infon key="name_7">surname:Mott;given-names:H. R.</infon><infon key="pub-id_pmid">14514689</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem</infon><infon key="type">ref</infon><infon key="volume">278</infon><infon key="year">2003</infon><offset>66544</offset><text>Molecular dissection of the interaction between the small G proteins Rac1 and RhoA and protein kinase C-related kinase 1 (PRK1)</text></passage><passage><infon key="fpage">2860</infon><infon key="lpage">2869</infon><infon key="name_0">surname:Hutchinson;given-names:C. L.</infon><infon key="name_1">surname:Lowe;given-names:P. N.</infon><infon key="name_2">surname:McLaughlin;given-names:S. H.</infon><infon key="name_3">surname:Mott;given-names:H. R.</infon><infon key="name_4">surname:Owen;given-names:D.</infon><infon key="pub-id_pmid">21351730</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry</infon><infon key="type">ref</infon><infon key="volume">50</infon><infon key="year">2011</infon><offset>66672</offset><text>Mutational analysis reveals a single binding interface between RhoA and its effector, PRK1</text></passage><passage><infon key="fpage">7999</infon><infon key="lpage">8011</infon><infon key="name_0">surname:Hutchinson;given-names:C. L.</infon><infon key="name_1">surname:Lowe;given-names:P. N.</infon><infon key="name_2">surname:McLaughlin;given-names:S. H.</infon><infon key="name_3">surname:Mott;given-names:H. R.</infon><infon key="name_4">surname:Owen;given-names:D.</infon><infon key="pub-id_pmid">24128008</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry</infon><infon key="type">ref</infon><infon key="volume">52</infon><infon key="year">2013</infon><offset>66763</offset><text>Differential binding of RhoA, RhoB, and RhoC to protein kinase C-related kinase (PRK) isoforms PRK1, PRK2, and PRK3: PRKs have the highest affinity for RhoB</text></passage><passage><infon key="fpage">1492</infon><infon key="lpage">1500</infon><infon key="name_0">surname:Modha;given-names:R.</infon><infon key="name_1">surname:Campbell;given-names:L. J.</infon><infon key="name_2">surname:Nietlispach;given-names:D.</infon><infon key="name_3">surname:Buhecha;given-names:H. R.</infon><infon key="name_4">surname:Owen;given-names:D.</infon><infon key="name_5">surname:Mott;given-names:H. R.</infon><infon key="pub-id_pmid">18006505</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem</infon><infon key="type">ref</infon><infon key="volume">283</infon><infon key="year">2008</infon><offset>66920</offset><text>The Rac1 polybasic region is required for interaction with its effector PRK1</text></passage><passage><infon key="fpage">113</infon><infon key="lpage">118</infon><infon key="name_0">surname:Kobashigawa;given-names:Y.</infon><infon key="name_1">surname:Kumeta;given-names:H.</infon><infon key="name_2">surname:Kanoh;given-names:D.</infon><infon key="name_3">surname:Inagaki;given-names:F.</infon><infon key="pub-id_pmid">19387844</infon><infon key="section_type">REF</infon><infon key="source">J. Biomol. NMR</infon><infon key="type">ref</infon><infon key="volume">44</infon><infon key="year">2009</infon><offset>66997</offset><text>The NMR structure of the TC10- and Cdc42-interacting domain of CIP4</text></passage><passage><infon key="fpage">29071</infon><infon key="lpage">29074</infon><infon key="name_0">surname:Burbelo;given-names:P. D.</infon><infon key="name_1">surname:Drechsel;given-names:D.</infon><infon key="name_2">surname:Hall;given-names:A.</infon><infon key="pub-id_pmid">7493928</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem</infon><infon key="type">ref</infon><infon key="volume">270</infon><infon key="year">1995</infon><offset>67065</offset><text>A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases</text></passage><passage><infon key="fpage">379</infon><infon key="lpage">383</infon><infon key="name_0">surname:Abdul-Manan;given-names:N.</infon><infon key="name_1">surname:Aghazadeh;given-names:B.</infon><infon key="name_2">surname:Liu;given-names:G. A.</infon><infon key="name_3">surname:Majumdar;given-names:A.</infon><infon key="name_4">surname:Ouerfelli;given-names:O.</infon><infon key="name_5">surname:Siminovitch;given-names:K. A.</infon><infon key="name_6">surname:Rosen;given-names:M. K.</infon><infon key="pub-id_pmid">10360578</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">399</infon><infon key="year">1999</infon><offset>67165</offset><text>Structure of Cdc42 in complex with the GTPase-binding domain of the “Wiskott-Aldrich syndrome” protein</text></passage><passage><infon key="fpage">1243</infon><infon key="lpage">1250</infon><infon key="name_0">surname:Owen;given-names:D.</infon><infon key="name_1">surname:Mott;given-names:H. R.</infon><infon key="name_2">surname:Laue;given-names:E. D.</infon><infon key="name_3">surname:Lowe;given-names:P. N.</infon><infon key="pub-id_pmid">10684602</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry</infon><infon key="type">ref</infon><infon key="volume">39</infon><infon key="year">2000</infon><offset>67272</offset><text>Residues in Cdc42 that specify binding to individual CRIB effector proteins</text></passage><passage><infon key="fpage">992</infon><infon key="lpage">999</infon><infon key="name_0">surname:Bailey;given-names:L. K.</infon><infon key="name_1">surname:Campbell;given-names:L. J.</infon><infon key="name_2">surname:Evetts;given-names:K. A.</infon><infon key="name_3">surname:Littlefield;given-names:K.</infon><infon key="name_4">surname:Rajendra;given-names:E.</infon><infon key="name_5">surname:Nietlispach;given-names:D.</infon><infon key="name_6">surname:Owen;given-names:D.</infon><infon key="name_7">surname:Mott;given-names:H. R.</infon><infon key="pub-id_pmid">18981177</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem</infon><infon key="type">ref</infon><infon key="volume">284</infon><infon key="year">2009</infon><offset>67348</offset><text>The structure of binder of Arl2 (BART) reveals a novel G protein binding domain: implications for function</text></passage><passage><infon key="fpage">7885</infon><infon key="lpage">7891</infon><infon key="name_0">surname:Thompson;given-names:G.</infon><infon key="name_1">surname:Owen;given-names:D.</infon><infon key="name_2">surname:Chalk;given-names:P. A.</infon><infon key="name_3">surname:Lowe;given-names:P. N.</infon><infon key="pub-id_pmid">9601050</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry</infon><infon key="type">ref</infon><infon key="volume">37</infon><infon key="year">1998</infon><offset>67455</offset><text>Delineation of the Cdc42/Rac-binding domain of p21-activated kinase</text></passage><passage><infon key="fpage">1692</infon><infon key="lpage">1704</infon><infon key="name_0">surname:Owen;given-names:D.</infon><infon key="name_1">surname:Campbell;given-names:L. J.</infon><infon key="name_2">surname:Littlefield;given-names:K.</infon><infon key="name_3">surname:Evetts;given-names:K. A.</infon><infon key="name_4">surname:Li;given-names:Z.</infon><infon key="name_5">surname:Sacks;given-names:D. B.</infon><infon key="name_6">surname:Lowe;given-names:P. N.</infon><infon key="name_7">surname:Mott;given-names:H. R.</infon><infon key="pub-id_pmid">17984089</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem</infon><infon key="type">ref</infon><infon key="volume">283</infon><infon key="year">2008</infon><offset>67523</offset><text>The IQGAP1-Rac1 and IQGAP1-Cdc42 interactions: interfaces differ between the complexes</text></passage><passage><infon key="name_0">surname:Watson;given-names:J. R.</infon><infon key="name_1">surname:Nietlispach;given-names:D.</infon><infon key="name_2">surname:Owen;given-names:D.</infon><infon key="name_3">surname:Mott;given-names:H. R.</infon><infon key="pub-id_doi">10.1007/s12104-016-9677-8</infon><infon key="section_type">REF</infon><infon key="source">Biomol. NMR Assign</infon><infon key="type">ref</infon><infon key="year">2016</infon><offset>67610</offset><text>1H, 13C and 15N resonance assignments of the Cdc42-binding domain of TOCA1</text></passage><passage><infon key="fpage">687</infon><infon key="lpage">696</infon><infon key="name_0">surname:Vranken;given-names:W. F.</infon><infon key="name_1">surname:Boucher;given-names:W.</infon><infon key="name_2">surname:Stevens;given-names:T. J.</infon><infon key="name_3">surname:Fogh;given-names:R. H.</infon><infon key="name_4">surname:Pajon;given-names:A.</infon><infon key="name_5">surname:Llinas;given-names:M.</infon><infon key="name_6">surname:Ulrich;given-names:E. L.</infon><infon key="name_7">surname:Markley;given-names:J. L.</infon><infon key="name_8">surname:Ionides;given-names:J.</infon><infon key="name_9">surname:Laue;given-names:E. D.</infon><infon key="pub-id_pmid">15815974</infon><infon key="section_type">REF</infon><infon key="source">Proteins</infon><infon key="type">ref</infon><infon key="volume">59</infon><infon key="year">2005</infon><offset>67685</offset><text>The CCPN data model for NMR spectroscopy: development of a software pipeline</text></passage><passage><infon key="fpage">381</infon><infon key="lpage">382</infon><infon key="name_0">surname:Rieping;given-names:W.</infon><infon key="name_1">surname:Habeck;given-names:M.</infon><infon key="name_2">surname:Bardiaux;given-names:B.</infon><infon key="name_3">surname:Bernard;given-names:A.</infon><infon key="name_4">surname:Malliavin;given-names:T. E.</infon><infon key="name_5">surname:Nilges;given-names:M.</infon><infon key="pub-id_pmid">17121777</infon><infon key="section_type">REF</infon><infon key="source">Bioinformatics</infon><infon key="type">ref</infon><infon key="volume">23</infon><infon key="year">2007</infon><offset>67762</offset><text>ARIA2: automated NOE assignment and data integration in NMR structure calculation</text></passage><passage><infon key="fpage">227</infon><infon key="lpage">241</infon><infon key="name_0">surname:Shen;given-names:Y.</infon><infon key="name_1">surname:Bax;given-names:A.</infon><infon key="pub-id_pmid">23728592</infon><infon key="section_type">REF</infon><infon key="source">J. Biomol. NMR</infon><infon key="type">ref</infon><infon key="volume">56</infon><infon key="year">2013</infon><offset>67844</offset><text>Protein backbone and side chain torsion angles predicted from NMR chemical shifts using artificial neural networks</text></passage><passage><infon key="name_0">surname:Hubbard;given-names:S.</infon><infon key="name_1">surname:Thornton;given-names:J.</infon><infon key="section_type">REF</infon><infon key="source">NACCESS</infon><infon key="type">ref</infon><infon key="year">1993</infon><offset>67959</offset></passage><passage><infon key="fpage">125</infon><infon key="lpage">147</infon><infon key="name_0">surname:Walrant;given-names:A.</infon><infon key="name_1">surname:Saxton;given-names:D. S.</infon><infon key="name_2">surname:Correia;given-names:G. P.</infon><infon key="name_3">surname:Gallop;given-names:J. L.</infon><infon key="pub-id_pmid">25997346</infon><infon key="section_type">REF</infon><infon key="source">Methods Cell Biol</infon><infon key="type">ref</infon><infon key="volume">128</infon><infon key="year">2015</infon><offset>67960</offset><text>Triggering actin polymerization in Xenopus egg extracts from phosphoinositide-containing lipid bilayers</text></passage><passage><infon key="fpage">512</infon><infon key="lpage">524</infon><infon key="name_0">surname:Lebensohn;given-names:A. M.</infon><infon key="name_1">surname:Kirschner;given-names:M. W.</infon><infon key="pub-id_pmid">19917258</infon><infon key="section_type">REF</infon><infon key="source">Mol. Cell</infon><infon key="type">ref</infon><infon key="volume">36</infon><infon key="year">2009</infon><offset>68064</offset><text>Activation of the WAVE complex by coincident signals controls actin assembly</text></passage><passage><infon key="fpage">384</infon><infon key="lpage">388</infon><infon key="name_0">surname:Mott;given-names:H. R.</infon><infon key="name_1">surname:Owen;given-names:D.</infon><infon key="name_2">surname:Nietlispach;given-names:D.</infon><infon key="name_3">surname:Lowe;given-names:P. N.</infon><infon key="name_4">surname:Manser;given-names:E.</infon><infon key="name_5">surname:Lim;given-names:L.</infon><infon key="name_6">surname:Laue;given-names:E. D.</infon><infon key="pub-id_pmid">10360579</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">399</infon><infon key="year">1999</infon><offset>68141</offset><text>Structure of the small G protein Cdc42 bound to the GTPase-binding domain of ACK</text></passage><passage><infon key="fpage">25478</infon><infon key="lpage">25489</infon><infon key="name_0">surname:Nakamura;given-names:K.</infon><infon key="name_1">surname:Man;given-names:Z.</infon><infon key="name_2">surname:Xie;given-names:Y.</infon><infon key="name_3">surname:Hanai;given-names:A.</infon><infon key="name_4">surname:Makyio;given-names:H.</infon><infon key="name_5">surname:Kawasaki;given-names:M.</infon><infon key="name_6">surname:Kato;given-names:R.</infon><infon key="name_7">surname:Shin;given-names:H.-W.</infon><infon key="name_8">surname:Nakayama;given-names:K.</infon><infon key="name_9">surname:Wakatsuki;given-names:S.</infon><infon key="pub-id_pmid">22679020</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem</infon><infon key="type">ref</infon><infon key="volume">287</infon><infon key="year">2012</infon><offset>68222</offset><text>Structural basis for membrane binding specificity of the Bin/Amphiphysin/Rvs (BAR) domain of Arfaptin-2 determined by Arl1 GTPase</text></passage><passage><infon key="fpage">759</infon><infon key="lpage">769</infon><infon key="name_0">surname:ten Klooster;given-names:J. P.</infon><infon key="name_1">surname:Jaffer;given-names:Z. M.</infon><infon key="name_2">surname:Chernoff;given-names:J.</infon><infon key="name_3">surname:Hordijk;given-names:P. L.</infon><infon key="pub-id_pmid">16492808</infon><infon key="section_type">REF</infon><infon key="source">J. Cell Biol</infon><infon key="type">ref</infon><infon key="volume">172</infon><infon key="year">2006</infon><offset>68352</offset><text>Targeting and activation of Rac1 are mediated by the exchange factor β-Pix</text></passage><passage><infon key="fpage">W500</infon><infon key="lpage">W502</infon><infon key="name_0">surname:Heinig;given-names:M.</infon><infon key="name_1">surname:Frishman;given-names:D.</infon><infon key="pub-id_pmid">15215436</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res</infon><infon key="type">ref</infon><infon key="volume">32</infon><infon key="year">2004</infon><offset>68430</offset><text>STRIDE: a web server for secondary structure assignment from known atomic coordinates of proteins</text></passage><passage><infon key="fpage">199</infon><infon key="lpage">211</infon><infon key="name_0">surname:Schanda;given-names:P.</infon><infon key="name_1">surname:Kupce;given-names:E.</infon><infon key="name_2">surname:Brutscher;given-names:B.</infon><infon key="pub-id_pmid">16341750</infon><infon key="section_type">REF</infon><infon key="source">J. Biomol. NMR</infon><infon key="type">ref</infon><infon key="volume">33</infon><infon key="year">2005</infon><offset>68528</offset><text>SOFAST-HMQC experiments for recording two-dimensional heteronuclear correlation spectra of proteins within a few seconds</text></passage><passage><infon key="fpage">985</infon><infon key="lpage">995</infon><infon key="name_0">surname:Fenwick;given-names:R. B.</infon><infon key="name_1">surname:Campbell;given-names:L. J.</infon><infon key="name_2">surname:Rajasekar;given-names:K.</infon><infon key="name_3">surname:Prasannan;given-names:S.</infon><infon key="name_4">surname:Nietlispach;given-names:D.</infon><infon key="name_5">surname:Camonis;given-names:J.</infon><infon key="name_6">surname:Owen;given-names:D.</infon><infon key="name_7">surname:Mott;given-names:H. R.</infon><infon key="pub-id_pmid">20696399</infon><infon key="section_type">REF</infon><infon key="source">Structure</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">2010</infon><offset>68649</offset><text>The RalB-RLIP76 complex reveals a novel mode of Ral-effector interaction</text></passage><passage><infon key="fpage">883</infon><infon key="lpage">897</infon><infon key="name_0">surname:de Vries;given-names:S. J.</infon><infon key="name_1">surname:van Dijk;given-names:M.</infon><infon key="name_2">surname:Bonvin;given-names:A. M. J. J.</infon><infon key="pub-id_pmid">20431534</infon><infon key="section_type">REF</infon><infon key="source">Nat. Protoc</infon><infon key="type">ref</infon><infon key="volume">5</infon><infon key="year">2010</infon><offset>68722</offset><text>The HADDOCK web server for data-driven biomolecular docking</text></passage><passage><infon key="fpage">18067</infon><infon key="lpage">18076</infon><infon key="name_0">surname:Rudolph;given-names:M. G.</infon><infon key="name_1">surname:Bayer;given-names:P.</infon><infon key="name_2">surname:Abo;given-names:A.</infon><infon key="name_3">surname:Kuhlmann;given-names:J.</infon><infon key="name_4">surname:Vetter;given-names:I. R.</infon><infon key="name_5">surname:Wittinghofer;given-names:A.</infon><infon key="pub-id_pmid">9660763</infon><infon key="section_type">REF</infon><infon key="source">J. Biol. Chem</infon><infon key="type">ref</infon><infon key="volume">273</infon><infon key="year">1998</infon><offset>68782</offset><text>The Cdc42/Rac interactive binding region motif of the Wiskott Aldrich syndrome protein (WASP) is necessary but not sufficient for tight binding to Cdc42 and structure formation</text></passage><passage><infon key="fpage">313</infon><infon key="lpage">324</infon><infon key="name_0">surname:Hemsath;given-names:L.</infon><infon key="name_1">surname:Dvorsky;given-names:R.</infon><infon key="name_2">surname:Fiegen;given-names:D.</infon><infon key="name_3">surname:Carlier;given-names:M.-F.</infon><infon key="name_4">surname:Ahmadian;given-names:M. R.</infon><infon key="pub-id_pmid">16246732</infon><infon key="section_type">REF</infon><infon key="source">Mol. Cell</infon><infon key="type">ref</infon><infon key="volume">20</infon><infon key="year">2005</infon><offset>68959</offset><text>An electrostatic steering mechanism of Cdc42 recognition by Wiskott-Aldrich syndrome proteins</text></passage><passage><infon key="fpage">14087</infon><infon key="lpage">14099</infon><infon key="name_0">surname:Elliot-Smith;given-names:A. E.</infon><infon key="name_1">surname:Owen;given-names:D.</infon><infon key="name_2">surname:Mott;given-names:H. R.</infon><infon key="name_3">surname:Lowe;given-names:P. N.</infon><infon key="pub-id_pmid">17999470</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry</infon><infon key="type">ref</infon><infon key="volume">46</infon><infon key="year">2007</infon><offset>69053</offset><text>Double mutant cycle thermodynamic analysis of the hydrophobic Cdc42-ACK protein-protein interaction</text></passage><passage><infon key="fpage">e78</infon><infon key="name_0">surname:Wang;given-names:J.</infon><infon key="name_1">surname:Lu;given-names:Q.</infon><infon key="name_2">surname:Lu;given-names:H. P.</infon><infon key="pub-id_pmid">16839193</infon><infon key="section_type">REF</infon><infon key="source">PLoS Comput. Biol</infon><infon key="type">ref</infon><infon key="volume">2</infon><infon key="year">2006</infon><offset>69153</offset><text>Single-molecule dynamics reveals cooperative binding-folding in protein recognition</text></passage><passage><infon key="fpage">12373</infon><infon key="lpage">12383</infon><infon key="name_0">surname:Elliot-Smith;given-names:A. E.</infon><infon key="name_1">surname:Mott;given-names:H. R.</infon><infon key="name_2">surname:Lowe;given-names:P. N.</infon><infon key="name_3">surname:Laue;given-names:E. D.</infon><infon key="name_4">surname:Owen;given-names:D.</infon><infon key="pub-id_pmid">16156650</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry</infon><infon key="type">ref</infon><infon key="volume">44</infon><infon key="year">2005</infon><offset>69237</offset><text>Specificity determinants on Cdc42 for binding its effector protein ACK</text></passage><passage><infon key="fpage">1233</infon><infon key="lpage">1243</infon><infon key="name_0">surname:Perkins;given-names:J. R.</infon><infon key="name_1">surname:Diboun;given-names:I.</infon><infon key="name_2">surname:Dessailly;given-names:B. H.</infon><infon key="name_3">surname:Lees;given-names:J. G.</infon><infon key="name_4">surname:Orengo;given-names:C.</infon><infon key="pub-id_pmid">20947012</infon><infon key="section_type">REF</infon><infon key="source">Structure</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">2010</infon><offset>69308</offset><text>Transient protein-protein interactions: structural, functional, and network properties</text></passage><passage><infon key="fpage">635</infon><infon key="lpage">648</infon><infon key="name_0">surname:Acuner Ozbabacan;given-names:S. E.</infon><infon key="name_1">surname:Engin;given-names:H. B.</infon><infon key="name_2">surname:Gursoy;given-names:A.</infon><infon key="name_3">surname:Keskin;given-names:O.</infon><infon key="pub-id_pmid">21676899</infon><infon key="section_type">REF</infon><infon key="source">Protein Eng. Des. Sel</infon><infon key="type">ref</infon><infon key="volume">24</infon><infon key="year">2011</infon><offset>69395</offset><text>Transient protein-protein interactions</text></passage><passage><infon key="fpage">883</infon><infon key="lpage">888</infon><infon key="name_0">surname:Schmid;given-names:E. M.</infon><infon key="name_1">surname:McMahon;given-names:H. T.</infon><infon key="pub-id_pmid">17713526</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">448</infon><infon key="year">2007</infon><offset>69434</offset><text>Integrating molecular and network biology to decode endocytosis</text></passage><passage><infon key="fpage">4371</infon><infon key="lpage">4383</infon><infon key="name_0">surname:Praefcke;given-names:G. J. K.</infon><infon key="name_1">surname:Ford;given-names:M. G. J.</infon><infon key="name_2">surname:Schmid;given-names:E. M.</infon><infon key="name_3">surname:Olesen;given-names:L. E.</infon><infon key="name_4">surname:Gallop;given-names:J. L.</infon><infon key="name_5">surname:Peak-Chew;given-names:S.-Y.</infon><infon key="name_6">surname:Vallis;given-names:Y.</infon><infon key="name_7">surname:Babu;given-names:M. M.</infon><infon key="name_8">surname:Mills;given-names:I. G.</infon><infon key="name_9">surname:McMahon;given-names:H. T.</infon><infon key="pub-id_pmid">15496985</infon><infon key="section_type">REF</infon><infon key="source">EMBO J</infon><infon key="type">ref</infon><infon key="volume">23</infon><infon key="year">2004</infon><offset>69498</offset><text>Evolving nature of the AP2 α-appendage hub during clathrin-coated vesicle endocytosis</text></passage><passage><infon key="fpage">519</infon><infon key="lpage">531</infon><infon key="name_0">surname:Höning;given-names:S.</infon><infon key="name_1">surname:Ricotta;given-names:D.</infon><infon key="name_2">surname:Krauss;given-names:M.</infon><infon key="name_3">surname:Späte;given-names:K.</infon><infon key="name_4">surname:Spolaore;given-names:B.</infon><infon key="name_5">surname:Motley;given-names:A.</infon><infon key="name_6">surname:Robinson;given-names:M.</infon><infon key="name_7">surname:Robinson;given-names:C.</infon><infon key="name_8">surname:Haucke;given-names:V.</infon><infon key="name_9">surname:Owen;given-names:D. J.</infon><infon key="pub-id_pmid">15916959</infon><infon key="section_type">REF</infon><infon key="source">Mol. Cell</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">2005</infon><offset>69588</offset><text>Phosphatidylinositol-(4,5)-bisphosphate regulates sorting signal recognition by the clathrin-associated adaptor complex AP2</text></passage><passage><infon key="fpage">5685</infon><infon key="lpage">5690</infon><infon key="name_0">surname:Leung;given-names:D. W.</infon><infon key="name_1">surname:Rosen;given-names:M. K.</infon><infon key="pub-id_pmid">15821030</infon><infon key="section_type">REF</infon><infon key="source">Proc. Natl. Acad. Sci. U.S.A</infon><infon key="type">ref</infon><infon key="volume">102</infon><infon key="year">2005</infon><offset>69712</offset><text>The nucleotide switch in Cdc42 modulates coupling between the GTPase-binding and allosteric equilibria of Wiskott-Aldrich syndrome protein</text></passage><passage><infon key="fpage">271</infon><infon key="lpage">285</infon><infon key="name_0">surname:Buck;given-names:M.</infon><infon key="name_1">surname:Xu;given-names:W.</infon><infon key="name_2">surname:Rosen;given-names:M. K.</infon><infon key="pub-id_pmid">15066431</infon><infon key="section_type">REF</infon><infon key="source">J. Mol. Biol</infon><infon key="type">ref</infon><infon key="volume">338</infon><infon key="year">2004</infon><offset>69851</offset><text>A two-state allosteric model for autoinhibition rationalizes WASP signal integration and targeting</text></passage><passage><infon key="fpage">73</infon><infon key="lpage">78</infon><infon key="name_0">surname:Miki;given-names:H.</infon><infon key="name_1">surname:Takenawa;given-names:T.</infon><infon key="pub-id_pmid">9473482</infon><infon key="section_type">REF</infon><infon key="source">Biochem. Biophys. Res. Commun</infon><infon key="type">ref</infon><infon key="volume">243</infon><infon key="year">1998</infon><offset>69950</offset><text>Direct binding of the verprolin-homology domain in N-WASP to actin is essential for cytoskeletal reorganization</text></passage></document></collection>
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+<collection><source>PMC</source><date>20201220</date><key>pmc.key</key><document><id>4937829</id><infon key="license">NO-CC CODE</infon><passage><infon key="article-id_doi">10.1038/nsmb.3237</infon><infon key="article-id_manuscript">NIHMS785109</infon><infon key="article-id_pmc">4937829</infon><infon key="article-id_pmid">27239796</infon><infon key="fpage">691</infon><infon key="issue">7</infon><infon key="license">Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
+</infon><infon key="lpage">697</infon><infon key="name_0">surname:Horowitz;given-names:Scott</infon><infon key="name_1">surname:Salmon;given-names:Loïc</infon><infon key="name_10">surname:Trievel;given-names:Raymond C.</infon><infon key="name_11">surname:Brooks;given-names:Charles L.;suffix:III</infon><infon key="name_12">surname:Bardwell;given-names:James CA</infon><infon key="name_2">surname:Koldewey;given-names:Philipp</infon><infon key="name_3">surname:Ahlstrom;given-names:Logan S.</infon><infon key="name_4">surname:Martin;given-names:Raoul</infon><infon key="name_5">surname:Quan;given-names:Shu</infon><infon key="name_6">surname:Afonine;given-names:Pavel V.</infon><infon key="name_7">surname:van den Bedem;given-names:Henry</infon><infon key="name_8">surname:Wang;given-names:Lili</infon><infon key="name_9">surname:Xu;given-names:Qingping</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">23</infon><infon key="year">2016</infon><offset>0</offset><text>Visualizing chaperone-assisted protein folding</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>47</offset><text>Challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding, where obtaining structural ensembles of chaperone:substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a novel structural biology approach based on X-ray crystallography, termed Residual Electron and Anomalous Density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy. This study resulted in a series of snapshots depicting the various folding states of Im7 while bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1106</offset><text>High-resolution structural models of protein-protein interactions are critical for obtaining mechanistic insights into biological processes. However, many protein-protein interactions are highly dynamic, making it difficult to obtain high-resolution data. Particularly challenging are interactions of intrinsically or conditionally disordered sections of proteins with their partner proteins. Recent advances in X-ray crystallography and NMR spectroscopy continue to improve our ability to analyze biomolecules that exist in multiple conformations. X-ray crystallography has historically provided valuable information on small-scale conformational changes, but observing large-amplitude heterogeneous conformational changes often falls beyond the reach of current crystallographic techniques. NMR can theoretically be used to determine heterogeneous ensembles, but in practice, this proves to be very challenging.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2020</offset><text>Despite the importance of understanding how proteins fold into their native state within the cell, our knowledge about this critical process remains limited. It is clear that molecular chaperones aid in protein folding. However, exactly how they facilitate the folding process is still being debated. Structural characterization of chaperone-assisted protein folding likely would help bring clarity to this question. Structural models of chaperone-substrate complexes have recently begun to provide information as to how a chaperone can recognize its substrate. However, the impact that chaperones have on their substrates, and how these interactions affect the folding process remain largely unknown. For most chaperones, it is still unclear whether the chaperone actively participates in and affects the folding of the substrate proteins, or merely provides a suitable microenvironment enabling the substrate to fold on its own. This is a truly fundamental question in the chaperone field, and one that has eluded the community largely because of the highly dynamic nature of the chaperone-substrate complexes.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3133</offset><text>To address this question, we investigated the ATP-independent Escherichia coli periplasmic chaperone Spy. Spy prevents protein aggregation and aids in protein folding under various stress conditions, including treatment with tannin and butanol. We originally discovered Spy by its ability to stabilize the protein-folding model Im7 in vivo and recently demonstrated that Im7 folds while associated with Spy. The crystal structure of Spy revealed that it forms a thin α-helical homodimeric cradle. Crosslinking and genetic experiments suggested that Spy interacts with substrates somewhere on its concave side. By using a novel X-ray crystallography-based approach to model disorder in crystal structures, we have now determined the high-resolution ensemble of the dynamic Spy:Im7 complex. This work provides a detailed view of chaperone-mediated protein folding and shows how substrates like Im7 find their native fold while bound to their chaperones.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>4089</offset><text>RESULTS</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>4097</offset><text>Crystallizing the Spy:Im7 complex</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4131</offset><text>We reasoned that to obtain crystals of complexes between Spy (domain boundaries in Supplementary Fig. 1) and its substrate proteins, our best approach was to identify crystallization conditions that yielded Spy crystals in the presence of protein substrates but not in their absence. We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. We found conditions in which all four substrates co-crystallized with Spy, but in which Spy alone did not yield crystals. Subsequent crystal washing and dissolution experiments confirmed the presence of the substrates in the co-crystals (Supplementary Fig. 2). The crystals diffracted to ~1.8 Å resolution. We used Spy:Im76-45 selenomethionine crystals for phasing with single-wavelength anomalous diffraction (SAD) experiments, and used this solution to build the well-ordered Spy portions of all four complexes. However, modeling of the substrate in the complex proved to be a substantial challenge, as the electron density of the substrate was discontinuous and fragmented. Even the minimal binding portion of Im7 (Im76-45) showed highly dispersed electron density (Fig. 1a). We hypothesized that the fragmented density was due to multiple, partially occupied conformations of the substrate bound within the crystal. Such residual density is typically not considered usable by traditional X-ray crystallography methods. Thus, we developed a new approach to interpret the chaperone-bound substrate in multiple conformations.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>5875</offset><text>READ: a strategy to visualize heterogeneous and dynamic biomolecules</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5944</offset><text>To determine the structure of the substrate portion of these Spy:substrate complexes, we conceived of an approach that we term READ, for Residual Electron and Anomalous Density. We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate. (2) We then labeled individual residues in the flexible regions of the substrate with the strong anomalous scatterer iodine, which serves to locate these residues in three-dimensional space using their anomalous density. (3) We performed molecular dynamics (MD) simulations to generate a pool of energetically reasonable conformations of the dynamic complex and (4) applied a sample-and-select algorithm to determine the minimal set of substrate conformations that fit both the residual and anomalous density. (5) Finally, we validated the ensemble using multiple statistical tests. Importantly, even though we only labeled a subset of the residues in the flexible regions of the substrate with iodine, the residual electron density can provide spatial information on many of the other flexible residues. These two forms of data are therefore complementary: by labeling individual residues, one can locate them to specific points in space. The electron density then allowed us to connect the labeled residues of the substrate by confining the protein chain within regions of detectable density. In this way, the two forms of data together were able to describe multiple conformations of the substrate within the crystal. As described in detail below, we developed the READ method to uncover the ensemble of conformations that the Spy-binding domain of Im7 (i.e., Im76-45) adopts while bound to Spy. However, we believe that READ will prove generally applicable to visualizing heterogeneous and dynamic complexes that have previously escaped detailed structural analysis.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>7950</offset><text>Collecting READ data for the Spy:Im76-45 complex</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7999</offset><text>To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3). Moreover, binding experiments indicated that Im76-45 comprises the entire Spy-binding region. To introduce the anomalous scatterer iodine, we replaced eight Im76-45 residues with the non-canonical amino acid 4-iodophenylalanine (pI-Phe). Its strong anomalous scattering allowed us to track the positions of these individual Im76-45 residues one at a time, potentially even if the residue was found in several locations in the same crystal. We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2). Consistent with our electron density map, we found that the majority of anomalous signals emerged in the cradle of Spy, implying that this is the likely Im7 substrate binding site. Consistent with the fragmented density, however, we observed multiple iodine positions for seven of the eight substituted residues. Together, these results indicated that the Im7 substrate binds Spy in multiple conformations.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>9410</offset><text>READ sample-and-select procedure</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9443</offset><text>To determine the structural ensemble that Im76-45 adopts while bound to Spy, we combined the residual electron density and the anomalous signals from our pI-Phe substituted Spy:Im76-45 complexes. To generate an accurate depiction of the chaperone-substrate interactions, we devised a selection protocol based on a sample-and-select procedure employed in NMR spectroscopy. This procedure iteratively constructs structural ensembles and then compares them to the experimental data. During each round of the selection, a genetic algorithm alters the ensemble and its agreement to the experimental data is re-evaluated. If successful, the selection identifies the smallest group of specific conformations that best fits the residual electron density and anomalous signals. The READ sample-and-select algorithm is diagrammed in Fig. 2.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10274</offset><text>Prior to performing the selection, we generated a large and diverse pool of chaperone-substrate complexes using coarse-grained MD simulations in a pseudo-crystal environment (Fig. 2 and Supplementary Fig. 4). The coarse-grained simulations are based on a single-residue resolution model for protein folding and were extended here to describe Spy-Im76-45 binding events (Online Methods). The initial conditions of the binding simulations are not biased toward a particular conformation of the substrate or any specific chaperone-substrate interaction (Online Methods). Im76-45 binds and unbinds to Spy throughout the simulations. This strategy allows a wide range of substrate conformations to interact with the chaperone. From the MD simulations, we extracted ~10,000 diverse Spy:Im76-45 complexes to be used by the ensuing selection. Each complex within this pool comprises one Spy dimer bound to a single Im76-45 substrate. This pool was then used by the selection algorithm to identify the minimal ensemble that best satisfies both the residual electron and anomalous crystallographic data.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11368</offset><text>The anomalous scattering portion of the selection uses our basic knowledge of pI-Phe geometry: the iodine is separated from its respective Cα atom in each coarse-grained conformer by 6.5 Å. The selection then picks ensembles that best reproduce the collection of iodine anomalous signals. Simultaneously, it uses the residual electron density to help choose ensembles. To make the electron density selection practical, we needed to develop a method to rapidly evaluate the agreement between the selected sub-ensembles and the experimental electron density on-the-fly during the selection procedure. To accomplish this task, we generated a compressed version of the experimental 2mFo−DFc electron density map for use in the selection. This process provided us with a target map that the ensuing selection tried to recapitulate. To reduce the extent of 3D space to be explored, this compressed map was created by only using density from regions of space significantly sampled by Im76-45 in the Spy:Im76-45 MD simulations. For each of the ~10,000 complexes in the coarse-grained MD pool, the electron density at the Cα positions of Im76-45 was extracted and used to construct an electron density map (Online Methods). These individual electron density maps from the separate conformers could then be combined (Fig. 2) and compared to the averaged experimental electron density map as part of the selection algorithm.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12790</offset><text>This approach allowed us to simultaneously use both the iodine anomalous signals and the residual electron density in the selection procedure. The selection resulted in small ensembles from the MD pool that best fit the READ data (Fig. 1c,d). Before analyzing the details of the Spy:Im76-45 complex, we first engaged in a series of validation tests to verify the ensemble and selection procedure (Supplementary Note 1, Figures 1c,d, Supplemental Figures 5-7). Combined, these validation tests confirmed that the selection procedure and selected six-member ensemble recapitulate the experimental data. Of note, the final six-membered ensemble was the largest ensemble that could simultaneously decrease the RFree and pass the 10-fold cross-validation test. This ensemble depicts the conformations that the substrate Im76-45 adopts while bound to the chaperone Spy (Fig. 3 Supplementary Movie 1, and Table 1).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13698</offset><text>Folding and interactions of Im7 while bound to Spy</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13749</offset><text>Our results showed that by using this novel READ approach, we were able to obtain structural information about the dynamic interaction of a chaperone with its substrate protein. We were particularly interested in finding answers to one of the most fundamental questions in chaperone biology—how does chaperone binding affect substrate structure and vice versa. By analyzing the individual structures of the six-member ensemble of Im76-45 bound to Spy, we observed that Im76-45 takes on several different conformations while bound. We found these conformations to be highly heterogeneous and to include unfolded, partially folded, and native-like states (Fig. 3). The ensemble primarily encompasses Im76-45 laying diagonally within the Spy cradle in several different orientations, but some conformations traverse as far as the tips or even extend over the side of the cradle (Figs. 3,4a).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14640</offset><text>We constructed a contact map of the complex, which shows the frequency of interactions for chaperone-substrate residue pairs (Fig. 4). We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46). The Spy-contacting residues comprise a mixture of charged, polar, and hydrophobic residues. Surprisingly, we noted that in the ensemble, Im76-45 interacts with only 38% of the hydrophobic residues in the Spy cradle, but interacts with 61% of the hydrophilic residues in the cradle. This mixture suggests the importance of both electrostatic and hydrophobic components in binding the Im76-45 ensemble. With respect to the substrate, we observed that nearly every residue in Im76-45 is in contact with Spy (Fig. 4a). However, we did notice that despite this uniformity, regions of Im76-45 preferentially interact with different regions in Spy (Fig. 4b). For example, the N-terminal half of Im76-45 binds more consistently in the Spy cradle, whereas the C-terminal half predominantly binds to the outer edges of Spy’s concave surface.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15807</offset><text>Not unexpectedly, we found that as Im76-45 progresses from the unfolded to the native state, its interactions with Spy shift accordingly. Whereas the least-folded Im76-45 pose in the ensemble forms the most hydrophobic contacts with Spy (Fig. 3), the two most-folded conformations form the fewest hydrophobic contacts (Fig. 3). This shift in contacts is likely due to hydrophobic residues of Im76-45 preferentially forming intra-molecular contacts upon folding (i.e., hydrophobic collapse), effectively removing themselves from the interaction sites. The diversity of conformations and binding sites observed here emphasizes the dynamic and heterogeneous nature of the chaperone-substrate ensemble. Although we do not yet have time resolution data of these various snapshots of Im76-45, this ensemble illustrates how a substrate samples its folding landscape while bound to a chaperone.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>16694</offset><text>Spy changes conformation upon substrate binding</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16742</offset><text>Comparing the structure of Spy in its substrate-bound and apo states revealed that the Spy dimer also undergoes significant conformational changes upon substrate binding (Fig. 5a and Supplementary Movie 2). Upon substrate binding, the Spy dimer twists 9° about its center relative to its apo form. This twist yields asymmetry and results in substantially different interaction patterns in the two Spy monomers (Fig. 4b). It is possible that this twist serves to increase heterogeneity in Spy by providing more binding poses. Additionally, we observed that the linker region (residues 47–57) of Spy, which participates in substrate interaction, becomes mostly disordered upon binding the substrate. This increased disorder might explain how Spy is able to recognize and bind different substrates and/or differing conformations of the same substrate. Importantly, we observed the same structural changes in Spy regardless of which of the four substrates was bound (Fig. 5b, Table 1). The RMSD between the well-folded sections of Spy in the four chaperone-substrate complexes was very small, less than 0.3 Å. Combined with competition experiments showing that the substrates compete in solution for Spy binding (Fig. 5c and Supplementary Fig. 8), we conclude that all the tested substrates share the same overall Spy binding site.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>18074</offset><text>DISCUSSION</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>18085</offset><text>To shed light on how chaperones interact with their substrates, we developed a novel structural biology method (READ) and applied it to determine a conformational ensemble of the chaperone Spy bound to substrate. As a substrate, we used Im76-45, the chaperone-interacting portion of the protein-folding model protein Im7. In the chaperone-bound ensemble, Im76-45 samples unfolded, partially folded, and native-like states. The ensemble provides an unprecedented description of the conformations that a substrate assumes while exploring its chaperone-associated folding landscape. This substrate-chaperone ensemble helps accomplish the longstanding goal of obtaining a detailed view of how a chaperone aids protein folding.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>18808</offset><text>We recently showed that Im7 can fold while remaining continuously bound to Spy. The high-resolution ensemble obtained here now provides insight into exactly how this occurs. The structures of our ensemble agree well with lower-resolution crosslinking data, which indicate that chaperone-substrate interactions primarily occur on the concave surface of Spy. The ensemble suggests a model in which Spy provides an amphipathic surface that allows substrate proteins to assume different conformations while bound to the chaperone. This model is consistent with previous studies postulating that the flexible binding of chaperones allows for substrate protein folding. The amphipathic concave surface of Spy likely facilitates this flexible binding and may be a crucial feature for Spy and potentially other chaperones, allowing them to bind multiple conformations of many different substrates.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>19698</offset><text>In contrast to Spy’s binding hotspots, Im76-45 displays substantially less specificity in its binding sites. Nearly all Im76-45 residues come in contact with Spy. Unfolded substrate conformers interact with Spy through both hydrophobic and hydrophilic interactions, whereas the binding of native-like states is mainly hydrophilic. This trend suggests that complex formation between an ATP-independent chaperone and its unfolded substrate may initially involve hydrophobic interactions, effectively shielding the exposed aggregation-sensitive hydrophobic regions in the substrate. Once the substrate begins to fold within this protected environment, it progressively buries its own hydrophobic residues, and its interactions with the chaperone shift towards becoming more electrostatic. Notably, the most frequent contacts between Spy and Im76-45 are charge-charge interactions. The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states. Residues Asp32 and Asp35 are close to each other in the folded state of Im7. This proximity likely causes electrostatic repulsion that destabilizes Im7’s native state. Interaction with Spy’s positively-charged residues likely relieves the charge repulsion between Asp32 and Asp35, promoting their compaction into a helical conformation. As inter-molecular hydrophobic interactions between Spy and the substrate become progressively replaced by intra-molecular interactions within the substrate, the affinity between chaperone and substrates could decrease, eventually leading to release of the folded client protein.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>21394</offset><text>Recently, we employed a genetic selection system to improve the chaperone activity of Spy. This selection resulted in “Super Spy” variants that were more effective at both preventing aggregation and promoting protein folding. In conjunction with our bound Im76-45 ensemble, these mutants now allowed us to investigate structural features important to chaperone function. Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both. Our ensemble revealed that two of the Super Spy mutations (H96L and Q100L) form part of the chaperone contact surface that binds to Im76-45 (Fig. 4a). Moreover, our co-structure suggests that the L32P substitution, which increases Spy’s flexibility, could operate by unhinging the N-terminal helix and effectively expanding the size of the disordered linker. This possibility is supported by the Spy:substrate structures, in which the linker region becomes more flexible compared to the apo state (Fig. 6a). This expansion would increase the structural plasticity for substrate binding. By sampling multiple conformations, this linker region may allow diverse substrate conformations to be accommodated.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>22639</offset><text>Other Super Spy mutations (F115I and F115L) caused increased flexibility but not tighter substrate binding. This residue does not directly contact Im76-45 in our READ-derived ensemble. Instead, when Spy is bound to substrate, F115 engages in close CH⋯π hydrogen bonds with Tyr104 (Fig. 6b). This interaction presumably reduces the mobility of the C-terminal helix. The F115I/L substitutions would replace these hydrogen bonds with hydrophobic interactions that have little angular dependence. As a result, the C-terminus, and possibly also the flexible linker, is likely to become more flexible and thus more accommodating of different conformations of substrates. Overall, comparison of our ensemble to the Super Spy variants provides specific examples to corroborate the importance of conformational flexibility in chaperone-substrate interactions.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>23493</offset><text>Despite extensive studies, exactly how complex chaperone machines help proteins fold remains controversial. Our study indicates that the chaperone Spy employs a simple surface binding approach that allows the substrate to explore various conformations and form transiently favorable interactions while being protected from aggregation. We speculate that many other chaperones could utilize a similar strategy. ATP and co-chaperone dependencies may have emerged later through evolution to better modulate and control chaperone action.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>24027</offset><text>In addition to insights into chaperone function, this work presents a new method for determining heterogeneous structural ensembles via a hybrid methodology of X-ray crystallography and computational modeling. Heterogeneous dynamic complexes or disordered regions of single proteins, once considered solely approachable by NMR spectroscopy, can now be visualized through X-ray crystallography. Consequently, this technique could enable structural characterization of many important dynamic and heterogeneous biomolecular systems.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>24557</offset><text>ONLINE METHODS</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24572</offset><text>For computational methods, including simulations of Spy-substrate interactions, binning the residual Im7 electron density, ensemble selection, validation tests, and contact map generation, please see Supplementary Note 1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24794</offset><text>Spy truncation mutants’ construction and in vitro and in vivo activity measurements</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24880</offset><text>To facilitate crystallization, we used Spy 29-124, a truncated Spy version that removes the unstructured N- and C-terminal tails (full length Spy is 138 amino acids). To determine if these alterations impact Spy’s chaperone activity in vitro, we performed in vitro chaperone activity assays and found that they had no significant effect; these deletions also had only a minor effect on Spy’s ability to stabilize Im7 in vivo (Supplementary Fig. 1). The in vitro activity of Spy 29-124 was assessed using the aldolase refolding assay as previously described. Briefly, in the denaturing step, 100 μM aldolase was denatured in buffer containing 6.6 M GdmCl, 40 mM HEPES pH 7.5, and 50 mM NaCl overnight at 22 °C (room temperature). In the refolding step, denatured aldolase was diluted to 3 μM in refolding buffer (40 mM HEPES, 150 mM NaCl, 5 mM DTT pH 7.5) in the presence of 6 μM WT Spy or Spy 29-124 (Spy:aldolase = 2:1). As a control, an identical experiment without Spy added was also performed. The refolding temperature was 37 °C with continuous shaking. The refolding status was monitored at different time points (1 min, 4 min, 10 min, and 20 min) and tested by diluting the refolding sample by 15-fold into the reaction buffer (0.15 mM NADH, 2 mM F1,6-DP, 1.8 U/ml GDH/TPI, 40 mM HEPES, and 150 mM NaCl pH 7.5) at 28 °C. The absorbance was monitored for 1.5 min at 340 nm. The percentage refolding was calculated and averaged over three repeats.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26343</offset><text>To determine the in vivo activity of the Spy mutants, the quantity of the unstable Im7 variant L53A I54A expressed in the periplasm was compared during Spy variant co-expression as previously described. Plasmid Spy (pTrc-spy) was used as the template for the construction of the variant plasmids of Spy for in vivo chaperone activity measurement (Supplementary Table 3). To use the native signal sequence of spy for the periplasmic export of the Spy variants, an NheI site was first introduced between the signal sequence and the mature protein coding region of Spy. The vector was then digested with NheI and BamHI, purified, and ligated with the linear fragments corresponding to truncated sequences (21–130, 24–130, 27–130, 30–130, and 33–130) of Spy.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>27108</offset><text>Cells containing a strain that expressed the unstable Im7 mutant IL53A I54A (pCDFTrc-ssIm7L53A I54A) were transformed with plasmids that expressed either WT or one of the five truncated Spy mutants and grown to mid-log phase in LB medium at 37 °C. Im7 L53A I54A and Spy expression were induced with various concentrations of IPTG for 2 h to compare the in vivo chaperone activity of WT Spy and the truncated Spy mutants at similar expression levels. Periplasmic fractions were prepared as previously described and were separated on 16% Tricine gel (Life Technologies Inc.). The bands corresponding to Spy and the C-terminal His-tagged Im7 were either directly visualized on Coomassie stained gels or determined by western blot using anti-His antibody (Abcam ab1187; validation provided on manufacturer’s website).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>27925</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>27961</offset><text>The gene for spy 29-124 was amplified from plasmid pET28sumo-spy with primer 1 (5′-CGC GGG ATC CTT CAA AGA CCT GAA CCT GAC CG-3′) and primer 2 (5′-CGC GCT CGA GTT ATG TCA GAC GCT TCT CAA AAT TAG C-3′), and was cloned into pET28sumo via BamHI and XhoI sites. The H96L variant was made by Phusion site-directed mutagenesis (New England Biolabs). WT and H96L Spy 29-124 were expressed and purified as described previously with the exception that Ni-HisTrap columns (GE Healthcare) were utilized instead of the Ni-NTA beads and mini-chromatography column. ULP1 cleavage occurred following elution from the Ni-HisTrap column overnight at 4 °C while dialyzing to 40 mM Tris, 300 mM NaCl, pH 8.0. After dialysis, Spy was passed over the HisTrap column to remove the cleaved SUMO tag (20 mM imidazole was left over from the dialysis). Cleavage of the SUMO tag leaves a single serine in position 28 of Spy. The flow-through was then concentrated and diluted 5 times with 20 mM Tris, pH 8 for further purification on a HiTrap Q column. Spy has an isoelectric point of 9.5 and therefore was collected in the flow-through. The flow-through containing Spy was concentrated and diluted 5-fold with 50 mM sodium phosphate at pH 6.5 and passed over a HiTrap SP column. Spy was then eluted with a gradient from 0 M to 1 M NaCl. Re-buffering to the final reaction buffer was accomplished by gel filtration, passing the pooled and concentrated fractions containing Spy over a HiLoad 75 column in 40 mM HEPES, 100 mM NaCl, pH 7.5. Fractions containing Spy were then concentrated, frozen in liquid nitrogen, and stored at −80 °C. WT Im7, Im7 L18A L19A L37A H40W, and Im7 L18A L19A L37A were purified by the same protocol as Spy, but without the SP column step. In addition to WT Im7 and these various Im7 mutants, co-crystallization experiments extensively utilized Im76-45, a minimal Spy-binding segment that encompasses the first two helices of Im7 and contains 46% of the total Im7 sequence. It displays partial helicity when free in solution (Supplementary Fig. 3). The 6-45 portion of Im7 (H2N-SISDYTEAEFVQLLKEIEKENVAATDDVLD VLLEHFVKIT-OH), 4-iodophenlyalanine variants, and a peptide corresponding to a portion of bovine alpha casein S1 148-177 (Ac-ELFRQFYQLDAYPSGAWYYVPLGTQYTDAP-amide) were obtained from New England Peptide at ≥ 95% purity. Anomalous signals for residues E12, E14, L19, and E21 substitutions were determined using a peptide containing Im7 6–26, which was also obtained from New England Peptide at ≥ 95% purity.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>30494</offset><text>Protein crystallization</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>30518</offset><text>Co-crystals of WT Spy 29-124 and Spy H96L 29-124 in complex with Im7 variants and casein were grown by vapor diffusion. 25–130 mg/ml dimer Spy was incubated with various Im7 or casein substrates at concentrations ranging from equimolar to three-fold excess substrate in 22%–33% PEG 3000, 0.88–1.0 M imidazole pH 8.0, and 40–310 mM zinc acetate at 20 °C. Crystals were flash frozen in liquid nitrogen using 35% PEG 3000 as a cryo-protectant. It is worthwhile to note that the flash freezing could somewhat bias the conformations observed in the crystal structure. However, we chose to freeze the crystal to provide us with the maximum capability to identify and interpret the iodine anomalous signals.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>31228</offset><text>Assessing presence of substrate in crystals</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31272</offset><text>Crystals were washed by sequential transfer between three to six 2 μl drops of mother liquor, incubating in each wash solution for 2–10 s in an effort to remove all surface bound and precipitated substrate protein before being dissolved for visualization by SDS-PAGE. Before loading, samples were boiled for 10 min in reducing loading buffer, and then loaded onto 16% Tricine gels. Wash samples and dissolved crystal samples were analyzed by Lumetein staining (Biotium) and Flamingo staining (Bio-Rad) per manufacturer’s instructions, and imaged using a FluorChem M Imager (ProteinSimple).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>31867</offset><text>X-ray crystallography</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31889</offset><text>Data were collected at the LS-CAT beamlines at the Advanced Photon Source at 100 K. SeMet and native Spy:Im76-45 crystals were collected at 12.7 keV and 9.7 keV, respectively. Spy:Casein 148-177, and Spy H96L:WT Im7 crystals were collected also collected at 12.7 keV. Data integration and scaling were performed with iMosflm and AIMLESS, respectively. As molecular replacement attempts using the previously published apo Spy structures (PDB IDs: 3O39 and 3OEO) were unsuccessful, the Spy:Im76-45 complex was solved using Se-SAD phasing with SeMet-Spy, followed by density modification and initial model building by AutoSol in Phenix. The initial model was completed and refined using the native Spy:Im76-45 complex data. The rest of the structures were built using the native Spy:Im76-45 structure as a molecular replacement search model. Refinements, including TLS refinement, were performed using COOT and Phenix. All refined structures were validated using the Molprobity server, with Clashscores ranking better than the 90th percentile for all structures. Structural figures were rendered using PyMOL and UCSF Chimera, and movies generated using UCSF Chimera. Several partially occupied zinc atoms were observed in the crystal structure. Although some of these zinc atoms could also potentially modelled as water molecules, doing so resulted in an increase in the RFree. Additionally, a section of density near His A96 that is potentially partially occupied by a combination of water, Spy linker region, and possibly zinc, was modelled as containing water molecules. Spy H96L:Im76-45 was employed for iodine anomalous scattering experiments due to increased robustness and reproducibility of the crystals.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>33599</offset><text>The expected anomalous scatterers in the structures were S in methionine residues of Spy, Zn from the crystallization buffer, and I in the single pI-Phe residue of each synthetic Im76-45 peptide. Each I site is expected to be partially occupied as Im76-45 had diffuse density corresponding to multiple, partially occupied conformers; the Zn sites also may be partially occupied. To identify I, S, and Zn atomic positions using anomalous scattering, datasets were collected at 6.5 keV and 14.0 keV at 100 K using the ID-D beamline at LS-CAT. Anomalous difference maps for initial anomalous signal screening were calculated with phases from a molecular replacement search using the native Spy:Im76-45 (with no Im76-45 built in) complex as the search model.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34354</offset><text>Anomalous difference maps calculated with the 14.0 keV data were used as controls to distinguish iodine from zinc atoms, as the iodine and zinc anomalous scattering factors are comparable at 14.0 keV, whereas at 6.5 keV, f″ is ~9-fold greater for iodine than for zinc. Anomalous differences were also collected and analyzed for a crystal of WT Spy 29-124:Im76-45 containing no iodine. The resulting anomalous difference map was inspected for peaks corresponding to sulfur, which were then excluded when selecting iodine peaks. Also, peaks that overlapped with Spy in the crystal lattice were excluded from analysis.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34972</offset><text>As an initial screen for placing iodine atoms in the 6.5 keV anomalous difference maps, the median methionine sulfur signal was used as a cutoff for each individual map to control for varying data quality between crystals. Then, all anomalous atoms were refined in Phenix using anomalous group refinement. Refined B-factor of placed iodine ions was then used to estimate the positional fluctuation of the anomalous signals. This positional fluctuation was used as estimated error in the ensuing selection. A summary of all the anomalous signal heights (Supplementary Table 1) and anomalous difference maps (Supplemental Dataset 1) are displayed at varying contour levels for maximum clarity of iodine and methionine peak heights.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>35702</offset><text>Substrate binding to Spy</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35727</offset><text>The dissociation constant of Im76-45 was determined via a fluorescence-based competition experiment with Im7 L18A L19A L37A H40W, and its ability to compete with casein 148-177 for Spy binding was tested. Im7 L18A L19A L37A H40W was chosen for competition experiments due to its tight binding (Supplementary Fig. 8) and substantial fluorescence change upon binding. This mutant binds to Spy tighter than Im7 L18A L19A L37A. 10 μM Spy 29-124 dimer was mixed with 10 μM Im7 L18A L19A L37A H40W or casein 148-177 to form a 1:1 complex in a buffer containing 40 mM HEPES pH 7.5 and 100 mM NaCl at 22 °C. Complex formation was monitored with a QuantaMaster 400 (Photon Technology International) using the tryptophan fluorescence of Im7 L18A L19A L37A H40W. Naturally tryptophan-free Im76-45 was then titrated into the complex to compete with Im7 L18A L19A L37A H40W for Spy binding. The observed fluorescence intensity at 350 nm was plotted as a function of the logarithm of the Im76-45 or casein 148-177 concentration. The data were fit for a one-site-binding competition model (OriginLab 9.1):   where A1 and A2 are the maximum and minimum asymptotes, respectively, and x is the concentration of Im76-45. x0 is the apparent KD for Im76-45 based on its ability to compete with Im7 L18A L19A L37A H40W. Using the KD of Im7 L18A L19A L37A H40W binding to Spy 29-124, we then calculated the KD for Im76-45 binding to Spy 29-124 using the Cheng-Prusoff equation:   where L is the concentration of Im7 L18A L19A L37A H40W and KD is the dissociation constant for Im7 L18A L19A L37A H40W binding to Spy. Due to interaction between higher oligomer states of Im76-45 and casein 148-177 (Supplementary Fig. 8), the competition curve was unable to be fit for casein 148-177 competing with Im76-45.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>37513</offset><text>The stoichiometry of binding of casein 148-177 and Spy was determined by tryptophan fluorescence of the casein upon Spy 29-124 addition. Increasing concentrations of Spy 29-124 were titrated to 20 μM of casein 148-177 in 40 mM HEPES (pH 7.5), 100 mM NaCl, at 22 °C. Complex formation was monitored with a QuantaMaster 400 (Photon Technology International) using the tryptophan fluorescence of casein 148-177. The observed fluorescence intensity at 339 nm was plotted as a function of the Spy 29-124 dimer concentration and fit with a quadratic equation using Origin 9.1 (OriginLab).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38098</offset><text>To determine the dissociation constant, increasing concentrations of Spy 29-124 were titrated to 0.25 μM of casein in 40 mM HEPES (pH 7.5), 100 mM NaCl, at 22 °C. Complex formation was monitored with a QuantaMaster 400 (Photon Technology International) using the tryptophan fluorescence of casein 148-177. The observed fluorescence intensity at 339 nm was corrected for dilution due to the titration and then plotted as a function of the Spy 29-124 dimer concentration. The data were fit using a square hyperbola function in Origin 9.1 (OriginLab):   where F is the recorded fluorescence signal, Fmax is the maximum fluorescence reached upon saturation of the complex, L is the concentration of free Spy in solution, KD is the dissociation constant, and C is a parameter for the offset. The calculated KD is an average of three independent repetitions. The measured dissociation constants for the different substrates ranged from 0.1 to 1 μM.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>39044</offset><text>Isothermal titration calorimetry (ITC)</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>39083</offset><text>Spy 29-124 and Im7-L18A L19A L37A H40W were dialyzed overnight against 40 mM HEPES, 100 mM NaCl, pH 7.5. 165 μM Spy dimer was loaded into a syringe and titrated into a cell containing 15 μM Im7 L18A L19 AL37A H40W at 25 °C in an iTC200 (Malvern Instruments) with an injection interval of 120 s and an initial delay time of 60 s. The solution was stirred at 1000 rpm, and the reference power was set to 6 μcal s−1 in high feedback mode. Data analysis was conducted using a plugin for Origin 7 (OriginLab), the software provided by the manufacturer.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>39637</offset><text>Analytical ultracentrifugation</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>39668</offset><text>Sedimentation velocity experiments for the Im76-45 and the bovine α-S1-casein peptide were performed using a Beckman Proteome Lab XL-I analytical ultracentrifuge (Beckman Coulter). Both peptides were first dialyzed against 40 mM HEPES, 100 mM NaCl, pH 7.5, then diluted to a concentration of 10 μM using the dialysis buffer. Samples were loaded into cells containing standard sector shaped 2-channel Epon centerpieces with 1.2 cm path-length (Beckman Coulter) and equilibrated to 22 °C for at least 1 h prior to sedimentation. All samples were spun at 48,000 rpm in a Beckman AN-50 Ti rotor, and the sedimentation of the protein was monitored continuously using interference optics, since the Im76-45 does not absorb strongly at 280 nm. Data analysis was conducted with SEDFIT (version 14.1), using the continuous c(s) distribution model. The confidence level for the maximum entropy (ME) regularization was set to 0.95. Buffer density and viscosity were calculated using SEDNTERP (http://sednterp.unh.edu/).</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>40682</offset><text>Supplementary Material</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>40705</offset><text>ACCESSION CODES</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>40721</offset><text>Structures and datasets in this work have been deposited in the PDB under the IDs 5INA, 5IOG, 5IOE, and 5IOA.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>40831</offset><text>AUTHOR CONTRIBUTIONS</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>40852</offset><text>Overall concept was conceived by S.H. and J.B. Experiments were designed by S.H., S.Q., J.B., R.T., H.B., and P.K. Experiments were performed by S.H., S.Q., P.K., R.M., and L.W. Analysis and computational modeling was designed by C.B., L.S., P.A., L.A., H.B., and S.H. Computational analysis was carried out by Q.X., S.H., L.S., L.A., P.A., P.K., and R.M. The manuscript was written primarily by S.H. and J.B., with assistance from L.S., L.A. and all other authors.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>41318</offset><text>COMPETING FINANCIAL INTERESTS</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>41348</offset><text>The authors declare no competing financial interests.</text></passage><passage><infon key="fpage">1225</infon><infon key="lpage">44</infon><infon key="name_0">surname:Keskin;given-names:O</infon><infon key="name_1">surname:Gursoy;given-names:A</infon><infon key="name_2">surname:Ma;given-names:B</infon><infon key="name_3">surname:Nussinov;given-names:R</infon><infon key="pub-id_pmid">18355092</infon><infon key="section_type">REF</infon><infon key="source">Chemical Reviews</infon><infon key="type">ref</infon><infon key="volume">108</infon><infon key="year">2008</infon><offset>41402</offset><text>Principles of protein-protein interactions: what are the preferred ways for proteins to interact?</text></passage><passage><infon key="fpage">16247</infon><infon key="lpage">16252</infon><infon key="name_0">surname:Fraser;given-names:JS</infon><infon key="pub-id_pmid">21918110</infon><infon key="section_type">REF</infon><infon key="source">Proceedings of the National Academy of Sciences of the United States of America</infon><infon key="type">ref</infon><infon key="volume">108</infon><infon key="year">2011</infon><offset>41500</offset><text>Accessing protein conformational ensembles using room-temperature X-ray crystallography</text></passage><passage><infon key="fpage">323</infon><infon key="lpage">31</infon><infon key="name_0">surname:Kay;given-names:LE</infon><infon key="pub-id_pmid">26707200</infon><infon key="section_type">REF</infon><infon key="source">J Mol Biol</infon><infon key="type">ref</infon><infon key="volume">428</infon><infon key="year">2016</infon><offset>41588</offset><text>New Views of Functionally Dynamic Proteins by Solution NMR Spectroscopy</text></passage><passage><infon key="fpage">126601</infon><infon key="name_0">surname:Salmon;given-names:L</infon><infon key="name_1">surname:Blackledge;given-names:M</infon><infon key="pub-id_pmid">26517337</infon><infon key="section_type">REF</infon><infon key="source">Rep Prog Phys</infon><infon key="type">ref</infon><infon key="volume">78</infon><infon key="year">2015</infon><offset>41660</offset><text>Investigating protein conformational energy landscapes and atomic resolution dynamics from NMR dipolar couplings: a review</text></passage><passage><infon key="fpage">10960</infon><infon key="lpage">10974</infon><infon key="name_0">surname:Blackledge;given-names:MJ</infon><infon key="pub-id_pmid">8218162</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry</infon><infon key="type">ref</infon><infon key="volume">32</infon><infon key="year">1993</infon><offset>41783</offset><text>Conformational Backbone Dynamics of the Cyclic Decapeptide Antamanide - Application of a New Multiconformational Search Algorithm-Based on Nmr Data</text></passage><passage><infon key="fpage">3181</infon><infon key="lpage">3185</infon><infon key="name_0">surname:Guerry;given-names:P</infon><infon key="section_type">REF</infon><infon key="source">Angewandte Chemie-International Edition</infon><infon key="type">ref</infon><infon key="volume">52</infon><infon key="year">2013</infon><offset>41931</offset><text>Mapping the Population of Protein Conformational Energy Sub-States from NMR Dipolar Couplings</text></passage><passage><infon key="fpage">255</infon><infon key="lpage">76</infon><infon key="name_0">surname:Jewett;given-names:AI</infon><infon key="name_1">surname:Shea;given-names:JE</infon><infon key="pub-id_pmid">19851829</infon><infon key="section_type">REF</infon><infon key="source">Cell Mol Life Sci</infon><infon key="type">ref</infon><infon key="volume">67</infon><infon key="year">2010</infon><offset>42025</offset><text>Reconciling theories of chaperonin accelerated folding with experimental evidence</text></passage><passage><infon key="fpage">98</infon><infon key="lpage">101</infon><infon key="name_0">surname:Mashaghi;given-names:A</infon><infon key="pub-id_pmid">23831649</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">500</infon><infon key="year">2013</infon><offset>42107</offset><text>Reshaping of the conformational search of a protein by the chaperone trigger factor</text></passage><passage><infon key="fpage">3571</infon><infon key="lpage">5</infon><infon key="name_0">surname:Buckle;given-names:AM</infon><infon key="name_1">surname:Zahn;given-names:R</infon><infon key="name_2">surname:Fersht;given-names:AR</infon><infon key="pub-id_pmid">9108017</infon><infon key="section_type">REF</infon><infon key="source">Proc Natl Acad Sci U S A</infon><infon key="type">ref</infon><infon key="volume">94</infon><infon key="year">1997</infon><offset>42191</offset><text>A structural model for GroEL-polypeptide recognition</text></passage><passage><infon key="fpage">923</infon><infon key="lpage">34</infon><infon key="name_0">surname:Martinez-Hackert;given-names:E</infon><infon key="name_1">surname:Hendrickson;given-names:WA</infon><infon key="pub-id_pmid">19737520</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">138</infon><infon key="year">2009</infon><offset>42244</offset><text>Promiscuous substrate recognition in folding and assembly activities of the trigger factor chaperone</text></passage><passage><infon key="fpage">1250494</infon><infon key="name_0">surname:Saio;given-names:T</infon><infon key="name_1">surname:Guan;given-names:X</infon><infon key="name_2">surname:Rossi;given-names:P</infon><infon key="name_3">surname:Economou;given-names:A</infon><infon key="name_4">surname:Kalodimos;given-names:CG</infon><infon key="pub-id_pmid">24812405</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">344</infon><infon key="year">2014</infon><offset>42345</offset><text>Structural basis for protein antiaggregation activity of the trigger factor chaperone</text></passage><passage><infon key="fpage">1042</infon><infon key="lpage">55</infon><infon key="name_0">surname:Joachimiak;given-names:LA</infon><infon key="name_1">surname:Walzthoeni;given-names:T</infon><infon key="name_2">surname:Liu;given-names:CW</infon><infon key="name_3">surname:Aebersold;given-names:R</infon><infon key="name_4">surname:Frydman;given-names:J</infon><infon key="pub-id_pmid">25416944</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">159</infon><infon key="year">2014</infon><offset>42431</offset><text>The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT</text></passage><passage><infon key="fpage">1354</infon><infon key="lpage">65</infon><infon key="name_0">surname:Chen;given-names:DH</infon><infon key="pub-id_pmid">23746846</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">153</infon><infon key="year">2013</infon><offset>42515</offset><text>Visualizing GroEL/ES in the act of encapsulating a folding protein</text></passage><passage><infon key="fpage">963</infon><infon key="lpage">74</infon><infon key="name_0">surname:Karagoz;given-names:GE</infon><infon key="pub-id_pmid">24581495</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">156</infon><infon key="year">2014</infon><offset>42582</offset><text>Hsp90-Tau complex reveals molecular basis for specificity in chaperone action</text></passage><passage><infon key="fpage">3078</infon><infon key="lpage">90</infon><infon key="name_0">surname:Dekker;given-names:C</infon><infon key="pub-id_pmid">21701561</infon><infon key="section_type">REF</infon><infon key="source">EMBO J</infon><infon key="type">ref</infon><infon key="volume">30</infon><infon key="year">2011</infon><offset>42660</offset><text>The crystal structure of yeast CCT reveals intrinsic asymmetry of eukaryotic cytosolic chaperonins</text></passage><passage><infon key="fpage">14</infon><infon key="lpage">9</infon><infon key="name_0">surname:Munoz;given-names:IG</infon><infon key="pub-id_pmid">21151115</infon><infon key="section_type">REF</infon><infon key="source">Nat Struct Mol Biol</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">2011</infon><offset>42759</offset><text>Crystal structure of the open conformation of the mammalian chaperonin CCT in complex with tubulin</text></passage><passage><infon key="fpage">415</infon><infon key="lpage">26</infon><infon key="name_0">surname:Elad;given-names:N</infon><infon key="pub-id_pmid">17499047</infon><infon key="section_type">REF</infon><infon key="source">Mol Cell</infon><infon key="type">ref</infon><infon key="volume">26</infon><infon key="year">2007</infon><offset>42858</offset><text>Topologies of a substrate protein bound to the chaperonin GroEL</text></passage><passage><infon key="fpage">6371</infon><infon key="lpage">6</infon><infon key="name_0">surname:Albert;given-names:A</infon><infon key="pub-id_pmid">20056599</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">285</infon><infon key="year">2010</infon><offset>42922</offset><text>Structure of GroEL in complex with an early folding intermediate of alanine glyoxylate aminotransferase</text></passage><passage><infon key="fpage">262</infon><infon key="lpage">9</infon><infon key="name_0">surname:Quan;given-names:S</infon><infon key="pub-id_pmid">21317898</infon><infon key="section_type">REF</infon><infon key="source">Nat Struct Mol Biol</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">2011</infon><offset>43026</offset><text>Genetic selection designed to stabilize proteins uncovers a chaperone called Spy</text></passage><passage><infon key="fpage">318</infon><infon key="lpage">24</infon><infon key="name_0">surname:Friel;given-names:CT</infon><infon key="name_1">surname:Smith;given-names:DA</infon><infon key="name_2">surname:Vendruscolo;given-names:M</infon><infon key="name_3">surname:Gsponer;given-names:J</infon><infon key="name_4">surname:Radford;given-names:SE</infon><infon key="pub-id_pmid">19252485</infon><infon key="section_type">REF</infon><infon key="source">Nat Struct Mol Biol</infon><infon key="type">ref</infon><infon key="volume">16</infon><infon key="year">2009</infon><offset>43107</offset><text>The mechanism of folding of Im7 reveals competition between functional and kinetic evolutionary constraints</text></passage><passage><infon key="fpage">1722</infon><infon key="lpage">38</infon><infon key="name_0">surname:Figueiredo;given-names:AM</infon><infon key="name_1">surname:Whittaker;given-names:SB</infon><infon key="name_2">surname:Knowling;given-names:SE</infon><infon key="name_3">surname:Radford;given-names:SE</infon><infon key="name_4">surname:Moore;given-names:GR</infon><infon key="pub-id_pmid">24123274</infon><infon key="section_type">REF</infon><infon key="source">Protein Sci</infon><infon key="type">ref</infon><infon key="volume">22</infon><infon key="year">2013</infon><offset>43215</offset><text>Conformational dynamics is more important than helical propensity for the folding of the all alpha-helical protein Im7</text></passage><passage><infon key="fpage">53</infon><infon key="lpage">8</infon><infon key="name_0">surname:Stull;given-names:F</infon><infon key="name_1">surname:Koldewey;given-names:P</infon><infon key="name_2">surname:Humes;given-names:JR</infon><infon key="name_3">surname:Radford;given-names:SE</infon><infon key="name_4">surname:Bardwell;given-names:JC</infon><infon key="pub-id_pmid">26619265</infon><infon key="section_type">REF</infon><infon key="source">Nat Struct Mol Biol</infon><infon key="type">ref</infon><infon key="volume">23</infon><infon key="year">2016</infon><offset>43334</offset><text>Substrate protein folds while it is bound to the ATP-independent chaperone Spy</text></passage><passage><infon key="fpage">2252</infon><infon key="lpage">2259</infon><infon key="name_0">surname:Kwon;given-names:E</infon><infon key="name_1">surname:Kim;given-names:DY</infon><infon key="name_2">surname:Gross;given-names:CA</infon><infon key="name_3">surname:Gross;given-names:JD</infon><infon key="name_4">surname:Kim;given-names:KK</infon><infon key="pub-id_pmid">20799348</infon><infon key="section_type">REF</infon><infon key="source">Protein Science</infon><infon key="type">ref</infon><infon key="volume">19</infon><infon key="year">2010</infon><offset>43413</offset><text>The crystal structure Escherichia coli Spy</text></passage><passage><infon key="fpage">e01584</infon><infon key="name_0">surname:Quan;given-names:S</infon><infon key="pub-id_pmid">24497545</infon><infon key="section_type">REF</infon><infon key="source">Elife</infon><infon key="type">ref</infon><infon key="volume">3</infon><infon key="year">2014</infon><offset>43456</offset><text>Super Spy variants implicate flexibility in chaperone action</text></passage><passage><infon key="fpage">689</infon><infon key="lpage">96</infon><infon key="name_0">surname:Creamer;given-names:LK</infon><infon key="name_1">surname:Richardson;given-names:T</infon><infon key="name_2">surname:Parry;given-names:DA</infon><infon key="pub-id_pmid">7305393</infon><infon key="section_type">REF</infon><infon key="source">Arch Biochem Biophys</infon><infon key="type">ref</infon><infon key="volume">211</infon><infon key="year">1981</infon><offset>43517</offset><text>Secondary structure of bovine alpha s1- and beta-casein in solution</text></passage><passage><infon key="fpage">6437</infon><infon key="lpage">6442</infon><infon key="name_0">surname:Chak;given-names:KF</infon><infon key="name_1">surname:Safo;given-names:MK</infon><infon key="name_2">surname:Ku;given-names:WY</infon><infon key="name_3">surname:Hsieh;given-names:SY</infon><infon key="name_4">surname:Yuan;given-names:HS</infon><infon key="section_type">REF</infon><infon key="source">Proceedings of the National Academy of Sciences</infon><infon key="type">ref</infon><infon key="volume">93</infon><infon key="year">1996</infon><offset>43585</offset><text>The crystal structure of the immunity protein of colicin E7 suggests a possible colicin-interacting surface</text></passage><passage><infon key="fpage">300</infon><infon key="lpage">318</infon><infon key="name_0">surname:Pashley;given-names:CL</infon><infon key="pub-id_pmid">22226836</infon><infon key="section_type">REF</infon><infon key="source">Journal of Molecular Biology</infon><infon key="type">ref</infon><infon key="volume">416</infon><infon key="year">2012</infon><offset>43693</offset><text>Conformational Properties of the Unfolded State of Im7 in Nondenaturing Conditions</text></passage><passage><infon key="fpage">72</infon><infon key="lpage">77</infon><infon key="name_0">surname:Burling;given-names:FT</infon><infon key="name_1">surname:Weis;given-names:WI</infon><infon key="name_2">surname:Flaherty;given-names:KM</infon><infon key="name_3">surname:Brunger;given-names:AT</infon><infon key="pub-id_pmid">8539602</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">271</infon><infon key="year">1996</infon><offset>43776</offset><text>Direct observation of protein solvation and discrete disorder with experimental crystallographic phases</text></passage><passage><infon key="fpage">1107</infon><infon key="lpage">1117</infon><infon key="name_0">surname:van den Bedem;given-names:H</infon><infon key="name_1">surname:Dhanik;given-names:A</infon><infon key="name_2">surname:Latombe;given-names:JC</infon><infon key="name_3">surname:Deacon;given-names:AM</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallographica Section D-Biological Crystallography</infon><infon key="type">ref</infon><infon key="volume">65</infon><infon key="year">2009</infon><offset>43880</offset><text>Modeling discrete heterogeneity in X-ray diffraction data by fitting multi-conformers</text></passage><passage><infon key="comment">Under Review</infon><infon key="name_0">surname:Salmon;given-names:L</infon><infon key="section_type">REF</infon><infon key="source">Journal of the American Chemical Society</infon><infon key="type">ref</infon><infon key="year">2015</infon><offset>43966</offset><text>Capturing a dynamic chaperone-substrate interaction using NMR-informed molecular modeling</text></passage><passage><infon key="fpage">850</infon><infon key="lpage">853</infon><infon key="name_0">surname:Brennan;given-names:S</infon><infon key="name_1">surname:Cowan;given-names:PL</infon><infon key="section_type">REF</infon><infon key="source">Review of Scientific Instruments</infon><infon key="type">ref</infon><infon key="volume">63</infon><infon key="year">1992</infon><offset>44056</offset><text>A Suite of Programs for Calculating X-Ray Absorption, Reflection, and Diffraction Performance for a Variety of Materials at Arbitrary Wavelengths</text></passage><passage><infon key="fpage">2351</infon><infon key="lpage">2361</infon><infon key="name_0">surname:Karanicolas;given-names:J</infon><infon key="name_1">surname:Brooks;given-names:CL;suffix:III</infon><infon key="pub-id_pmid">12237457</infon><infon key="section_type">REF</infon><infon key="source">Protein Science</infon><infon key="type">ref</infon><infon key="volume">11</infon><infon key="year">2002</infon><offset>44202</offset><text>The origins of asymmetry in the folding transition states of protein L and protein G</text></passage><passage><infon key="fpage">945</infon><infon key="lpage">957</infon><infon key="name_0">surname:Jewett;given-names:AI</infon><infon key="name_1">surname:Shea;given-names:JE</infon><infon key="pub-id_pmid">16987526</infon><infon key="section_type">REF</infon><infon key="source">Journal of Molecular Biology</infon><infon key="type">ref</infon><infon key="volume">363</infon><infon key="year">2006</infon><offset>44287</offset><text>Folding on the chaperone: Yield enhancement through loose binding</text></passage><passage><infon key="fpage">517</infon><infon key="lpage">25</infon><infon key="name_0">surname:Bardwell;given-names:JC</infon><infon key="name_1">surname:Jakob;given-names:U</infon><infon key="pub-id_pmid">23018052</infon><infon key="section_type">REF</infon><infon key="source">Trends Biochem Sci</infon><infon key="type">ref</infon><infon key="volume">37</infon><infon key="year">2012</infon><offset>44353</offset><text>Conditional disorder in chaperone action</text></passage><passage><infon key="fpage">359</infon><infon key="lpage">66</infon><infon key="name_0">surname:Quan;given-names:S</infon><infon key="name_1">surname:Hiniker;given-names:A</infon><infon key="name_2">surname:Collet;given-names:JF</infon><infon key="name_3">surname:Bardwell;given-names:JC</infon><infon key="pub-id_pmid">23299746</infon><infon key="section_type">REF</infon><infon key="source">Methods Mol Biol</infon><infon key="type">ref</infon><infon key="volume">966</infon><infon key="year">2013</infon><offset>44394</offset><text>Isolation of bacteria envelope proteins</text></passage><passage><infon key="fpage">1560</infon><infon key="lpage">4</infon><infon key="name_0">surname:Fischer;given-names:M</infon><infon key="name_1">surname:Shoichet;given-names:BK</infon><infon key="name_2">surname:Fraser;given-names:JS</infon><infon key="pub-id_pmid">26032594</infon><infon key="section_type">REF</infon><infon key="source">Chembiochem</infon><infon key="type">ref</infon><infon key="volume">16</infon><infon key="year">2015</infon><offset>44434</offset><text>One Crystal, Two Temperatures: Cryocooling Penalties Alter Ligand Binding to Transient Protein Sites</text></passage><passage><infon key="fpage">271</infon><infon key="lpage">81</infon><infon key="name_0">surname:Battye;given-names:TG</infon><infon key="name_1">surname:Kontogiannis;given-names:L</infon><infon key="name_2">surname:Johnson;given-names:O</infon><infon key="name_3">surname:Powell;given-names:HR</infon><infon key="name_4">surname:Leslie;given-names:AG</infon><infon key="pub-id_pmid">21460445</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">67</infon><infon key="year">2011</infon><offset>44535</offset><text>iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM</text></passage><passage><infon key="fpage">235</infon><infon key="lpage">42</infon><infon key="name_0">surname:Winn;given-names:MD</infon><infon key="pub-id_pmid">21460441</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">67</infon><infon key="year">2011</infon><offset>44615</offset><text>Overview of the CCP4 suite and current developments</text></passage><passage><infon key="fpage">213</infon><infon key="lpage">221</infon><infon key="name_0">surname:Adams;given-names:PD</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallographica Section D-Biological Crystallography</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>44667</offset><text>PHENIX: a comprehensive Python-based system for macromolecular structure solution</text></passage><passage><infon key="fpage">486</infon><infon key="lpage">501</infon><infon key="name_0">surname:Emsley;given-names:P</infon><infon key="name_1">surname:Lohkamp;given-names:B</infon><infon key="name_2">surname:Scott;given-names:WG</infon><infon key="name_3">surname:Cowtan;given-names:K</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallographica Section D-Biological Crystallography</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>44749</offset><text>Features and development of Coot</text></passage><passage><infon key="fpage">12</infon><infon key="lpage">21</infon><infon key="name_0">surname:Chen;given-names:VB</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallographica Section D-Biological Crystallography</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>44782</offset><text>MolProbity: all-atom structure validation for macromolecular crystallography</text></passage><passage><infon key="section_type">REF</infon><infon key="source">The PyMOL Molecular Graphics System, Version 1.3r1</infon><infon key="type">ref</infon><infon key="year">2010</infon><offset>44859</offset></passage><passage><infon key="fpage">1605</infon><infon key="lpage">1612</infon><infon key="name_0">surname:Pettersen;given-names:EF</infon><infon key="pub-id_pmid">15264254</infon><infon key="section_type">REF</infon><infon key="source">Journal of Computational Chemistry</infon><infon key="type">ref</infon><infon key="volume">25</infon><infon key="year">2004</infon><offset>44860</offset><text>UCSF chimera - A visualization system for exploratory research and analysis</text></passage><passage><infon key="fpage">352</infon><infon key="lpage">367</infon><infon key="name_0">surname:Afonine;given-names:PV</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallographica Section D-Biological Crystallography</infon><infon key="type">ref</infon><infon key="volume">D68</infon><infon key="year">2012</infon><offset>44936</offset><text>Towards automated crystallographic structure refinement with phenix.refine</text></passage><passage><infon key="fpage">1606</infon><infon key="lpage">1619</infon><infon key="name_0">surname:Schuck;given-names:P</infon><infon key="pub-id_pmid">10692345</infon><infon key="section_type">REF</infon><infon key="source">Biophysical Journal</infon><infon key="type">ref</infon><infon key="volume">78</infon><infon key="year">2000</infon><offset>45011</offset><text>Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling</text></passage><passage><infon key="file">nihms785109f1.jpg</infon><infon key="id">F1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>45129</offset><text>Crystallographic data and ensemble selection. (a) 2mFo−DFc omit map of residual Im76-45 and flexible linker electron density contoured at 0.5 σ. This is the residual density that is used in the READ selection. (b) Composites of iodine positions detected from anomalous signals using pI-Phe substitutions, colored and numbered by sequence. Multiple iodine positions were detected for most residues. Agreement to the residual Im76-45 electron density (c) and anomalous iodine signals (d) for ensembles of varying size generated by randomly choosing from the MD pool (blue) and from the selection procedure (black). The agreement from back-calculating a subset of data excluded from the selection procedure is shown by the red curve (cross-validation). The cost function, χ2, decreases as the agreement to the experimental data increases and is defined in the Online Methods.</text></passage><passage><infon key="file">nihms785109f2.jpg</infon><infon key="id">F2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>46008</offset><text>Flowchart of the READ sample-and-select process.</text></passage><passage><infon key="file">nihms785109f3.jpg</infon><infon key="id">F3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>46057</offset><text>Spy:Im76-45 ensemble, arranged by RMSD to native state of Im76-45. Although the six-membered ensemble from the READ selection should be considered only as an ensemble, for clarity, the individual conformers are shown separately here. Spy is depicted as a gray surface and the Im76-45 conformer is shown as orange balls. Atoms that were either not directly selected in the READ procedure, or whose position could not be justified based on agreement with the residual electron density were removed, leading to non-contiguous sections. Dashed lines connect non-contiguous segments of the Im76-45 substrate. Residues of the Spy flexible linker region that fit the residual electron density are shown as larger gray spheres. Shown below each ensemble member is the RMSD of each conformer to the native state of Im76-45, as well as the percentage of contacts between Im76-45 and Spy that are hydrophobic.</text></passage><passage><infon key="file">nihms785109f4.jpg</infon><infon key="id">F4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>46956</offset><text>Contact maps of Spy:Im76-45 complex. (a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures. For clarity, Im76-45 is represented with a single conformation. The frequency plotted is calculated as the average contact frequency from Spy to every residue of Im76-45 and vice-versa. As the residues involved in contacts are more evenly distributed in Im76-45 compared to Spy, its contact map was amplified. (b) Detailed contact maps of Spy:Im76-45. Contacts to the two Spy monomers are depicted separately. Note that the flexible linker region of Spy (residues 47–57) is not represented in the 2D contact maps.</text></passage><passage><infon key="file">nihms785109f5.jpg</infon><infon key="id">F5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>47612</offset><text>Spy conformation changes upon substrate binding. (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). Error bars depict standard deviations of n=3 technical replicates.</text></passage><passage><infon key="file">nihms785109f6.jpg</infon><infon key="id">F6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>48143</offset><text>Flexibility of Spy linker region and effect of Super Spy mutants. (a) The Spy linker region adopts one dominant conformation in its apo state (PDB ID 3039, gray), but expands and adopts multiple conformations in bound states (green). (b) F115 and L32 tether Spy’s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility. The Super Spy mutants F115L, F115I, and L32P are proposed to gain activity by increasing the flexibility or size of this linker region. L32, F115, and Y104 are rendered in purple to illustrate residues that are most affected by Super Spy mutations; CH⋯π hydrogen bonds are depicted by orange dashes.</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>48804</offset><text>Crystallography Statistics</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;void&quot; rules=&quot;none&quot;&gt;&lt;thead&gt;&lt;tr&gt;&lt;th valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;th valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SeMet Spy:Im7&lt;sub&gt;6-45&lt;/sub&gt;&lt;/th&gt;&lt;th valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Spy:Im7&lt;sub&gt;6-45&lt;/sub&gt;&lt;/th&gt;&lt;th valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Spy:Casein 148-177, substrate not modeled&lt;/th&gt;&lt;th valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Spy H96L:Im7 L18A L19A L37A, substrate not modeled&lt;/th&gt;&lt;th valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Spy H96L:WT Im7, substrate not modeled&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PDB ID&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5INA&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5IOG&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5IOE&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5IOA&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot; align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot;&gt;&lt;bold&gt;Data collection&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Space group&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;P4&lt;sub&gt;1&lt;/sub&gt;22&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;P4&lt;sub&gt;1&lt;/sub&gt;22&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;P4&lt;sub&gt;1&lt;/sub&gt;22&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;P4&lt;sub&gt;1&lt;/sub&gt;22&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;P4&lt;sub&gt;1&lt;/sub&gt;22&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot; align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot;&gt;Cell dimensions&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt; &lt;italic&gt;a&lt;/italic&gt;, &lt;italic&gt;b&lt;/italic&gt;, &lt;italic&gt;c&lt;/italic&gt; (Å)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;42.9, 42.9, 259.3&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;42.9, 42.9, 260.2&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;43.0, 43.0, 258.2&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;43.1, 43.1, 258.7&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;43.1, 43.14, 260.2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt; &lt;italic&gt;α&lt;/italic&gt;, &lt;italic&gt;β&lt;/italic&gt;, &lt;italic&gt;γ&lt;/italic&gt; (°)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90, 90, 90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution (Å)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;64.82–2.44(2.53–2.44)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;30.50–1.79(1.83–1.79)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;36.88–1.77 (1.80–1.77)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;30.48–1.87(1.91–1.87)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;33.21–1.87(1.91–1.87)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;merge&lt;/sub&gt; (%)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;10.6(36)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;8.2(108)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.2(134)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;8.4(152)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;9.6(249)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;I&lt;/italic&gt;/σ(&lt;italic&gt;I)&lt;/italic&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;15.1(6.8)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;7.0(1.1)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;15.3(1.6)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;13.8(1.8)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;13.2(1.3)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Completeness (%)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100(100)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;94.0(90.1)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.9(99.5)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100(100)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;96.8(93.1)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Redundancy&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;15.6(15.6)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4.3(4.2)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;8.7(8.2)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;9.6(9.4)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;8.2(8.2)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;CC1/2&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.998(0.689)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.999(0.745)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.999(0.676)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.998(0.606)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot; align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot;&gt;&lt;bold&gt;Refinement&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution (Å)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.79&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.77&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.87&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.87&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;No. of Reflections&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;22583&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;25052&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;21505&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;20838&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;work/&lt;/sub&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.22/0.23&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.21/0.24&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.22/0.24&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.21/0.25&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;No. of Atoms&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1765&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1669&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1715&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1653&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt; Protein&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1586&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1493&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1541&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1444&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt; Ligand/ion&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;30&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;56&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;60&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;30&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt; Water&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;149&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;120&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;114&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;179&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;B-factors&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;49.4&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;48.5&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;47.4&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;39.2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt; Protein&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;49.0&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;47.5&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;46.3&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;38.3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt; Ligand/ion&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;48.6&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;65.9&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;80.4&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;62.9&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt; Water&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;54.2&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;51.9&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;44.5&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;42.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;r.m.s. Deviations&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt; Bond lengths (Å)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.013&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.013&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.013&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.014&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt; Bond angles (º)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.24&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.30&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.24&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.39&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>48831</offset><text>	SeMet Spy:Im76-45	Spy:Im76-45	Spy:Casein 148-177, substrate not modeled	Spy H96L:Im7 L18A L19A L37A, substrate not modeled	Spy H96L:WT Im7, substrate not modeled	 	PDB ID		5INA	5IOG	5IOE	5IOA	 	Data collection	 	Space group	P4122	P4122	P4122	P4122	P4122	 	Cell dimensions	 	 a, b, c (Å)	42.9, 42.9, 259.3	42.9, 42.9, 260.2	43.0, 43.0, 258.2	43.1, 43.1, 258.7	43.1, 43.14, 260.2	 	 α, β, γ (°)	90, 90, 90	90, 90, 90	90, 90, 90	90, 90, 90	90, 90, 90	 	Resolution (Å)	64.82–2.44(2.53–2.44)	30.50–1.79(1.83–1.79)	36.88–1.77 (1.80–1.77)	30.48–1.87(1.91–1.87)	33.21–1.87(1.91–1.87)	 	Rmerge (%)	10.6(36)	8.2(108)	6.2(134)	8.4(152)	9.6(249)	 	I/σ(I)	15.1(6.8)	7.0(1.1)	15.3(1.6)	13.8(1.8)	13.2(1.3)	 	Completeness (%)	100(100)	94.0(90.1)	99.9(99.5)	100(100)	96.8(93.1)	 	Redundancy	15.6(15.6)	4.3(4.2)	8.7(8.2)	9.6(9.4)	8.2(8.2)	 	CC1/2		0.998(0.689)	0.999(0.745)	0.999(0.676)	0.998(0.606)	 	Refinement	 	Resolution (Å)		1.79	1.77	1.87	1.87	 	No. of Reflections		22583	25052	21505	20838	 	Rwork/Rfree		0.22/0.23	0.21/0.24	0.22/0.24	0.21/0.25	 	No. of Atoms		1765	1669	1715	1653	 	 Protein		1586	1493	1541	1444	 	 Ligand/ion		30	56	60	30	 	 Water		149	120	114	179	 	B-factors		49.4	48.5	47.4	39.2	 	 Protein		49.0	47.5	46.3	38.3	 	 Ligand/ion		48.6	65.9	80.4	62.9	 	 Water		54.2	51.9	44.5	42.1	 	r.m.s. Deviations						 	 Bond lengths (Å)		0.013	0.013	0.013	0.014	 	 Bond angles (º)		1.24	1.30	1.24	1.39	 	</text></passage></document></collection>
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+<collection><source>PMC</source><date>20201216</date><key>pmc.key</key><document><id>4968113</id><infon key="license">CC BY-NC</infon><passage><infon key="alt-title">A. TEPLYAKOV ET AL. MABS</infon><infon key="article-id_doi">10.1080/19420862.2016.1190060</infon><infon key="article-id_pmc">4968113</infon><infon key="article-id_pmid">27210805</infon><infon key="article-id_publisher-id">1190060</infon><infon key="fpage">1045</infon><infon key="issue">6</infon><infon key="kwd">Antibody structure CDR canonical structure CDR H3 phage library VH:VL packing</infon><infon key="license">This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.</infon><infon key="lpage">1063</infon><infon key="name_0">surname:Teplyakov;given-names:Alexey</infon><infon key="name_1">surname:Obmolova;given-names:Galina</infon><infon key="name_2">surname:Malia;given-names:Thomas J.</infon><infon key="name_3">surname:Luo;given-names:Jinquan</infon><infon key="name_4">surname:Muzammil;given-names:Salman</infon><infon key="name_5">surname:Sweet;given-names:Raymond</infon><infon key="name_6">surname:Almagro;given-names:Juan Carlos</infon><infon key="name_7">surname:Gilliland;given-names:Gary L.</infon><infon key="section_type">TITLE</infon><infon key="title">KEYWORDS</infon><infon key="type">front</infon><infon key="volume">8</infon><infon key="year">2016</infon><offset>0</offset><text>Structural diversity in a human antibody germline library</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>58</offset><text>ABSTRACT</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>67</offset><text>To support antibody therapeutic development, the crystal structures of a set of 16 germline variants composed of 4 different kappa light chains paired with 4 different heavy chains have been determined. All four heavy chains of the antigen-binding fragments (Fabs) have the same complementarity-determining region (CDR) H3 that was reported in an earlier Fab structure. The structure analyses include comparisons of the overall structures, canonical structures of the CDRs and the VH:VL packing interactions. The CDR conformations for the most part are tightly clustered, especially for the ones with shorter lengths. The longer CDRs with tandem glycines or serines have more conformational diversity than the others. CDR H3, despite having the same amino acid sequence, exhibits the largest conformational diversity. About half of the structures have CDR H3 conformations similar to that of the parent; the others diverge significantly. One conclusion is that the CDR H3 conformations are influenced by both their amino acid sequence and their structural environment determined by the heavy and light chain pairing. The stem regions of 14 of the variant pairs are in the ‘kinked’ conformation, and only 2 are in the extended conformation. The packing of the VH and VL domains is consistent with our knowledge of antibody structure, and the tilt angles between these domains cover a range of 11 degrees. Two of 16 structures showed particularly large variations in the tilt angles when compared with the other pairings. The structures and their analyses provide a rich foundation for future antibody modeling and engineering efforts.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1705</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1718</offset><text>At present, therapeutic antibodies are the largest class of biotherapeutic proteins that are in clinical trials. The use of monoclonal antibodies as therapeutics began in the early 1980s, and their composition has transitioned from murine antibodies to generally less immunogenic humanized and human antibodies. The technologies currently used to obtain human antibodies include transgenic mice containing human antibody repertoires, cloning directly from human B cells, and in vitro selection from antibody libraries using various display technologies. Once a candidate antibody is identified, protein engineering is usually required to produce a molecule with the right biophysical and functional properties. All engineering efforts are guided by our understanding of the atomic structures of antibodies. In such efforts, the crystal structure of the specific antibody may not be available, but modeling can be used to guide the engineering efforts. Today's antibody modeling approaches, which normally focus on the variable region, are being developed by the application of structural principles and insights that are evolving as our knowledge of antibody structures continues to expand.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2909</offset><text>Our current structural knowledge of antibodies is based on a multitude of studies that used many techniques to gain insight into the functional and structural properties of this class of macromolecule. Five different antibody isotypes occur, IgG, IgD, IgE, IgA and IgM, and each isotype has a unique role in the adaptive immune system. IgG, IgD and IgE isotypes are composed of 2 heavy chains (HCs) and 2 light chains (LCs) linked through disulfide bonds, while IgA and IgM are double and quintuple versions of antibodies, respectively. Isotypes IgG, IgD and IgA each have 4 domains, one variable (V) and 3 constant (C) domains, while IgE and IgM each have the same 4 domains along with an additional C domain. These multimeric forms are linked with an additional J chain. The LCs that associate with the HCs are divided into 2 functionally indistinguishable classes, κ and λ. Both κ and λ polypeptide chains are composed of a single V domain and a single C domain.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3893</offset><text>The heavy and light chains are composed of structural domains that have ∼110 amino acid residues. These domains have a common folding pattern often referred to as the “immunoglobulin fold,” formed by the packing together of 2 anti-parallel β-sheets. All immunoglobulin chains have an N-terminal V domain followed by 1 to 4 C domains, depending upon the chain type. In antibodies, the heavy and light chain V domains pack together forming the antigen combining site. This site, which interacts with the antigen (or target), is the focus of current antibody modeling efforts. This interaction site is composed of 6 complementarity-determining regions (CDRs) that were identified in early antibody amino acid sequence analyses to be hypervariable in nature, and thus are responsible for the sequence and structural diversity of our antibody repertoire.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4750</offset><text>The sequence diversity of the CDR regions presents a substantial challenge to antibody modeling. However, an initial structural analysis of the combining sites of the small set of structures of immunoglobulin fragments available in the 1980s found that 5 of the 6 hypervariable loops or CDRs had canonical structures (a limited set of main-chain conformations). A CDR canonical structure is defined by its length and conserved residues located in the hypervariable loop and framework residues (V-region residues that are not part of the CDRs). Furthermore, studies of antibody sequences revealed that the total number of canonical structures are limited for each CDR, indicating possibly that antigen recognition may be affected by structural restrictions at the antigen-binding site. Later studies found that the CDR loop length is the primary determining factor of antigen-binding site topography because it is the primary factor for determining a canonical structure. Additional efforts have led to our current understanding that the LC CDRs L1, L2, and L3 have preferred sets of canonical structures based on length and amino acid sequence composition. This was also found to be the case for the H1 and H2 CDRs. Classification schemes for the canonical structures of these 5 CDRs have emerged and evolved as the number of depositions in the Protein Data Bank of Fab fragments of antibodies grow. Recently, a comprehensive CDR classification scheme was reported identifying 72 clusters of conformations observed in antibody structures. The knowledge and predictability of these CDR canonical structures have greatly advanced antibody modeling efforts.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6405</offset><text>In contrast to CDRs L1, L2, L3, H1 and H2, no canonical structures have been observed for CDR H3, which is the most variable in length and amino acid sequence. Some clustering of conformations was observed for the shortest lengths; however, for the longer loops, only the portions nearest the framework (torso, stem or anchor region) were found to have defined conformations. In the torso region, 2 primary groups could be identified, which led to sequence-based rules that can predict with some degree of reliability the conformation of the stem region. The “kinked” or “bulged” conformation is the most prevalent, but an “extended” or “non-bulged” conformation is also, but less frequently, observed. The cataloging and development of the rules for predicting the conformation of the anchor region of CDR H3 continue to be refined, producing new insight into the CDR H3 conformations and new tools for antibody engineering.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>7347</offset><text>Current antibody modeling approaches take advantage of the most recent advances in homology modeling, the evolving understanding of the CDR canonical structures, the emerging rules for CDR H3 modeling and the growing body of antibody structural data available from the PDB. Recent antibody modeling assessments show continued improvement in the quality of the models being generated by a variety of modeling methods. Although antibody modeling is improving, the latest assessment revealed a number of challenges that need to be overcome to provide accurate 3-dimensional models of antibody V regions, including accuracies in the modeling of CDR H3. The need for improvement in this area was also highlighted in a recent study reporting an approach and results that may influence future antibody modeling efforts. One important finding of the antibody modeling assessments was that errors in the structural templates that are used as the basis for homology models can propagate into the final models, producing inaccuracies that may negatively influence the predictive nature of the V region model.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>8445</offset><text>To support antibody engineering and therapeutic development efforts, a phage library was designed and constructed based on a limited number of scaffolds built with frequently used human germ-line IGV and IGJ gene segments that encode antigen combining sites suitable for recognition of peptides and proteins. This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). Selection of these genes was based on the high frequency of their use and their cognate canonical structures that were found binding to peptides and proteins, as well as their ability to be expressed in bacteria and displayed on filamentous phage. The implementation of the library involves the diversification of the human germline genes to mimic that found in natural human libraries.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>9350</offset><text>The crystal structure determinations and structural analyses of all germline Fabs in the library described above along with the structures of a fourth HC germline, IGHV3-53 (H3-53), paired with the 4 LCs of the library have been carried out to support antibody therapeutic development. All 16 HCs of the Fabs have the same CDR H3 that was reported in an earlier Fab structure. This is the first systematic study of the same VH and VL structures in the context of different pairings. The structure analyses include comparisons of the overall structures, canonical structures of the L1, L2, L3, H1 and H2 CDRs, the structures of all CDR H3s, and the VH:VL packing interactions. The structures and their analyses provide a foundation for future antibody engineering and structure determination efforts.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>10150</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10158</offset><text>Crystal structures</text></passage><passage><infon key="file">d37e422.xml</infon><infon key="id">t0001</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>10177</offset><text>Crystal data, X-ray data, and refinement statistics.</text></passage><passage><infon key="file">d37e422.xml</infon><infon key="id">t0001</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;colgroup&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;/colgroup&gt;&lt;thead&gt;&lt;tr&gt;&lt;th align=&quot;left&quot;&gt;Fab&lt;hr/&gt;&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;H1-69:L1-39&lt;hr/&gt;&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;H1-69:L3-11&lt;hr/&gt;&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;H1-69:L3-20&lt;hr/&gt;&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;H1-69:L4-1&lt;hr/&gt;&lt;/th&gt;&lt;/tr&gt;&lt;tr&gt;&lt;th align=&quot;left&quot;&gt;&lt;italic&gt;PDB identifier&lt;/italic&gt;&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;5I15&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;5I16&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;5I17&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;5I18&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Crystal Data&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Crystallization Solution&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Buffer, pH&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.1 M MES- pH 6.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.1 M MES pH 6.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.1 M MES, pH 6.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.1 M HEPES, pH 7.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Precipitant&lt;xref rid=&quot;t1fn0001&quot; ref-type=&quot;fn&quot;&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5 M Na Formate&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;25% PEG 3350&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2.0 M Amm Sulfate&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;10% PEG 8000&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Additive&lt;xref rid=&quot;t1fn0001&quot; ref-type=&quot;fn&quot;&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.2 M Na Formate&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5% MPD&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;8% EG&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Space Group&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;P3&lt;sub&gt;1&lt;/sub&gt;21&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;C2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;P422&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;P42&lt;sub&gt;1&lt;/sub&gt;2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Molecules/AU&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Unit Cell&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; a(Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;129.2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;212.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;152.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;120.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; b(Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;129.2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;55.1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;152.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;120.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; c(Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;91.8&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;80.3&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;123.4&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;64.2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; β(°)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;90.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;97.8&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;90.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;90.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; γ(°)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;120.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;90.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;90.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;90.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; V&lt;sub&gt;m&lt;/sub&gt; (Å&lt;sup&gt;3&lt;/sup&gt;/Da)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;4.67&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2.44&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;3.77&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2.39&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Solvent Content (%)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;74&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;50&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;67&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;48&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;X-Ray Data&lt;xref rid=&quot;t1fn0002&quot; ref-type=&quot;fn&quot;&gt;&lt;sup&gt;2&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Resolution (Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;30-2.6 (2.7-2.6)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;30.0-1.9 (1.95-1.9)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;30.0-3.3 (3.4-3.3)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;30-1.9 (2.0-1.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Measured Reflections&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;136,745 (8,650)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;241,145 (16,580)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;237,504 (15,007)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;801,080 (19,309)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Unique Reflections&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;27,349 (1,730)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;71,932 (5,198)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;22,379 (1,590)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;35,965 (2,194)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Completeness (%)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;99.3 (98.7)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;99.0 (97.3)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;99.5 (96.8)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;98.5 (82.8)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Redundancy&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5.0 (5.0)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;3.4 (3.2)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;10.6 (9.4)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;22.3 (8.8)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; R&lt;sub&gt;merge&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.048 (0.522)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.044 (0.245)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.086 (0.536)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.093 (0.231)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; &amp;lt; I/σ &amp;gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;21.2 (3.9)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;17.8 (4.7)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;25.5 (4.5)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;29.2 (8.1)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; B-factor (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;60.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;33.2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;61.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;19.6&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Refinement&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Resolution (Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;15-2.6&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;15-1.9&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;15-3.3&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;15-1.9&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Number of Reflections&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;26,238&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;70,346&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;21,197&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;34,850&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Number of All Atoms&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;3,224&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;6,975&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;6,398&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;3,695&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Number of Waters&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;472&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;399&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; R-factor (%)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;20.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;19.2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;20.2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;16.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; R-free (%)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;24.1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;22.2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;24.7&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;21.3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;RMSD&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Bond Lengths (Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.006&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.005&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.005&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.008&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Bond Angles (°)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1.2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1.1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Mean B-factor (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;65.3&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;34.4&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;80.1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;20.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Ramachandran Plot (%)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Outliers&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.9&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Favored&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;92.3&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;96.9&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;93.1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;96.9&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>10230</offset><text>Fab	H1-69:L1-39	H1-69:L3-11	H1-69:L3-20	H1-69:L4-1	 	PDB identifier	5I15	5I16	5I17	5I18	 	Crystal Data	 	 	 	 	 	Crystallization Solution	 	 	 	 	 	 Buffer, pH	0.1 M MES- pH 6.5	0.1 M MES pH 6.5	0.1 M MES, pH 6.5	0.1 M HEPES, pH 7.5	 	 Precipitant1	5 M Na Formate	25% PEG 3350	2.0 M Amm Sulfate	10% PEG 8000	 	 Additive1	 	0.2 M Na Formate	5% MPD	8% EG	 	 Space Group	P3121	C2	P422	P4212	 	 Molecules/AU	1	2	2	1	 	 Unit Cell	 	 	 	 	 	 a(Å)	129.2	212.0	152.5	120.0	 	 b(Å)	129.2	55.1	152.5	120.0	 	 c(Å)	91.8	80.3	123.4	64.2	 	 β(°)	90.0	97.8	90.0	90.0	 	 γ(°)	120.0	90.0	90.0	90.0	 	 Vm (Å3/Da)	4.67	2.44	3.77	2.39	 	 Solvent Content (%)	74	50	67	48	 	X-Ray Data2	 	 	 	 	 	 Resolution (Å)	30-2.6 (2.7-2.6)	30.0-1.9 (1.95-1.9)	30.0-3.3 (3.4-3.3)	30-1.9 (2.0-1.9)	 	 Measured Reflections	136,745 (8,650)	241,145 (16,580)	237,504 (15,007)	801,080 (19,309)	 	 Unique Reflections	27,349 (1,730)	71,932 (5,198)	22,379 (1,590)	35,965 (2,194)	 	 Completeness (%)	99.3 (98.7)	99.0 (97.3)	99.5 (96.8)	98.5 (82.8)	 	 Redundancy	5.0 (5.0)	3.4 (3.2)	10.6 (9.4)	22.3 (8.8)	 	 Rmerge	0.048 (0.522)	0.044 (0.245)	0.086 (0.536)	0.093 (0.231)	 	 &lt; I/σ &gt;	21.2 (3.9)	17.8 (4.7)	25.5 (4.5)	29.2 (8.1)	 	 B-factor (Å2)	60.5	33.2	61.0	19.6	 	Refinement	 	 	 	 	 	 Resolution (Å)	15-2.6	15-1.9	15-3.3	15-1.9	 	 Number of Reflections	26,238	70,346	21,197	34,850	 	 Number of All Atoms	3,224	6,975	6,398	3,695	 	 Number of Waters	2	472	0	399	 	 R-factor (%)	20.5	19.2	20.2	16.7	 	 R-free (%)	24.1	22.2	24.7	21.3	 	RMSD	 	 	 	 	 	 Bond Lengths (Å)	0.006	0.005	0.005	0.008	 	 Bond Angles (°)	1.2	1.1	1.0	1.1	 	 Mean B-factor (Å2)	65.3	34.4	80.1	20.0	 	Ramachandran Plot (%)	 	 	 	 	 	 Outliers	0.0	0.0	0.9	0.0	 	 Favored	92.3	96.9	93.1	96.9	 	</text></passage><passage><infon key="file">d37e422.xml</infon><infon key="id">t0001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>12072</offset><text>Abbreviations: Amm, ammonium;EG, ethylene glycol; PEG, polyethylene glycol.</text></passage><passage><infon key="file">d37e422.xml</infon><infon key="id">t0001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>12148</offset><text>Values for high-resolution shell are in parentheses.</text></passage><passage><infon key="file">d37e892.xml</infon><infon key="id">t0001</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>12201</offset><text>(Continued) Crystal data, X-ray data, and refinement statistics.</text></passage><passage><infon key="file">d37e892.xml</infon><infon key="id">t0001</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;colgroup&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;/colgroup&gt;&lt;thead&gt;&lt;tr&gt;&lt;th align=&quot;left&quot;&gt;Fab&lt;hr/&gt;&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;H3-23:L1-39&lt;hr/&gt;&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;H3-23:L3-11&lt;hr/&gt;&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;H3-23:L3-20&lt;hr/&gt;&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;H3-23:L4-1&lt;hr/&gt;&lt;/th&gt;&lt;/tr&gt;&lt;tr&gt;&lt;th align=&quot;left&quot;&gt;&lt;italic&gt; PDB identifier&lt;/italic&gt;&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;5I19&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;5I1A&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;5I1C&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;5I1D&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Crystal Data&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Crystallization Solution&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Buffer, pH&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;No Buffer&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.1 M Na Acetate, pH 4.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.1 M MES, pH 6.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.1 M HEPES, pH 7.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Precipitant&lt;xref rid=&quot;t1fn0003&quot; ref-type=&quot;fn&quot;&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;20% PEG 3350&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2.0 M Amm Sulfate&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;16% PEG 3350&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2.0 M Amm Sulfate&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Additive&lt;xref rid=&quot;t1fn0003&quot; ref-type=&quot;fn&quot;&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.2 M Li Citrate&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5% PEG 400&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.2 M Amm Acetate&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2% PEG 400&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Space Group&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;P4&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;P2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;P6&lt;sub&gt;2&lt;/sub&gt;22&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;P2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Molecules/AU&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Unit Cell&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; a(Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;96.6&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;60.9&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;121.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;62.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; b(Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;96.6&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;110.6&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;121.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;111.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; c(Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;105.4&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;158.9&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;160.4&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;160.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; β(°)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;90&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;90&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;90&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; V&lt;sub&gt;m&lt;/sub&gt; (Å&lt;sup&gt;3&lt;/sup&gt;/Da)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2.60&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2.82&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;3.60&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2.90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Solvent Content (%)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;53&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;56&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;66&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;57&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;X-Ray Data&lt;xref rid=&quot;t1fn0004&quot; ref-type=&quot;fn&quot;&gt;&lt;sup&gt;2&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Resolution (Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;30-2.8 (2.9-2.8)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;30-2.0 (2.1-2.0)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;30-2.25 (2.3-2.25)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;30-2.0 (2.1-2.0)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Measured Reflections&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;177,681 (12,072)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;351,312 (8,634)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;887,349 (59,919)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;873,523 (49,118)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Unique Reflections&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;12,678 (899)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;58,989 (2,870)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;32,572 (2,300)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;75,540 (5,343)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Completeness (%)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;99.5 (97.4)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;80.9 (54.2)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;96.9 (94.8)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;99.7 (96.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Redundancy&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;14.0 (13.4)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;6.0 (3.0)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;27.2 (26.1)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;11.6 (9.2)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; R&lt;sub&gt;merge&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.091 (0.594)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.066 (0.204)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.086 (0.478)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.094 (0.488)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; &amp;lt; I/σ &amp;gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;31.2 (5.1)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;20.4 (4.6)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;37.0 (10.4)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;21.6 (5.0)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; B-factor (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;42.8&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;27.1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;33.7&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;29.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Refinement&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Resolution (Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;15-2.8&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;15-2.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;15-2.25&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;15-2.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Number of Reflections&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;11,972&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;57,599&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;31,411&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;74,238&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Number of All Atoms&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;3,234&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;6,948&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;3,472&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;7,210&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Number of Waters&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;416&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;222&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;635&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; R-factor (%)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;23.9&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;20.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;22.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;21.6&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; R-free (%)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;31.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;25.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;26.6&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;25.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;RMSD&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Bond Lengths (Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.009&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.010&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.005&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.008&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Bond Angles (°)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1.3&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1.3&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Mean B-factor (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;48.4&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;36.7&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;47.7&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;46.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Ramachandran Plot (%)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Outliers&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Favored&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;92.3&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;96.8&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;97.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;97.6&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>12266</offset><text>Fab	H3-23:L1-39	H3-23:L3-11	H3-23:L3-20	H3-23:L4-1	 	 PDB identifier	5I19	5I1A	5I1C	5I1D	 	Crystal Data	 	 	 	 	 	Crystallization Solution	 	 	 	 	 	 Buffer, pH	No Buffer	0.1 M Na Acetate, pH 4.5	0.1 M MES, pH 6.5	0.1 M HEPES, pH 7.5	 	 Precipitant1	20% PEG 3350	2.0 M Amm Sulfate	16% PEG 3350	2.0 M Amm Sulfate	 	 Additive1	0.2 M Li Citrate	5% PEG 400	0.2 M Amm Acetate	2% PEG 400	 	 Space Group	P41212	P212121	P6222	P212121	 	 Molecules/AU	1	2	1	2	 	Unit Cell	 	 	 	 	 	 a(Å)	96.6	60.9	121.5	62.7	 	 b(Å)	96.6	110.6	121.5	111.0	 	 c(Å)	105.4	158.9	160.4	160.0	 	 β(°)	90	90	90	90	 	 Vm (Å3/Da)	2.60	2.82	3.60	2.90	 	 Solvent Content (%)	53	56	66	57	 	X-Ray Data2	 	 	 	 	 	 Resolution (Å)	30-2.8 (2.9-2.8)	30-2.0 (2.1-2.0)	30-2.25 (2.3-2.25)	30-2.0 (2.1-2.0)	 	 Measured Reflections	177,681 (12,072)	351,312 (8,634)	887,349 (59,919)	873,523 (49,118)	 	 Unique Reflections	12,678 (899)	58,989 (2,870)	32,572 (2,300)	75,540 (5,343)	 	 Completeness (%)	99.5 (97.4)	80.9 (54.2)	96.9 (94.8)	99.7 (96.9)	 	 Redundancy	14.0 (13.4)	6.0 (3.0)	27.2 (26.1)	11.6 (9.2)	 	 Rmerge	0.091 (0.594)	0.066 (0.204)	0.086 (0.478)	0.094 (0.488)	 	 &lt; I/σ &gt;	31.2 (5.1)	20.4 (4.6)	37.0 (10.4)	21.6 (5.0)	 	 B-factor (Å2)	42.8	27.1	33.7	29.4	 	Refinement	 	 	 	 	 	 Resolution (Å)	15-2.8	15-2.0	15-2.25	15-2.0	 	 Number of Reflections	11,972	57,599	31,411	74,238	 	 Number of All Atoms	3,234	6,948	3,472	7,210	 	 Number of Waters	0	416	222	635	 	 R-factor (%)	23.9	20.5	22.0	21.6	 	 R-free (%)	31.5	25.5	26.6	25.1	 	RMSD	 	 	 	 	 	 Bond Lengths (Å)	0.009	0.010	0.005	0.008	 	 Bond Angles (°)	1.3	1.3	1.0	1.1	 	 Mean B-factor (Å2)	48.4	36.7	47.7	46.4	 	Ramachandran Plot (%)	 	 	 	 	 	 Outliers	0.0	0.0	0.0	0.0	 	 Favored	92.3	96.8	97.5	97.6	 	</text></passage><passage><infon key="file">d37e892.xml</infon><infon key="id">t0001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>14105</offset><text>Abbreviations: Amm, ammonium; PEG, polyethylene glycol.</text></passage><passage><infon key="file">d37e892.xml</infon><infon key="id">t0001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>14161</offset><text>Values for high-resolution shell are in parentheses.</text></passage><passage><infon key="file">d37e1371.xml</infon><infon key="id">t0001</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>14214</offset><text>(Continued) Crystal data, X-ray data, and refinement statistics.</text></passage><passage><infon key="file">d37e1371.xml</infon><infon key="id">t0001</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;colgroup&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;/colgroup&gt;&lt;thead&gt;&lt;tr&gt;&lt;th align=&quot;left&quot;&gt;Fab&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;H3-53:L1-39&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;H3-53:L3-11&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;H3-53:L3-20&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;H3-53:L4-1&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;italic&gt;PDB indentifier&lt;/italic&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1E&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1G&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1H&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1I&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Crystal Data&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Crystallization Solution&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Buffer, pH&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;No buffer&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.1 M Na Acetate pH 4.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.1 M Na Acetate pH 4.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.1M MES, pH 6.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Precipitant&lt;xref rid=&quot;t1fn0005&quot; ref-type=&quot;fn&quot;&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;16% PEG 3350&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;25% PEG 3350&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;19% PEG 4000&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;17% PEG 3350&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Additive&lt;xref rid=&quot;t1fn0005&quot; ref-type=&quot;fn&quot;&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.2 M Amm Sulfate 5% Dioxane&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.2 M Li&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.2 M Amm Sulfate&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.2 M Na Formate, 5% MPD&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Space Group&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;P6&lt;sub&gt;5&lt;/sub&gt;22&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;P6&lt;sub&gt;5&lt;/sub&gt;22&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;P6&lt;sub&gt;5&lt;/sub&gt;22&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;P3&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Molecules/AU&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Unit Cell&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; a(Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;89.4&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;88.1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;89.4&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;68.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; b(Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;89.4&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;88.1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;89.4&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;68.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; c(Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;212.4&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;219.6&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;211.7&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;95.6&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; β(°)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;90&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;90&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;90&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; γ(°)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;120&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;120&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;120&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;120&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; V&lt;sub&gt;m&lt;/sub&gt; (Å&lt;sup&gt;3&lt;/sup&gt;/Da)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2.57&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2.64&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2.57&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2.64&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Solvent Content (%)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;52&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;53&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;52&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;53&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;X-Ray Data&lt;xref rid=&quot;t1fn0006&quot; ref-type=&quot;fn&quot;&gt;&lt;sup&gt;2&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Resolution (Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;30-2.7 (2.8-2.7)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;30-2.3 (2.4-2.3)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;30-2.2 (2.3-2.0)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;30-2.5 (2.6-2.5)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Measured Reflections&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;297,367 (19,369)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;333,739 (8,008)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;381,125 (1,591)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;137,992 (9,883)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Unique Reflections&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;14,402 (1,003)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;21,683 (1,135)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;24,323 (964)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;16,727 (1,227)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Completeness (%)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;99.6 (96.8)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;93.8 (68.4)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;95.3 (52.0)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;98.6 (98.1)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Redundancy&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;20.6 (19.3)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;15.4 (7.1)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;15.7 (1.7)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;8.2 (8.1)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; R&lt;sub&gt;merge&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.095 (0.451)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.057 (0.324)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.062 (0.406)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.047 (0.445)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; &amp;lt; I/σ &amp;gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;38.3 (8.1)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;36.7 (5.5)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;36.2 (1.6)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;31.6 (5.6)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; B-factor (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;33.2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;37.3&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;33.7&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;54.8&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Refinement&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Resolution (Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;15-2.7&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;15-2.3&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;15-2.2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;15-2.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Number of Reflections&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;13,583&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;20,255&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;24,962&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;15,811&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Number of All Atoms&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;3,335&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;3,271&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;3,298&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;3,239&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Number of Waters&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;88&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;70&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;71&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;21&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; R-factor (%)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;19.1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;29.8&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;22.8&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;25.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; R-free (%)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;26.4&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;38.3&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;26.6&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;33.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;RMSD&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Bond Lengths (Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.008&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.005&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.005&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.006&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Bond Angles (°)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1.2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Mean B-factor (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;49.1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;46.3&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;51.7&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;88.9&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Ramachandran Plot (%)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Outliers&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1.2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Favored&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;96.7&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;97.1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;96.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;90.9&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>14279</offset><text>Fab	H3-53:L1-39	H3-53:L3-11	H3-53:L3-20	H3-53:L4-1	 	PDB indentifier	5I1E	5I1G	5I1H	5I1I	 	Crystal Data	 	 	 	 	 	Crystallization Solution	 	 	 	 	 	 Buffer, pH	No buffer	0.1 M Na Acetate pH 4.5	0.1 M Na Acetate pH 4.5	0.1M MES, pH 6.5	 	 Precipitant1	16% PEG 3350	25% PEG 3350	19% PEG 4000	17% PEG 3350	 	 Additive1	0.2 M Amm Sulfate 5% Dioxane	0.2 M Li2SO4	0.2 M Amm Sulfate	0.2 M Na Formate, 5% MPD	 	 Space Group	P6522	P6522	P6522	P31	 	 Molecules/AU	1	1	1	1	 	Unit Cell	 	 	 	 	 	 a(Å)	89.4	88.1	89.4	68.1	 	 b(Å)	89.4	88.1	89.4	68.1	 	 c(Å)	212.4	219.6	211.7	95.6	 	 β(°)	90	90	90	90	 	 γ(°)	120	120	120	120	 	 Vm (Å3/Da)	2.57	2.64	2.57	2.64	 	 Solvent Content (%)	52	53	52	53	 	X-Ray Data2	 	 	 	 	 	 Resolution (Å)	30-2.7 (2.8-2.7)	30-2.3 (2.4-2.3)	30-2.2 (2.3-2.0)	30-2.5 (2.6-2.5)	 	 Measured Reflections	297,367 (19,369)	333,739 (8,008)	381,125 (1,591)	137,992 (9,883)	 	 Unique Reflections	14,402 (1,003)	21,683 (1,135)	24,323 (964)	16,727 (1,227)	 	 Completeness (%)	99.6 (96.8)	93.8 (68.4)	95.3 (52.0)	98.6 (98.1)	 	 Redundancy	20.6 (19.3)	15.4 (7.1)	15.7 (1.7)	8.2 (8.1)	 	 Rmerge	0.095 (0.451)	0.057 (0.324)	0.062 (0.406)	0.047 (0.445)	 	 &lt; I/σ &gt;	38.3 (8.1)	36.7 (5.5)	36.2 (1.6)	31.6 (5.6)	 	 B-factor (Å2)	33.2	37.3	33.7	54.8	 	Refinement	 	 	 	 	 	 Resolution (Å)	15-2.7	15-2.3	15-2.2	15-2.5	 	 Number of Reflections	13,583	20,255	24,962	15,811	 	 Number of All Atoms	3,335	3,271	3,298	3,239	 	 Number of Waters	88	70	71	21	 	 R-factor (%)	19.1	29.8	22.8	25.0	 	 R-free (%)	26.4	38.3	26.6	33.7	 	RMSD	 	 	 	 	 	 Bond Lengths (Å)	0.008	0.005	0.005	0.006	 	 Bond Angles (°)	1.2	1.0	1.0	1.1	 	 Mean B-factor (Å2)	49.1	46.3	51.7	88.9	 	Ramachandran Plot (%)	 	 	 	 	 	 Outliers	0.2	0.2	0.2	1.2	 	 Favored	96.7	97.1	96.5	90.9	 	</text></passage><passage><infon key="file">d37e1371.xml</infon><infon key="id">t0001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>16142</offset><text>Abbreviations: Amm, ammonium; PEG, polyethylene glycol.</text></passage><passage><infon key="file">d37e1371.xml</infon><infon key="id">t0001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>16198</offset><text>Values for high-resolution shell are in parentheses.</text></passage><passage><infon key="file">d37e1852.xml</infon><infon key="id">t0001</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>16251</offset><text>(Continued) Crystal data, X-ray data, and refinement statistics.</text></passage><passage><infon key="file">d37e1852.xml</infon><infon key="id">t0001</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;colgroup&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;/colgroup&gt;&lt;thead&gt;&lt;tr&gt;&lt;th align=&quot;left&quot;&gt;Fab&lt;hr/&gt;&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;H5-51:L1-39&lt;hr/&gt;&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;H5-51:L3-11&lt;hr/&gt;&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;H5-51:L3-20&lt;hr/&gt;&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;H5-51:L4-1&lt;hr/&gt;&lt;/th&gt;&lt;/tr&gt;&lt;tr&gt;&lt;th align=&quot;left&quot;&gt;&lt;italic&gt; PDB identifier&lt;/italic&gt;&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;4KMT&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;5I1J&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;5I1K&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;5I1L&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Crystal Data&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Crystallization Solution&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Buffer, pH&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.1 M CHES, pH 9.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.1 M Tris, pH 8.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.1 M CHES, pH 9.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.1 M Tris, pH 8.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Precipitant&lt;xref rid=&quot;t1fn0007&quot; ref-type=&quot;fn&quot;&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1.8 M Amm Sulfate&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;25% PEG 3350&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1.0 M Amm Sulfate&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;24% PEG 3350&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Additive&lt;xref rid=&quot;t1fn0007&quot; ref-type=&quot;fn&quot;&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5% dioxane&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.2 M MgCl&lt;sub&gt;2&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.2 M Amm Sulfate&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Space Group&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;P2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;P2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;P2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;P2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Molecules/AU&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Unit Cell&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; a(Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;63.7&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;64.1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;63.8&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;106.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; b(Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;73.8&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;73.8&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;74.1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;38.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; c(Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;103.1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;103.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;103.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;112.3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; β(°)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;90&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;90&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;90&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;100.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; V&lt;sub&gt;m&lt;/sub&gt; (Å&lt;sup&gt;3&lt;/sup&gt;/Da)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2.53&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2.56&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2.54&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;2.28&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Solvent Content (%)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;51&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;52&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;51&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;46&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;X-Ray Data&lt;xref rid=&quot;t1fn0008&quot; ref-type=&quot;fn&quot;&gt;&lt;sup&gt;2&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Resolution (Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;30-2.1 (2.2-2.1)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;30-2.5 (2.6-2.5)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;30-1.65 (1.7-1.65)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;30-1.95 (2.0-1.95)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Measured Reflections&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;131,839 (6,655)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;120,521 (7,988)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;246,750 (4,142)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;320,324 (12,119)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Unique Reflections&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;27,026 (1,885)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;17,286 (1,236)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;53,058 (2,141)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;61,554 (3,243)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Completeness (%)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;93.6 (89.8)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;99.7 (97.3)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;89.8 (49.8)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;94.4 (67.1)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Redundancy&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;4.9 (3.5)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;7.0 (6.5)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;4.7 (1.9)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5.2 (3.7)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; R&lt;sub&gt;merge&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.079 (0.278)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.080 (0.281)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.034 (0.131)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.060 (0.395)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; &amp;lt; I/σ &amp;gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;16.8 (5.7)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;21.1(6.9)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;27.5 (5.8)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;19.7 (3.1)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; B-factor (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;26.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;27.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;21.6&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;31.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Refinement&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Resolution (Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;15-2.1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;15-2.5&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;15-1.65&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;15-1.95&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Number of Reflections&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;25,857&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;16,328&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;51,882&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;60,181&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Number of All Atoms&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;3,676&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;3,454&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;3,814&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;7,175&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Number of Waters&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;302&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;196&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;527&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;445&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; R-factor (%)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;17.1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;17.7&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;17.2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;19.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; R-free (%)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;22.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;25.8&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;19.7&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;25.8&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;RMSD&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Bond Lengths (Å)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.006&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.009&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.005&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.009&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Bond Angles (°)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1.3&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1.3&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;1.3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Mean B-factor (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;25.2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;38.2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;20.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;19.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;Ramachandran Plot (%)&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Outliers&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.0&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; Favored&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;98.4&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;97.9&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;98.1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;98.0&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>16316</offset><text>Fab	H5-51:L1-39	H5-51:L3-11	H5-51:L3-20	H5-51:L4-1	 	 PDB identifier	4KMT	5I1J	5I1K	5I1L	 	Crystal Data	 	 	 	 	 	Crystallization Solution	 	 	 	 	 	 Buffer, pH	0.1 M CHES, pH 9.5	0.1 M Tris, pH 8.5	0.1 M CHES, pH 9.5	0.1 M Tris, pH 8.5	 	 Precipitant1	1.8 M Amm Sulfate	25% PEG 3350	1.0 M Amm Sulfate	24% PEG 3350	 	 Additive1	5% dioxane	0.2 M MgCl2	 	0.2 M Amm Sulfate	 	 Space Group	P212121	P212121	P212121	P21	 	 Molecules/AU	1	1	1	2	 	Unit Cell	 	 	 	 	 	 a(Å)	63.7	64.1	63.8	106.0	 	 b(Å)	73.8	73.8	74.1	38.0	 	 c(Å)	103.1	103.0	103.0	112.3	 	 β(°)	90	90	90	100.4	 	 Vm (Å3/Da)	2.53	2.56	2.54	2.28	 	 Solvent Content (%)	51	52	51	46	 	X-Ray Data2	 	 	 	 	 	 Resolution (Å)	30-2.1 (2.2-2.1)	30-2.5 (2.6-2.5)	30-1.65 (1.7-1.65)	30-1.95 (2.0-1.95)	 	 Measured Reflections	131,839 (6,655)	120,521 (7,988)	246,750 (4,142)	320,324 (12,119)	 	 Unique Reflections	27,026 (1,885)	17,286 (1,236)	53,058 (2,141)	61,554 (3,243)	 	 Completeness (%)	93.6 (89.8)	99.7 (97.3)	89.8 (49.8)	94.4 (67.1)	 	 Redundancy	4.9 (3.5)	7.0 (6.5)	4.7 (1.9)	5.2 (3.7)	 	 Rmerge	0.079 (0.278)	0.080 (0.281)	0.034 (0.131)	0.060 (0.395)	 	 &lt; I/σ &gt;	16.8 (5.7)	21.1(6.9)	27.5 (5.8)	19.7 (3.1)	 	 B-factor (Å2)	26.0	27.0	21.6	31.4	 	Refinement	 	 	 	 	 	 Resolution (Å)	15-2.1	15-2.5	15-1.65	15-1.95	 	 Number of Reflections	25,857	16,328	51,882	60,181	 	 Number of All Atoms	3,676	3,454	3,814	7,175	 	 Number of Waters	302	196	527	445	 	 R-factor (%)	17.1	17.7	17.2	19.4	 	 R-free (%)	22.0	25.8	19.7	25.8	 	RMSD	 	 	 	 	 	 Bond Lengths (Å)	0.006	0.009	0.005	0.009	 	 Bond Angles (°)	1.0	1.3	1.3	1.3	 	 Mean B-factor (Å2)	25.2	38.2	20.0	19.5	 	Ramachandran Plot (%)	 	 	 	 	 	 Outliers	0.0	0.0	0.0	0.0	 	 Favored	98.4	97.9	98.1	98.0	 	</text></passage><passage><infon key="file">d37e1852.xml</infon><infon key="id">t0001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>18144</offset><text>Abbreviations: Amm, ammonium; PEG, polyethylene glycol.</text></passage><passage><infon key="file">d37e1852.xml</infon><infon key="id">t0001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>18200</offset><text>Values for high-resolution shell are in parentheses.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18253</offset><text>The crystal structures of a germline library composed of 16 Fabs generated by combining 4 HCs (H1-69, H3-23, H3-53 and H5-51) and 4 LCs (L1-39, L3-11, L3-20 and L4-1) have been determined. The Fab heavy and light chain sequences for the variants numbered according to Chothia are shown in Fig. S1. The four different HCs all have the same CDR H3 sequence, ARYDGIYGELDF. Crystallization of the 16 Fabs was previously reported. Three sets of the crystals were isomorphous with nearly identical unit cells (Table 1). These include (1) H3-23:L3-11 and H3-23:L4-1 in P212121, (2) H3-53:L1-39, H3-53:L3-11 and H3-53:L3-20 in P6522, and (3) H5-51:L1-39, H5-51:L3-11 and H5-51:L3-20 in P212121. Crystallization conditions for the 3 groups are also similar, but not identical (Table 1). Variations occur in the pH (buffer) and the additives, and, in group 3, PEG 3350 is the precipitant for one variants while ammonium sulfate is the precipitant for the other two. The similarity in the crystal forms is attributed in part to cross-seeding using the microseed matrix screening for groups 2 and 3.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19344</offset><text>The crystal structures of the 16 Fabs have been determined at resolutions ranging from 3.3 Å to 1.65 Å (Table 1). The number of Fab molecules in the crystallographic asymmetric unit varies from 1 (for 12 Fabs) to 2 (for 4 Fabs). Overall the structures are fairly complete, and, as can be expected, the models for the higher resolution structures are more complete than those for the lower resolution structures (Table S1). Invariably, the HCs have more disorder than the LCs. For the LC, the disorder is observed at 2 of the C-terminal residues with few exceptions. Apart from the C-terminus, only a few surface residues in LC are disordered.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19993</offset><text>The HCs feature the largest number of disordered residues, with the lower resolution structures having the most. The C-terminal residues including the 6xHis tags are disordered in all 16 structures. In addition to these, 2 primary disordered stretches of residues are observed in a number of structures (Table S1). One involves the loop connecting the first 2 β-strands of the constant domain (in all Fabs except H3-23:L1-39, H3-23:L3-11 and H3-53:L1-39). The other is located in CDR H3 (in H5-51:L3-11, H5-51:L3-20 and in one of 2 copies of H3-23:L4-1). CDR H1 and CDR H2 also show some degree of disorder, but to a lesser extent.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>20628</offset><text>CDR canonical structures</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20653</offset><text>Several CDR definitions have evolved over decades of antibody research. Depending on the focus of the study, the CDR boundaries differ slightly between various definitions. In this work, we use the CDR definition of North et al., which is similar to that of Martin with the following exceptions: 1) CDRs H1 and H3 begin immediately after the Cys; and 2) CDR L2 includes an additional residue at the N-terminal side, typically Tyr.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>21085</offset><text>CDR H1</text></passage><passage><infon key="file">kmab-08-06-1190060-g001.jpg</infon><infon key="id">f0001</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>21092</offset><text>The superposition of CDR H1 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51.</text></passage><passage><infon key="file">t0002.xml</infon><infon key="id">t0002</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>21216</offset><text>Canonical structures.</text></passage><passage><infon key="file">t0002.xml</infon><infon key="id">t0002</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;colgroup&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;/colgroup&gt;&lt;thead&gt;&lt;tr&gt;&lt;th align=&quot;left&quot;&gt;Pairs&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;PDB&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;CDR H1&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;CDR H2&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;CDR H3&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H1-69&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt;KASGGTFSSYAIS&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;GIIPIFGTAN&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;ARYDGIYGELDF&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H1-69&lt;/bold&gt;:L1-39&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I15&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H1-13-4&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H2-10-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H3-12-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H1-69&lt;/bold&gt;:L3-11&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I16&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H1-13-1/H1-13-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H2-10-1/H2-10-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H3-12-1/H3-12-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H1-69&lt;/bold&gt;:L3-20&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I17&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H1-13-3/H1-13-6&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H2-10-1/NA&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H3-12-1/H3-12-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H1-69&lt;/bold&gt;:L4-1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I18&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H1-13-10&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H2-10-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H3-12-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H3-23&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt;AASGFTFSSYAMS&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;AISGSGGSTY&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;AKYDGIYDGIYGELDF&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H3-23&lt;/bold&gt;:L1-39&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I19&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H1-13-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H2-10-2&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H3-12-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H3-23&lt;/bold&gt;:L3-11&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1A&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H1-13-1/H1-13-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H2-10-2/H2-10-2&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H3-12-1/H3-12-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H3-23&lt;/bold&gt;:L3-20&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1C&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H1-13-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H2-10-2&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H3-12-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H3-23&lt;/bold&gt;:L4-1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1D&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H1-13-1/H1-13-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H2-10-2/H2-10-2&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H3-12-1/NA&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H3-53&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt;AASGFTVSSNYMS&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;VIYSGGSTY&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;ARYDGIYGELDF&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H3-53&lt;/bold&gt;:L1-39&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1E&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H1-13-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H2-9-3&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H3-12-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H3-53&lt;/bold&gt;:L3-11&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1G&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H1-13-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H2-9-3&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H3-12-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H3-53&lt;/bold&gt;:L3-20&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1H&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H1-13-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H2-9-3&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H3-12-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H3-53&lt;/bold&gt;:L4-1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1I&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H1-13-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H2-9-3&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;NA&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H5-51&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt;KGSGYSFTSYWIG&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;IIYPGDSDTR&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;ARYDGIYGELDF&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H5-51&lt;/bold&gt;:L1-39&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;4KMT&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H1-13-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H2-10-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H3-12-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H5-51&lt;/bold&gt;:L3-11&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1J&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H1-13-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H2-10-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;NA&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H5-51&lt;/bold&gt;:L3-20&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1K&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H1-13-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H2-10-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;NA&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;H5-51&lt;/bold&gt;:L4-1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1L&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H1-13-1/H1-13-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H2-10-1/H2-10-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;H3-12-1/H3-12-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;CDR L1&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;CDR L2&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;CDR L3&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;L1-39&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt;RASQSISSYLN&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;YAASSLQS&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;QQSYSTPLT&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;H1-69:&lt;bold&gt;L1-39&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I15&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L1-11-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L2-8-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L3-9-cis7-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;H3-23:&lt;bold&gt;L1-39&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I19&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L1-11-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L2-8-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L3-9-cis7-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;H3-53:&lt;bold&gt;L1-39&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1E&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L1-11-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L2-8-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L3-9-cis7-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;H5-51:&lt;bold&gt;L1-39&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;4KMT&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L1-11-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L2-8-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L3-9-cis7-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;L3-11&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt;RASQSVSSYLA&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;YDASNRAT&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;QQRSNWPLT&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;H1-69:&lt;bold&gt;L3-11&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I16&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L1-11-1/L1-11-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L2-8-1/L2-8-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L3-9-cis7-1/L3-9-cis7-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;H3-23:&lt;bold&gt;L3-11&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1A&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L1-11-1/L1-11-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L2-8-1/L2-8-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L3-9-cis7-1/L3-9-cis7-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;H3-53:&lt;bold&gt;L3-11&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1G&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L1-11-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L2-8-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L3-9-cis7-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;H5-51:&lt;bold&gt;L3-11&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1J&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L1-11-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L2-8-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L3-9-cis7-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;L3-20&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt;RASQSVSSSYLA&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;YGASSRAT&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;QQYGSSPLT&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;H1-69:&lt;bold&gt;L3-20&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I17&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L1-12-2/L1-12-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L2-8-1/L2-8-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L3-9-cis7-1/L3-9-cis7-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;H3-23:&lt;bold&gt;L3-20&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1C&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L1-12-2&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L2-8-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L3-9-cis7-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;H3-53:&lt;bold&gt;L3-20&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1H&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L1-12-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L2-8-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L3-9-cis7-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;H5-51:&lt;bold&gt;L3-20&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1K&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L1-12-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L2-8-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L3-9-cis7-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;&lt;bold&gt;L4-1&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;left&quot;&gt; &lt;/td&gt;&lt;td align=&quot;left&quot;&gt;KSSQSVLYSSNNKNYLA&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;YWASTRES&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;QQYYSTPLT&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;H1-69:&lt;bold&gt;L4-1&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I18&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L1-17-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L2-8-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L3-9-cis7-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;H3-23:&lt;bold&gt;L4-1&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1D&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L1-17-1/L1-17-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L2-8-1/L2-8-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L3-9-cis7-1/L3-9-cis7-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;H3-53:&lt;bold&gt;L4-1&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1I&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L1-17-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L2-8-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L3-9-cis7-1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;H5-51:&lt;bold&gt;L4-1&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1L&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L1-17-1/L1-17-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L2-8-1/L2-8-1&lt;/td&gt;&lt;td align=&quot;left&quot;&gt;L3-9-cis7-1/L3-9-cis7-1&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>21238</offset><text>Pairs	PDB	CDR H1	CDR H2	CDR H3	 	H1-69	 	KASGGTFSSYAIS	GIIPIFGTAN	ARYDGIYGELDF	 	H1-69:L1-39	5I15	H1-13-4	H2-10-1	H3-12-1	 	H1-69:L3-11	5I16	H1-13-1/H1-13-1	H2-10-1/H2-10-1	H3-12-1/H3-12-1	 	H1-69:L3-20	5I17	H1-13-3/H1-13-6	H2-10-1/NA	H3-12-1/H3-12-1	 	H1-69:L4-1	5I18	H1-13-10	H2-10-1	H3-12-1	 	H3-23	 	AASGFTFSSYAMS	AISGSGGSTY	AKYDGIYDGIYGELDF	 	H3-23:L1-39	5I19	H1-13-1	H2-10-2	H3-12-1	 	H3-23:L3-11	5I1A	H1-13-1/H1-13-1	H2-10-2/H2-10-2	H3-12-1/H3-12-1	 	H3-23:L3-20	5I1C	H1-13-1	H2-10-2	H3-12-1	 	H3-23:L4-1	5I1D	H1-13-1/H1-13-1	H2-10-2/H2-10-2	H3-12-1/NA	 	H3-53	 	AASGFTVSSNYMS	VIYSGGSTY	ARYDGIYGELDF	 	H3-53:L1-39	5I1E	H1-13-1	H2-9-3	H3-12-1	 	H3-53:L3-11	5I1G	H1-13-1	H2-9-3	H3-12-1	 	H3-53:L3-20	5I1H	H1-13-1	H2-9-3	H3-12-1	 	H3-53:L4-1	5I1I	H1-13-1	H2-9-3	NA	 	H5-51	 	KGSGYSFTSYWIG	IIYPGDSDTR	ARYDGIYGELDF	 	H5-51:L1-39	4KMT	H1-13-1	H2-10-1	H3-12-1	 	H5-51:L3-11	5I1J	H1-13-1	H2-10-1	NA	 	H5-51:L3-20	5I1K	H1-13-1	H2-10-1	NA	 	H5-51:L4-1	5I1L	H1-13-1/H1-13-1	H2-10-1/H2-10-1	H3-12-1/H3-12-1	 	 	 	CDR L1	CDR L2	CDR L3	 	L1-39	 	RASQSISSYLN	YAASSLQS	QQSYSTPLT	 	H1-69:L1-39	5I15	L1-11-1	L2-8-1	L3-9-cis7-1	 	H3-23:L1-39	5I19	L1-11-1	L2-8-1	L3-9-cis7-1	 	H3-53:L1-39	5I1E	L1-11-1	L2-8-1	L3-9-cis7-1	 	H5-51:L1-39	4KMT	L1-11-1	L2-8-1	L3-9-cis7-1	 	L3-11	 	RASQSVSSYLA	YDASNRAT	QQRSNWPLT	 	H1-69:L3-11	5I16	L1-11-1/L1-11-1	L2-8-1/L2-8-1	L3-9-cis7-1/L3-9-cis7-1	 	H3-23:L3-11	5I1A	L1-11-1/L1-11-1	L2-8-1/L2-8-1	L3-9-cis7-1/L3-9-cis7-1	 	H3-53:L3-11	5I1G	L1-11-1	L2-8-1	L3-9-cis7-1	 	H5-51:L3-11	5I1J	L1-11-1	L2-8-1	L3-9-cis7-1	 	L3-20	 	RASQSVSSSYLA	YGASSRAT	QQYGSSPLT	 	H1-69:L3-20	5I17	L1-12-2/L1-12-1	L2-8-1/L2-8-1	L3-9-cis7-1/L3-9-cis7-1	 	H3-23:L3-20	5I1C	L1-12-2	L2-8-1	L3-9-cis7-1	 	H3-53:L3-20	5I1H	L1-12-1	L2-8-1	L3-9-cis7-1	 	H5-51:L3-20	5I1K	L1-12-1	L2-8-1	L3-9-cis7-1	 	L4-1	 	KSSQSVLYSSNNKNYLA	YWASTRES	QQYYSTPLT	 	H1-69:L4-1	5I18	L1-17-1	L2-8-1	L3-9-cis7-1	 	H3-23:L4-1	5I1D	L1-17-1/L1-17-1	L2-8-1/L2-8-1	L3-9-cis7-1/L3-9-cis7-1	 	H3-53:L4-1	5I1I	L1-17-1	L2-8-1	L3-9-cis7-1	 	H5-51:L4-1	5I1L	L1-17-1/L1-17-1	L2-8-1/L2-8-1	L3-9-cis7-1/L3-9-cis7-1	 	</text></passage><passage><infon key="file">t0002.xml</infon><infon key="id">t0002</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>23317</offset><text>CDRs are defined using the Dunbrack convention [12]. Assignments for 2 copies of the Fab in the asymmetric unit are given for 5 structures. No assignment (NA) for CDRs with missing residues.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23508</offset><text>The four HCs feature CDR H1 of the same length, and their sequences are highly similar (Table 2). The CDR H1 backbone conformations for all variants for each of the HCs are shown in Fig. 1. Three of the HCs, H3-23, H3-53 and H5-51, have the same canonical structure, H1-13-1, and the backbone conformations are tightly clustered for each set of Fab structures as reflected in the rmsd values (Fig. 1B-D). Some deviation is observed for H3-53, mostly due to H3-53:L4-1, which exhibits a significant degree of disorder in CDR H1. The electron density for the backbone is weak and discontinuous, and completely missing for several side chains.   </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24155</offset><text>The CDR H1 structures with H1-69 shown in Fig. 1A are quite variable, both for the structures with different LCs and for the copies of the same Fab in the asymmetric unit, H1-69:L3-11 and H1-69:L3-20. In total, 6 independent Fab structures produce 5 different canonical structures, namely H1-13-1, H1-13-3, H1-13-4, H1-13-6 and H1-13-10. A major difference of H1-69 from the other germlines in the experimental data set is the presence of Gly instead of Phe or Tyr at position 27 (residue 5 of 13 in CDR H1). Glycine introduces the possibility of a higher degree of conformational flexibility that undoubtedly translates to the differences observed, and contributes to the elevated thermal parameters for the atoms in the amino acid residues in this region.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>24914</offset><text>CDR H2</text></passage><passage><infon key="file">kmab-08-06-1190060-g002.jpg</infon><infon key="id">f0002</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>24921</offset><text>The superposition of CDR H2 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>25045</offset><text>The canonical structures of CDR H2 have fairly consistent conformations (Table 2, Fig. 2). Each of the 4 HCs adopts only one canonical structure regardless of the pairing LC. Germlines H1-69 and H5-51 have the same canonical structure assignment H2-10-1, H3-23 has H2-10-2, and H3-53 has H2-9-3. The conformations for all of these CDR H2s are tightly clustered (Fig. 2). In one case, in the second Fab of H1-69:L3-20, CDR H2 is partially disordered (Δ55-60).  </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>25510</offset><text>Although three of the germlines have CDR H2 of the same length, 10 residues, they adopt 2 distinctively different conformations depending mostly on the residue at position 71 from the so-called CDR H4. Arg71 in H3-23 fills the space between CDRs H2 and H4, and defines the conformation of the tip of CDR H2 so that residue 54 points away from the antigen binding site. Germlines H1-69 and H5-51 are unique in the human repertoire in having an Ala at position 71 that leaves enough space for H-Pro52a to pack deeper against CDR H4 so that the following residues 53 and 54 point toward the putative antigen.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>26116</offset><text>Conformations of CDR H2 in H1-69 and H5-51, both of which have canonical structure H2-10-1, show little deviation within each set of 4 structures. However, there is a significant shift of the CDR as a rigid body when the 2 sets are superimposed. Most likely this is the result of interaction of CDR H2 with CDR H1, namely with the residue at position 33 (residue 11 of 13 in CDR H1). Germline H1-69 has Ala at position 33 whereas in H5-51 position 33 is occupied by a bulky Trp, which stacks against H-Tyr52 and drives CDR H2 away from the center.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>26664</offset><text>CDR L1</text></passage><passage><infon key="file">kmab-08-06-1190060-g003.jpg</infon><infon key="id">f0003</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>26671</offset><text>The superposition of CDR L1 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>26794</offset><text>The four LC CDRs L1 feature 3 different lengths (11, 12 and 17 residues) having a total of 4 different canonical structure assignments. Of these LCs, L1-39 and L3-11 have the same canonical structure, L1-11-1, and superimpose very well (Fig. 3A, B). For the remaining 2, L3-20 has 2 different assignments, L1-12-1 and L1-12-2, while L4-1 has a single assignment, L1-17-1.  </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>27169</offset><text>L4-1 has the longest CDR L1, composed of 17 amino acid residues (Fig. 3D). Despite this, the conformations are tightly clustered (rmsd is 0.20 Å). The backbone conformations of the stem regions superimpose well. Some changes in conformation occur between residues 30a and 30f (residues 8 and 13 of 17 in CDR L1). This is the tip of the loop region, which appears to have similar conformations that fan out the structures because of the slight differences in torsion angles in the backbone near Tyr30a and Lys30f.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>27685</offset><text>L3-20 is the most variable in CDR L1 among the 4 germlines as indicated by an rmsd of 0.54 Å (Fig. 3C). Two structures, H3-53:L3-20 and H5-51:L3-20 are assigned to canonical structure L1-12-1 with virtually identical backbone conformations. The third structure, H3-23:L3-20, has CDR L1 as L1-12-2, which deviates from L1-12-1 at residues 29-32, i.e., at the site of insertion with respect to the 11-residue CDR. The fourth member of the set, H1-69:L3-20, was crystallized with 2 Fabs in the asymmetric unit. The conformation of CDR L1 in these 2 Fabs is slightly different, and both conformations fall somewhere between L1-12-1 and L1-12-2. This reflects the lack of accuracy in the structure due to low resolution of the X-ray data (3.3 Å).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>28432</offset><text>CDR L2</text></passage><passage><infon key="file">kmab-08-06-1190060-g004.jpg</infon><infon key="id">f0004</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28439</offset><text>The superposition of CDR L2 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>28562</offset><text>All four LCs have CDR L2 of the same length and canonical structure, L2-8-1 (Table 2). The CDR L2 conformations for each of the LCs paired with the 4 HCs are clustered more tightly than any of the other CDRs (rmsd values are in the range 0.09-0.16 Å), and all 4 sets have virtually the same conformation despite the sequence diversity of the loop. No significant conformation outliers are observed (Fig. 4).  </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>28976</offset><text>CDR L3</text></passage><passage><infon key="file">kmab-08-06-1190060-g005.jpg</infon><infon key="id">f0005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28983</offset><text>The superposition of CDR L3 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>29106</offset><text>As with CDR L2, all 4 LCs have CDR L3 of the same length and canonical structure, L3-9-cis7-1 (Table 2). The conformations of CDR L3 for L1-39, L3-11, and particularly for L320, are not as tightly clustered as those of L4-1 (Fig. 5). The slight conformational variability occurs in the region of amino acid residues 90-92, which is in contact with CDR H3.  </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>29466</offset><text>CDR H3 conformational diversity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>29498</offset><text>As mentioned earlier, all 16 Fabs have the same CDR H3, for which the amino acid sequence is derived from the anti-CCL2 antibody CNTO 888. The loop and the 2 β-strands of the CDR H3 in this ‘parent’ structure are stabilized by H-bonds between the carbonyl oxygen and peptide nitrogen atoms in the 2 strands. An interesting feature of these CDR H3 structures is the presence of a water molecule that interacts with the peptide nitrogens and carbonyl oxygens near the bridging loop connecting the 2 β-strands. This water is present in both the bound (4DN4) and unbound (4DN3) forms of CNTO 888. The stem region of CDR H3 in the parental Fab is in a ‘kinked’ conformation, in which the indole nitrogen of Trp103 forms a hydrogen bond with the carbonyl oxygen of Leu100b. The carboxyl group of Asp101 forms a salt bridge with Arg94. These interactions are illustrated in Fig. S2.</text></passage><passage><infon key="file">kmab-08-06-1190060-g006.jpg</infon><infon key="id">f0006</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>30385</offset><text>Ribbon representations of (A) the superposition of all CDR H3s of the structures with complete backbone traces. (B) The CDR H3s rotated 90° about the y axis of the page. The structure of each CDR H3 is represented with a different color.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>30624</offset><text>Despite having the same amino acid sequence in all variants, CDR H3 has the highest degree of structural diversity and disorder of all of the CDRs in the experimental set. Three of the 21 Fab structures (including multiple copies in the asymmetric unit), H5-51:L3-11, H551:L3-20 and H3-23:L4-1 (one of the 2 Fabs), have missing (disordered) residues at the apex of the CDR loop. Another four of the Fabs, H3-23:L1-39, H3-53:L1-39, H3-53:L3-11 and H3-53:L4-1 have missing side-chain atoms. The variations in CDR H3 conformation are illustrated in Fig. 6 for the 18 Fab structures that have ordered backbone atoms.  </text></passage><passage><infon key="file">kmab-08-06-1190060-g007.jpg</infon><infon key="id">f0007</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>31240</offset><text>A comparison of representatives of the “kinked” and “extended” structures. (A) The “kinked” CDR H3 of H1-69:L3-11 with purple carbon atoms and yellow dashed lines connecting the H-bond pairs for Leu100b O and Trp103 NE1, Arg94 NE and Asp101 OD1, and Arg94 NH2 and Asp101 OD2. (B) The “extended” CDR H3 of H1-69:L3-20 with green carbon atoms and yellow dashed lines connecting the H-bond pairs for Asp101 OD1 and OD2 and Trp103 NE1.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>31688</offset><text>In 10 of the 18 Fab structures, H1-69:L1-39, H1-69:L3-11 (2 Fabs), H1-69:L4-1, H3-23:L3-11 (2 Fabs), H3-23:L3-20, H3-53:L3-11, H3-53:L3-20 and H5-51:L1-39, the CDRs have similar conformations to that found in 4DN3. The bases of these structures have the ‘kinked’ conformation with the H-bond between Trp103 and Leu100b. A representative CDR H3 structure for H1-69:L1-39 illustrating this is shown in Fig. 7A. The largest backbone conformational deviation for the set is at Tyr99, where the C=O is rotated by 90° relative to that observed in 4DN3. Also, it is worth noting that only one of these structures, H1-69:L4-1, has the conserved water molecule in CDR H3 observed in the 4DN3 and 4DN4 structures. In fact, it is the only Fab in the set that has a water molecule present at this site. The CDR H3 for this structure is shown in Fig. S3.  </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>32539</offset><text>The remaining 8 Fabs can be grouped into 5 different conformational classes. Three of the Fabs, H3-23:L1-39, H3-23:L4-1 and H3-53:L1-39, have distinctive conformations. The stem regions in these 3 cases are in the ‘kinked’ conformation consistent with that observed for 4DN3. The five remaining Fabs, H5-51:L4-1 (2 copies), H1-69:L3-20 (2 copies) and H3-53:L4-1, have 3 different CDR H3 conformations (Fig. S4). The stem regions of CDR H3 for the H5-51:L4-1 Fabs are in the ‘kinked’ conformation while, surprisingly, those of the H1-69:L3-20 pair and H3-53:L4-1 are in the ‘extended’ conformation (Fig. 7B).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>33161</offset><text>VH:VL domain packing</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>33182</offset><text>The VH and VL domains have a β-sandwich structure (also often referred as a Greek key motif) and each is composed of a 4-stranded and a 5-stranded antiparallel β-sheets. The two domains pack together such that the 5-stranded β-sheets, which have hydrophobic surfaces, interact with each other bringing the CDRs from both the VH and VL domains into close proximity. The domain packing of the variants was assessed by computing the domain interface interactions, the VH:VL tilt angles, the buried surface area and surface complementarity. The results of these analyses are shown in Tables 3, 4 and S2.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_3</infon><offset>33791</offset><text>VH:VL interface amino acid residue interactions</text></passage><passage><infon key="file">kmab-08-06-1190060-g008.jpg</infon><infon key="id">f0008</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>33839</offset><text>The conserved VH:VL interactions as viewed along the VH/VL axis. The VH residues are in blue, the VL residues are in orange.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>33964</offset><text>The VH:VL interface is pseudosymmetric, and involves 2 stretches of the polypeptide chain from each domain, namely CDR3 and the framework region between CDRs 1 and 2. These stretches form antiparallel β-hairpins within the internal 5-stranded β-sheet. There are a few principal inter-domain interactions that are conserved not only in the experimental set of 16 Fabs, but in all human antibodies. They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). With the exception of L-Ala43, all other residues are conserved in human germlines. Position 43 may be alternatively occupied by Ser, Val or Pro (as in L4-1), but the hydrophobic interaction with H-Tyr91 is preserved. These core interactions provide enough stability to the VH:VL dimer so that additional VH-VL contacts can tolerate amino acid sequence variations in CDRs H3 and L3 that form part of the VH:VL interface.  </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>35026</offset><text>In total, about 20 residues are involved in the VH:VL interactions on each side (Fig. S5). Half of them are in the framework regions and those residues (except residue 61 in HC, which is actually in CDR2 in Kabat's definition) are conserved in the set of 16 Fabs. The side chain conformations of these conserved residues are also highly similar. One notable exception is H-Trp47, which exhibits 2 conformations of the indole ring. In most of the structures, it has the χ2 angle of ∼80°, while the ring is flipped over (χ2 = −100°) in H5-51:L3:11 and H5-51:L3-20. Interestingly, these are the only 2 structures with residues missing in CDR H3 because of disorder, although both structures are determined at high resolution and the rest of the structure is well defined. Apparently, residues flanking CDR H3 in the 2 VH:VL pairings are inconsistent with any stable conformation of CDR H3, which translates into a less restricted conformational space for some of them, including H-Trp47.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>36020</offset><text>VH:VL tilt angles</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>36038</offset><text>The relative orientation of VH and VL has been measured in a number of different ways. Presented here are the results of 2 different approaches for determining the orientation of one domain relative to the other.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>36251</offset><text>The first approach uses ABangles, the results of which are shown in Table S2. The four LCs all are classified as Type A because they have a proline at position 44, and the results for each orientation parameter are within the range of values of this type reported by Dunbar and co-workers. In fact, the parameter values for the set of 16 Fabs are in the middle of the distribution observed for 351 non-redundant antibody structures determined at 3.0 Å resolution or better. The only exception is HC1, which is shifted toward smaller angles with the mean value of 70.8° as compared to the distribution centered at 72° for the entire PDB. This probably reflects the invariance of CDR H3 in the current set as opposed to the CDR H3 diversity in the PDB.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>37007</offset><text>The second approach used for comparing tilt angles involved computing the difference in the tilt angles between all pairs of structures. For structures with 2 copies of the Fab in the asymmetric unit, only one structure was used. The differences between independent Fabs in the same structure are 4.9° for H1-69:L3-20, 1.6° for H1-69:L3-11, 1.4° for H3-23:L4-1, 3.3° for H3-23:L3-11, and 2.5° for H5-51:L4-1. With the exception of H1-69:L3-20, the angles are within the range of 2-3° as are observed in the identical structures in the PDB. In H1-69:L3-20, one of the Fabs is substantially disordered so that part of CDR H2 (the outer β-strand, residues 55-60) is completely missing. This kind of disorder may compromise the integrity of the VH domain and its interaction with the VL. Indeed, this Fab has the largest twist angle HC2 within the experimental set that exceeds the mean value by 2.5 standard deviations (Table S2).</text></passage><passage><infon key="file">kmab-08-06-1190060-g009.jpg</infon><infon key="id">f0009</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>37943</offset><text>An illustration of the difference in tilt angle for 2 pairs of variants by the superposition of the VH domains of (A) H1-69:L3-20 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 10.5°) and (B) H1-69:L4-1 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 1.6°).</text></passage><passage><infon key="file">t0003.xml</infon><infon key="id">t0003</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>38256</offset><text>Differences in VH:VL tilt angles.</text></passage><passage><infon key="file">t0003.xml</infon><infon key="id">t0003</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;colgroup&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;/colgroup&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt; &lt;hr/&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;H1-69:L1-39&lt;hr/&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;H1-69:L3-11&lt;hr/&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;H1-69:L3-20&lt;hr/&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;H1-69:L4-1&lt;hr/&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;H3-23:L1-39&lt;hr/&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;H3-23:L3-11&lt;hr/&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;H3-23:L3-20&lt;hr/&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;H3-23:L4-1&lt;hr/&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;H3-53:L1-39&lt;hr/&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;H3-53:L3-11&lt;hr/&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;H3-53:L3-20&lt;hr/&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;H3-53:L4-1&lt;hr/&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;H5-51:L1-39&lt;hr/&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;H5-51:L3-11&lt;hr/&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;H5-51:L3-20&lt;hr/&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;H5-51:L4-1&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H1-69:L1-39&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;2.1&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;8.9&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;1.1&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;4.2&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;3.0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;9.5&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;1.5&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;3.3&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.6&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.1&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;1.6&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;1.8&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;2.9&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;2.4&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;5.2&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H1-69:L3-11&lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;7.3&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;2.9&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;2.5&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;2.0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;8.4&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;1.3&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;2.6&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;2.9&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.2&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;1.8&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.9&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;4.6&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;4.4&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;5.0&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H1-69:L3-20&lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;9.2&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;5.0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;8.7&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;7.4&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;7.6&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;8.9&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;8.6&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;9.4&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;7.9&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;10.5&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;10.1&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;11.0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;9.7&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H1-69:L4-1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;4.6&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.9&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;10.1&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;1.8&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;4.4&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;4.7&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;4.1&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;2.3&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;1.6&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;2.5&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;2.3&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;6.2&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H3-23:L1-39&lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;4.0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;8.0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;2.8&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;4.8&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;4.8&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;5.4&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.6&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;6.0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;6.2&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;6.6&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;6.6&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H3-23:L3-11&lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;9.3&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;2.1&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;2.9&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.3&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.3&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;4.6&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;5.8&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;5.0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;5.2&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H3-23:L3-20&lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;8.9&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;7.9&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;7.0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;7.6&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;7.9&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;10.5&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;9.7&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;10.7&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;6.2&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H3-23:L4-1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.6&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.8&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.7&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;1.5&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.2&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.7&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.8&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;5.6&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H3-53:L1-39&lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;1.0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;1.6&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;2.9&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;4.6&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;5.3&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;4.8&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.1&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H3-53:L3-11&lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;1.3&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;2.9&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;4.8&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;5.2&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;5.0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;2.3&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H3-53:L3-20&lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;2.5&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.8&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;4.2&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.9&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;2.2&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H3-53:L4-1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;2.9&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;3.3&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;4.2&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H5-51:L1-39&lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;1.9&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;0.6&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;5.8&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H5-51:L3-11&lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;1.9&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;5.7&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H5-51:L3-20&lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;0&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;&lt;bold&gt;5.8&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H5-51:L4-1&lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;0&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>38290</offset><text> 	H1-69:L1-39	H1-69:L3-11	H1-69:L3-20	H1-69:L4-1	H3-23:L1-39	H3-23:L3-11	H3-23:L3-20	H3-23:L4-1	H3-53:L1-39	H3-53:L3-11	H3-53:L3-20	H3-53:L4-1	H5-51:L1-39	H5-51:L3-11	H5-51:L3-20	H5-51:L4-1	 	H1-69:L1-39	0	2.1	8.9	1.1	4.2	3.0	9.5	1.5	3.3	3.6	3.1	1.6	1.8	2.9	2.4	5.2	 	H1-69:L3-11	 	0	7.3	2.9	2.5	2.0	8.4	1.3	2.6	2.9	3.2	1.8	3.9	4.6	4.4	5.0	 	H1-69:L3-20	 	 	0	9.2	5.0	8.7	7.4	7.6	8.9	8.6	9.4	7.9	10.5	10.1	11.0	9.7	 	H1-69:L4-1	 	 	 	0	4.6	3.9	10.1	1.8	4.4	4.7	4.1	2.3	1.6	2.5	2.3	6.2	 	H3-23:L1-39	 	 	 	 	0	4.0	8.0	2.8	4.8	4.8	5.4	3.6	6.0	6.2	6.6	6.6	 	H3-23:L3-11	 	 	 	 	 	0	9.3	3.0	2.1	2.9	3.3	3.3	4.6	5.8	5.0	5.2	 	H3-23:L3-20	 	 	 	 	 	 	0	8.9	7.9	7.0	7.6	7.9	10.5	9.7	10.7	6.2	 	H3-23:L4-1	 	 	 	 	 	 	 	0	3.6	3.8	3.7	1.5	3.2	3.7	3.8	5.6	 	H3-53:L1-39	 	 	 	 	 	 	 	 	0	1.0	1.6	2.9	4.6	5.3	4.8	3.1	 	H3-53:L3-11	 	 	 	 	 	 	 	 	 	0	1.3	2.9	4.8	5.2	5.0	2.3	 	H3-53:L3-20	 	 	 	 	 	 	 	 	 	 	0	2.5	3.8	4.2	3.9	2.2	 	H3-53:L4-1	 	 	 	 	 	 	 	 	 	 	 	0	2.9	3.0	3.3	4.2	 	H5-51:L1-39	 	 	 	 	 	 	 	 	 	 	 	 	0	1.9	0.6	5.8	 	H5-51:L3-11	 	 	 	 	 	 	 	 	 	 	 	 	 	0	1.9	5.7	 	H5-51:L3-20	 	 	 	 	 	 	 	 	 	 	 	 	 	 	0	5.8	 	H5-51:L4-1	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	0	 	</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>39582</offset><text>The differences in the tilt angle are shown for all pairs of V regions in Table 3. They range from 0.6° to 11.0°. The smallest differences in the tilt angle are between the Fabs in isomorphous crystal forms. The largest deviations in the tilt angle, up to 11.0°, are found for 2 structures, H1-69:L3-20 and H3-23:L3-20, that stand out from the other Fabs. One of the 2 structures, H1-69:L3-20, has its CDR H3 in the ‘extended’ conformation; the other structure has it in the ‘kinked’ conformation. Two examples illustrating large (10.5°) and small (1.6°) differences in the tilt angles are shown in Fig. 9.   </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>40207</offset><text>VH:VL buried surface area and complementarity</text></passage><passage><infon key="file">t0004.xml</infon><infon key="id">t0004</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>40253</offset><text>VH:VL surface areas and surface complementarity.</text></passage><passage><infon key="file">t0004.xml</infon><infon key="id">t0004</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;colgroup&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;/colgroup&gt;&lt;thead&gt;&lt;tr&gt;&lt;th align=&quot;left&quot;&gt;Chain Pairs&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;PDB&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;Contact surfaceVH (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;Contact surfaceVL (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;Interface(Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;Surface complementarity&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;H1-69:L1-39&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I15&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;727&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;771&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;749&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;0.743&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;H1-69:L3-11&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I16&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;802&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;870&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;836&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;0.762&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;H1-69:L3-20&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I17&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;713&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;736&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;725&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;0.723&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;H1-69:L4-1&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I18&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;729&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;736&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;733&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;0.734&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H3-23:L1-39&lt;xref ref-type=&quot;bibr&quot; rid=&quot;cit0001&quot;&gt;&lt;sup&gt;&lt;bold&gt;1&lt;/bold&gt;&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I19&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;795&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;817&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;806&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;0.722&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;H3-23:L3-11&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1A&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;822&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;834&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;828&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;0.725&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;H3-23:L3-20&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1C&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;670&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;698&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;684&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;0.676&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;H3-23:L4-1&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1D&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;743&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;770&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;757&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;0.708&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H3-53:L1-39&lt;xref ref-type=&quot;bibr&quot; rid=&quot;cit0001&quot;&gt;&lt;sup&gt;&lt;bold&gt;1&lt;/bold&gt;&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1E&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;698&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;719&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;709&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.712&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H3-53:L3-11&lt;xref ref-type=&quot;bibr&quot; rid=&quot;cit0001&quot;&gt;&lt;sup&gt;&lt;bold&gt;1&lt;/bold&gt;&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1G&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;747&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;758&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;753&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.690&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;H3-53:L3-20&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1H&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;743&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;735&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;739&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;0.687&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;H3-53:L4-1&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1I&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;689&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;693&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;691&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;0.711&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;H5-51:L1-39&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;4KMT&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;761&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;808&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;785&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;0.728&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H5-51:L3-11&lt;xref ref-type=&quot;bibr&quot; rid=&quot;cit0002&quot;&gt;&lt;sup&gt;&lt;bold&gt;2&lt;/bold&gt;&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1J&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;648&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;714&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;681&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.717&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;H5-51:L3-20&lt;xref ref-type=&quot;bibr&quot; rid=&quot;cit0002&quot;&gt;&lt;sup&gt;&lt;bold&gt;2&lt;/bold&gt;&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1K&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;622&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;643&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;633&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;0.740&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;center&quot;&gt;&lt;bold&gt;H5-51:L4-1&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;5I1L&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;790&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;792&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;791&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;0.704&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>40302</offset><text>Chain Pairs	PDB	Contact surfaceVH (Å2)	Contact surfaceVL (Å2)	Interface(Å2)	Surface complementarity	 	H1-69:L1-39	5I15	727	771	749	0.743	 	H1-69:L3-11	5I16	802	870	836	0.762	 	H1-69:L3-20	5I17	713	736	725	0.723	 	H1-69:L4-1	5I18	729	736	733	0.734	 	H3-23:L1-39	5I19	795	817	806	0.722	 	H3-23:L3-11	5I1A	822	834	828	0.725	 	H3-23:L3-20	5I1C	670	698	684	0.676	 	H3-23:L4-1	5I1D	743	770	757	0.708	 	H3-53:L1-39	5I1E	698	719	709	0.712	 	H3-53:L3-11	5I1G	747	758	753	0.690	 	H3-53:L3-20	5I1H	743	735	739	0.687	 	H3-53:L4-1	5I1I	689	693	691	0.711	 	H5-51:L1-39	4KMT	761	808	785	0.728	 	H5-51:L3-11	5I1J	648	714	681	0.717	 	H5-51:L3-20	5I1K	622	643	633	0.740	 	H5-51:L4-1	5I1L	790	792	791	0.704	 	</text></passage><passage><infon key="file">t0004.xml</infon><infon key="id">t0004</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>40999</offset><text>Some side chain atoms in CDR H3 are missing.</text></passage><passage><infon key="file">t0004.xml</infon><infon key="id">t0004</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>41044</offset><text>Residues in CDR H3 are missing: YGE in H5-51:L3-11, GIY in H5-51:L3-20.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>41116</offset><text>The results of the PISA contact surface calculation and surface complementarity calculation are shown in Table 4. The interface areas are calculated as the average of the VH and VL contact surfaces. Six of the 16 structures have CDR H3 side chains or complete residues missing, and therefore their interfaces are much smaller than in the other 10 structures with complete CDRs (the results are provided for all Fabs for completeness). Among the complete structures, the interface areas range from 684 to 836 Å2. Interestingly, the 2 structures that have the largest tilt angle differences with the other variants, H3-23:L3-20 and H1-69:L3-20, have the smallest VH:VL interfaces, 684 and 725 Å2, respectively. H3-23:L3-20 is also unique in that it has the lowest value (0.676) of surface complementarity.  </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_3</infon><offset>41927</offset><text>Stability of germline pairings</text></passage><passage><infon key="file">t0005.xml</infon><infon key="id">t0005</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>41958</offset><text>Melting temperatures for the 16 Fabs.</text></passage><passage><infon key="file">t0005.xml</infon><infon key="id">t0005</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;colgroup&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;col width=&quot;86.4pt&quot; align=&quot;left&quot;/&gt;&lt;/colgroup&gt;&lt;thead&gt;&lt;tr&gt;&lt;th align=&quot;left&quot;&gt; &lt;/th&gt;&lt;th align=&quot;center&quot;&gt;L3-20&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;L4-1&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;L3-11&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;L1-39&lt;/th&gt;&lt;th align=&quot;center&quot;&gt;HC average&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;H1-69&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;73.6&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;74.8&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;75.6&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;80.3&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;76.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;H3-23&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;74.8&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;75.2&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;4.8&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;81.5&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;76.6&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;H3-53&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;68.4&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;68.0&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;71.5&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;73.9&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;70.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;H5-51&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;68.4&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;68.4&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;71.9&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;77.0&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;71.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot;&gt;LC average&lt;/td&gt;&lt;td align=&quot;center&quot;&gt;71.3&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;71.6&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;73.5&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot;&gt;78.2&lt;/td&gt;&lt;td align=&quot;center&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>41996</offset><text> 	L3-20	L4-1	L3-11	L1-39	HC average	 	H1-69	73.6	74.8	75.6	80.3	76.1	 	H3-23	74.8	75.2	4.8	81.5	76.6	 	H3-53	68.4	68.0	71.5	73.9	70.5	 	H5-51	68.4	68.4	71.9	77.0	71.4	 	LC average	71.3	71.6	73.5	78.2	 	 	</text></passage><passage><infon key="file">t0005.xml</infon><infon key="id">t0005</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>42203</offset><text>Colors: blue (Tm &lt; 70°C), green (70°C &lt; Tm &lt; 73°C), yellow (73°C &lt; Tm &lt; 78°C), orange (Tm &gt; 78°C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>42308</offset><text>Melting temperatures (Tm) were measured for all Fabs using differential scanning calorimetry (Table 5). It appears that for each given LC, the Fabs with germlines H1-69 and H3-23 are substantially more stable than those with germlines H3-53 and H5-51. In addition, L1-39 provides a much higher degree of stabilization than the other 3 LC germlines when combined with any of the HCs. As a result, the Tm for pairs H1-69:L1-39 and H3-23:L1-39 is 12-13° higher than for pairs H3-53:L3-20, H3-53:L4-1, H5-51:L3-20 and H5-51:L4-1.  </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>42838</offset><text>These findings correlate well with the degree of conformational disorder observed in the crystal structures. Parts of CDR H3 main chain are completely disordered, and were not modeled in Fabs H5-51:L3-20 and H5-51:L3-11 that have the lowest Tms in the set. No electron density is observed for a number of side chains in CDRs H3 and L3 in all Fabs with germline H3-53, which indicates loose packing of the variable domains. All those molecules are relatively unstable, as is reflected in their low Tms.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>43340</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>43351</offset><text>This is the first report of a systematic structural investigation of a phage germline library. The 16 Fab structures offer a unique look at all pairings of 4 different HCs (H1-69, H3-23, H3-53, and H5-51) and 4 different LCs (L1-39, L3-11, L3-20 and L4-1), all with the same CDR H3. The structural data set taken as a whole provides insight into how the backbone conformations of the CDRs of a specific heavy or light chain vary when it is paired with 4 different light or heavy chains, respectively. A large variability in the CDR conformations for the sets of HCs and LCs is observed. In some cases the CDR conformations for all members of a set are virtually identical, for others subtle changes occur in a few members of a set, and in some cases larger deviations are observed within a set. The five variants that crystallized with 2 copies of the Fab in the asymmetric unit serve somewhat as controls for the influence of crystal packing on the conformations of the CDRs. In four of the 5 structures the CDR conformations are consistent. In only one case, that of H1-69:L3-20 (the lowest resolution structure), do we see differences in the conformations of the 2 copies of CDRs H1 and L1. This variability is likely a result of 2 factors, crystal packing interactions and internal instability of the variable domain.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>44673</offset><text>For the CDRs with canonical structures, the largest changes in conformation occur for CDR H1 of H1-69 and H3-53. The other 2 HCs, H3-23 and H5-51, have canonical structures that are remarkably well conserved (Fig. 1). Of the 4 HCs, H1-69 has the greatest number of canonical structure assignments (Table 2). H1-69 is unique in having a pair of glycine residues at positions 26 and 27, which provide more conformational freedom in CDR H1. Besides IGHV1-69, only the germlines of the VH4 family possess double glycines in CDR H1, and it will be interesting to see if they are also conformationally unstable.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>45281</offset><text>Having all 16 VH:VL pairs with the same CDR H3 provides some insights into why molecular modeling efforts of CDR H3 have proven so difficult. As mentioned in the Results section, this data set is composed of 21 Fabs, since 5 of the 16 variants have 2 Fab copies in the asymmetric unit. For the 18 Fabs with complete backbone atoms for CDR H3, 10 have conformations similar to that of the parent, while the others have significantly different conformations (Fig. 6). Thus, it is likely that the CDR H3 conformation is dependent upon 2 dominating factors: 1) amino acid sequence; and 2) VH and VL context. More than half of the variants retain the conformation of the parent despite having differences in the VH:VL pairing. This subset includes 2 structures with 2 copies of the Fab in the asymmetric unit, all of which are nearly identical in conformation. This provides an internal control showing a consistency in the conformations. The remaining 8 structures exhibit “non-parental” conformations, indicating that the VH and VL context can also be a dominating factor influencing CDR H3. Importantly, there are 5 distinctive conformations in this subset. This subset also has 2 structures with 2 Fab copies in the asymmetric unit. Each pair has nearly identical conformations providing an internal check on the consistency of the conformations. Interestingly, as described earlier, these 2 pairs differ in the stem regions with the H1-69:L3-20 pair in the ‘extended’ conformation and H5-51:L4-1 pair in the ‘kinked’ conformation. The conformations are different from each other, as well as from the parent.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>46902</offset><text>The CDR H3 conformational analysis shows that, for each set of variants of one HC paired with the 4 different LCs, both “parental” and “non-parental” conformations are observed. The same variability is observed for the sets of variants composed of one LC paired with each of the 4 HCs. Thus, no patterns of conformational preference for a particular HC or LC emerge to shed any direct light on what drives the conformational differences. This finding supports the hypothesis of Weitzner et al. that the H3 conformation is controlled both by its sequence and its environment.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>47486</offset><text>In looking at a possible correlation between the tilt angle and the conformation of CDR H3, no clear trends are observed. Two variants, H1-69:L3-20 and H3-23:L3-20, have the largest differences in the tilt angles compared to other variants as seen in Table 3. The absolute VH:VL orientation parameters for the 2 Fabs (Table S2) show significant deviation in HL, LC1 and HC2 values (2-3 standard deviations from the mean). One of the variants, H3-23:L3-20, has the CDR H3 conformation similar to the parent, but the other, H1-69:L3-20, is different.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>48037</offset><text>As noted in the Results section, the 2 variants, H1-69:L3-20 and H3-23:L3-20, are outliers in terms of the tilt angle; at the same time, both have the smallest VH:VL interface. These smaller interfaces may perhaps translate to a significant deviation in how VH is oriented relative to VL than the other variants. These deviations from the other variants can also be seen to some extent in VH:VL orientation parameters in Table S2, as well as in the smaller number of residues involved in the VH:VL interfaces of these 2 variants (Fig. S5). These differences undoubtedly influence the conformation of the CDRs, in particular CDR H1 (Fig. 1A) and CDR L1 (Fig. 3C), especially with the tandem glycines and multiple serines present, respectively.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>48784</offset><text>Pairing of different germlines yields antibodies with various degrees of stability. As indicated by the melting temperatures, germlines H1-69 and H3-23 for HC and germline L1-39 for LC produce more stable Fabs compared to the other germlines in the experimental set. Structural determinants of the differential stability are not always easy to decipher. One possible explanation of the clear preference of LC germline L1-39 is that CDR L3 has smaller residues at positions 91 and 94, allowing for more room to accommodate CDR H3. Other germlines have bulky residues, Tyr, Arg and Trp, at these positions, whereas L1-39 has Ser and Thr. Various combinations of germline sequences for VL and VH impose certain constraints on CDR H3, which has to adapt to the environment. A more compact CDR L3 may be beneficial in this situation.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>49613</offset><text>At the other end of the stability range is LC germline L3-20, which yields antibodies with the lowest Tms. While pairings with H3-53 and H5-51 may be safely called a mismatch, those with H1-69 and H3-23 have Tms about 5-6° higher. Curiously, the 2 Fabs, H1-69:L3-20 and H3-23:L3-20, deviate markedly in their tilt angles from the rest of the panel. It is possible that by adopting extreme tilt angles the structure modulates CDR H3 and its environment, which apparently cannot be achieved solely by conformational rearrangement of the CDR. Note that most of the VH:VL interface residues are invariant; therefore, significant change of the tilt angle must come with a penalty in free energy. Yet, for the 2 antibodies, the total gain in stability merits the domain repacking.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>50389</offset><text>Overall, the stability of the Fab, as measured by Tm, is a result of the mutual adjustment of the HC and LC variable domains and adjustment of CDR H3 to the VH:VL cleft. The final conformation represents an energetic minimum; however, in most cases it is very shallow, so that a single mutation can cause a dramatic rearrangement of the structure.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>50737</offset><text>In summary, the analysis of this structural library of germline variants composed of all pairs of 4 HCs and 4LCs, all with the same CDR H3, offers some unique insights into antibody structure and how pairing and sequence may influence, or not, the canonical structures of the L1, L2, L3, H1 and H2 CDRs. Comparison of the CDR H3s reveals a large set of variants with conformations similar to the parent, while a second set has significant conformational variability, indicating that both the sequence and the structural context define the CDR H3 conformation. Quite unexpectedly, 2 of the variants, H1-69:L3-20 and H3-53:L4-1, have the ‘extended’ stem region differing from the other 14 that have a ‘kinked’ stem region. Why this is the case is unclear at present. These data reveal the difficulty of modeling CDR H3 accurately, as shown again in Antibody Modeling Assessment II. Furthermore, antibody CDRs, H3 in particular, may go through conformational changes upon binding their targets, making structural prediction for docking purposes an even more difficult task. Fortunately, for most applications of antibody modeling, such as engineering affinity and biophysical properties, an accurate CDR H3 structure is not always necessary. For those applications where accurate CDR structures are essential, such as docking, the results in this work demonstrate the importance of experimental structures. With the recent advances in expression and crystallization methods, Fab structures can be obtained rapidly.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>52257</offset><text>The set of 16 germline Fab structures offers a unique dataset to facilitate software development for antibody modeling. The results essentially support the underlying idea of canonical structures, indicating that most CDRs with germline sequences tend to adopt predefined conformations. From this point of view, a novel approach to design combinatorial antibody libraries would be to cover the range of CDR conformations that may not necessarily coincide with the germline usage in the human repertoire. This would insure more structural diversity, leading to a more diverse panel of antibodies that would bind to a broad spectrum of targets.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>52900</offset><text>Materials and methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>52922</offset><text>Fab production, purification and crystallization</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>52971</offset><text>The production, purification and crystallization of the Fabs reported in this article were described previously. Briefly, the 16 Fabs were produced by combining 4 different HC and 4 different LC germline constructs. The human HC germlines were IGHV1-69 (H1-69), IGHV3-23 (H3-23), IGHV3-53 (H3-53) and IGHV5-51 (H5-51) in the IMGT nomenclature. The human LC germlines were IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1) corresponding to O12, L6, A27 and B3 in the V-BASE nomenclature. CDR H3 of the anti-CCL2 antibody CNTO 888 with the amino acid sequence ARYDGIYGELDF  was used in all Fab constructs. The J region genes were IGHJ1 for the HC and IGKJ1 for the LC for all Fabs. Human IgG1 and κ constant regions were used in all Fab constructs. A 6xHis tag was added to the C-terminus of the HC to facilitate purification.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>53822</offset><text>The Fabs were expressed in HEK 293E cells and purified by affinity and size-exclusion chromatography. For crystallization, the Fabs were dialyzed into 20 mM Tris buffer, pH 7.4, with 50 mM NaCl and concentrated to 12-18 mg/mL. Automated crystallization screening was carried out using the vapor diffusion method at 20°C with an Oryx4 (Douglas Instruments) or a Mosquito (TTP Labtech) crystallization robot in a sitting drop format using Corning 3550 plates. Initial screening was carried out with an in-house 192-well screen optimized for Fab crystallization and the Hampton 96-well Crystal Screen HT (Hampton Research). For the majority of the Fabs, the crystallization protocol employed microseed matrix screening using self-seeding or cross-seeding approaches. A summary of the final crystallization conditions for each of the Fabs is presented in Table 1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>54687</offset><text>X-ray data collection</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>54709</offset><text>For 13 of the Fab crystals, X-ray data collection was carried out at Janssen Research and Development, LLC using a Rigaku MicroMax™-007HF microfocus X-ray generator equipped with a Saturn 944 CCD detector and an X-stream™ 2000 cryocooling system (Rigaku), and for the remaining 3, X-ray data collection was carried out at the Advanced Photon Source (APS) synchrotron at Argonne National Laboratory using the IMCA 17-ID beamline with a Pilatus 6M detector. For X-ray data collection, the Fab crystals were soaked for a few seconds in a cryo-protectant solution containing the corresponding mother liquor supplemented with 17-25% glycerol (Table S1). The crystals for which data were collected in-house were flash cooled in the stream of nitrogen at 100 K. Crystals sent to the APS were flash cooled in liquid nitrogen prior to shipping them to the synchrotron. Diffraction data for all variants were processed with the program XDS. X-ray data statistics are given in Table 1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>55691</offset><text>Structure determination</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>55715</offset><text>A summary of the methods used in the structure solution and refinement of the 16 Fabs is presented in Table S1. Twelve of the structures were solved by molecular replacement with Phaser using different combinations of search models for the VH, VL and constant domains. Four of the structures, H3-53:L1-39, H3-53:L3-11, H5-51:L1-39 and H5-51:L3-11, were solved by direct replacement followed by rigid body refinement with REFMAC. All structures were refined using REFMAC. Model adjustments were carried out using the program Coot. The refinement statistics are given in Table 1. Other crystallographic calculations were performed with the CCP4 suite of programs. The structural figures were prepared using the PyMOL Molecular Graphics System, Version 1.0 (Schrödinger, LLC).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>56492</offset><text>Structural analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>56512</offset><text>The canonical structure assignments (Table 2) were made using PyIgClassify, an online canonical structure classification tool (http://dunbrack2.fccc.edu/pyigclassify/) that uses the rules set forth by Dunbrack and coworkers.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>56738</offset><text>The conformational variability within the CDRs was assessed by calculating the root-mean-square deviation (rmsd) from the average structure that was generated after superposition of all structures of the set using the main-chain atoms of the CDR in question. The rmsd was calculated for all main-chain atoms (N, CA, C, O) of the CDR.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>57072</offset><text>The contact surface areas of the VH and VL domains at the VH:VL inteface were computed with the CCP4 program PISA. The surface complementarity of the VH and VL domains was computed using the CCP4 program SC.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>57280</offset><text>VH:VL tilt angles</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>57298</offset><text>The orientation of the VH domain with respect to the VL domain was assessed using 2 different approaches. The first approach calculates the 6 VH:VL orientation parameters that describe the VH:VL relationship according to Dunbar and co-workers using a script downloaded from the website (http://opig.stats.ox.ac.uk/webapps/abangle). The six parameters include 5 angles, HL, H1, H2, L1 and L2, and a distance, dc. These parameters are derived by first defining 2 planes, one for each domain, based on core residues in the domains. The distance between the planes, dc, is determined along a vector between the planes that is used to establish a consistent coordinate system. The torsion angle between the domains, HL, is much like the VH:VL packing angle defined by Abhinandan and Martin. The tilt of one domain relative to the other is defined by the HC1 and LC1 angles, and the twist of one domain relative to the other is defined by the HC2 and LC2 angles.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>58255</offset><text>The second approach calculates the difference in the tilt angle between pairs of Fvs, which reflects the relative orientation between the VH and VL domains. The difference with respect to the reference structure is calculated by sequential root-mean-square superposition of the VL and VH domains using β-sheet core Cα positions (Chothia numbering scheme): 3–13, 18–25, 33–38, 43–49, 61–67, 70–76, 85–90, 97–103 for VL; 3–7, 18–24, 34–40, 44–51, 56–59, 67–72, 77–82a, 87–94, 102–110 for VH. The κ angle in the spherical polar angular system (ω, ϕ, κ) of the latter transformation is the difference in the tilt angle.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>58911</offset><text>Differential scanning calorimetry</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>58945</offset><text>DSC experiments were performed on a VP-capillary DSC system (MicroCal Inc., Northampton, MA) in which temperature differences between the reference and sample cell are continuously measured and calibrated to power units. Samples were heated from 10°C to 95°C at a heating rate of 60°C/hour. The pre-scan time was 15 minutes and the filtering period was 10 seconds. The concentration used in the DSC experiments was about 0.4 mg/mL in phosphate-buffered saline. Analysis of the resulting thermograms was performed using MicroCal Origin 7 software. Melting temperature of proteins was determined by deconvolution of the DSC scans using non-2 state model in the MicroCal Origin 7 software. Scans were deconvoluted using a non-2 state model with either 1-step transition or 2-step transition depending on the number of resolved peaks observed in a scan.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>59799</offset><text>Accession numbers</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>59817</offset><text>Atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers 4KMT, 5I15, 5I16, 5I17, 5I18, 5I19, 5I1A, 5I1C, 5I1D, 5I1E, 5I1G, 5I1H, 5I1I, 5I1J, 5I1K and 5I1L.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>60025</offset><text>Supplementary Material</text></passage><passage><infon key="section_type">COMP_INT</infon><infon key="type">title_1</infon><offset>60048</offset><text>Disclosure of potential conflicts of interest</text></passage><passage><infon key="section_type">COMP_INT</infon><infon key="type">paragraph</infon><offset>60094</offset><text>No potential conflicts of interest were disclosed.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>60145</offset><text>References</text></passage><passage><infon key="fpage">197</infon><infon key="lpage">204</infon><infon key="name_0">surname:Reichert;given-names:JM</infon><infon key="pub-id_doi">10.1080/19420862.2015.1125583</infon><infon key="pub-id_pmid">26651519</infon><infon key="section_type">REF</infon><infon key="source">MAbs</infon><infon key="type">ref</infon><infon key="volume">8</infon><infon key="year">2016</infon><offset>60156</offset><text>Antibodies to watch in 2016</text></passage><passage><infon key="fpage">211</infon><infon key="lpage">250</infon><infon key="name_0">surname:Wu;given-names:TT</infon><infon key="name_1">surname:Kabat;given-names:EA</infon><infon key="pub-id_doi">10.1084/jem.132.2.211</infon><infon key="pub-id_pmid">5508247</infon><infon key="section_type">REF</infon><infon key="source">J Exp Med</infon><infon key="type">ref</infon><infon key="volume">132</infon><infon key="year">1970</infon><offset>60184</offset><text>An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity</text></passage><passage><infon key="fpage">901</infon><infon key="lpage">917</infon><infon key="name_0">surname:Chothia;given-names:C</infon><infon key="name_1">surname:Lesk;given-names:AM</infon><infon key="pub-id_doi">10.1016/0022-2836(87)90412-8</infon><infon key="pub-id_pmid">3681981</infon><infon key="section_type">REF</infon><infon key="source">J Mol Biol</infon><infon key="type">ref</infon><infon key="volume">196</infon><infon key="year">1987</infon><offset>60338</offset><text>Canonical structures for the hypervariable regions of immunoglobulins</text></passage><passage><infon key="fpage">877</infon><infon key="lpage">883</infon><infon key="name_0">surname:Chothia;given-names:C</infon><infon key="name_1">surname:Lesk;given-names:AM</infon><infon key="name_2">surname:Tramontano;given-names:A</infon><infon key="name_3">surname:Levitt;given-names:M</infon><infon key="name_4">surname:Smith-Gill;given-names:SJ</infon><infon key="name_5">surname:Air;given-names:G</infon><infon key="name_6">surname:Sheriff;given-names:S</infon><infon key="name_7">surname:Padlan;given-names:EA</infon><infon key="name_8">surname:Davies;given-names:D</infon><infon key="name_9">surname:Tulip;given-names:WR</infon><infon key="pub-id_doi">10.1038/342877a0</infon><infon key="pub-id_pmid">2687698</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">342</infon><infon key="year">1989</infon><offset>60408</offset><text>Conformations of immunoglobulin hypervariable regions</text></passage><passage><infon key="fpage">799</infon><infon key="lpage">817</infon><infon key="name_0">surname:Chothia;given-names:C</infon><infon key="name_1">surname:Lesk;given-names:AM</infon><infon key="name_2">surname:Gherardi;given-names:E</infon><infon key="name_3">surname:Tomlinson;given-names:IM</infon><infon key="name_4">surname:Walter;given-names:G</infon><infon key="name_5">surname:Marks;given-names:JD</infon><infon key="name_6">surname:Llewelyn;given-names:MB</infon><infon key="name_7">surname:Winter;given-names:G</infon><infon key="pub-id_doi">10.1016/0022-2836(92)90224-8</infon><infon key="pub-id_pmid">1404389</infon><infon key="section_type">REF</infon><infon key="source">J Mol Biol</infon><infon key="type">ref</infon><infon key="volume">227</infon><infon key="year">1992</infon><offset>60462</offset><text>Structural repertoire of the human VH segments</text></passage><passage><infon key="fpage">4628</infon><infon key="lpage">4638</infon><infon key="name_0">surname:Tomlinson;given-names:IM</infon><infon key="name_1">surname:Cox;given-names:JP</infon><infon key="name_2">surname:Herardi;given-names:GE</infon><infon key="name_3">surname:Lesk;given-names:AM</infon><infon key="name_4">surname:Chothia;given-names:C</infon><infon key="pub-id_pmid">7556106</infon><infon key="section_type">REF</infon><infon key="source">EMBO J</infon><infon key="type">ref</infon><infon key="volume">14</infon><infon key="year">1995</infon><offset>60509</offset><text>The structural repertoire of the human V kappa domain</text></passage><passage><infon key="fpage">497</infon><infon key="lpage">504</infon><infon key="name_0">surname:Vargas-Madrazo;given-names:E</infon><infon key="name_1">surname:Lara-Ochoa;given-names:F</infon><infon key="name_2">surname:Almagro;given-names:JC</infon><infon key="pub-id_doi">10.1006/jmbi.1995.0633</infon><infon key="pub-id_pmid">7490765</infon><infon key="section_type">REF</infon><infon key="source">J Mol Biol</infon><infon key="type">ref</infon><infon key="volume">254</infon><infon key="year">1995</infon><offset>60563</offset><text>Canonical structure repertoire of the antigen-binding site of immunoglobulins suggests strong geometrical restrictions associated to the mechanism of immune recognition</text></passage><passage><infon key="fpage">927</infon><infon key="lpage">948</infon><infon key="name_0">surname:Al-Lazikani;given-names:B</infon><infon key="name_1">surname:Lesk;given-names:AM</infon><infon key="name_2">surname:Chothia;given-names:C</infon><infon key="pub-id_doi">10.1006/jmbi.1997.1354</infon><infon key="pub-id_pmid">9367782</infon><infon key="section_type">REF</infon><infon key="source">J Mol Biol</infon><infon key="type">ref</infon><infon key="volume">273</infon><infon key="year">1997</infon><offset>60732</offset><text>Standard conformations for the canonical structures of immunoglobulins</text></passage><passage><infon key="fpage">337</infon><infon key="lpage">354</infon><infon key="name_0">surname:Collis;given-names:AV</infon><infon key="name_1">surname:Brouwer;given-names:AP</infon><infon key="name_2">surname:Martin;given-names:AC</infon><infon key="pub-id_doi">10.1016/S0022-2836(02)01222-6</infon><infon key="pub-id_pmid">12488099</infon><infon key="section_type">REF</infon><infon key="source">J Mol Biol</infon><infon key="type">ref</infon><infon key="volume">325</infon><infon key="year">2003</infon><offset>60803</offset><text>Analysis of the antigen combining site: correlations between length and sequence composition of the hypervariable loops and the nature of the antigen</text></passage><passage><infon key="fpage">235</infon><infon key="lpage">242</infon><infon key="name_0">surname:Berman;given-names:HM</infon><infon key="name_1">surname:Westbrook;given-names:J</infon><infon key="name_2">surname:Feng;given-names:Z</infon><infon key="name_3">surname:Gilliland;given-names:G</infon><infon key="name_4">surname:Bhat;given-names:TN</infon><infon key="name_5">surname:Weissig;given-names:H</infon><infon key="name_6">surname:Shindyalov;given-names:IN</infon><infon key="name_7">surname:Bourne;given-names:PE</infon><infon key="pub-id_doi">10.1093/nar/28.1.235</infon><infon key="pub-id_pmid">10592235</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res</infon><infon key="type">ref</infon><infon key="volume">28</infon><infon key="year">2000</infon><offset>60953</offset><text>The Protein Data Bank</text></passage><passage><infon key="fpage">800</infon><infon key="lpage">815</infon><infon key="name_0">surname:Martin;given-names:AC</infon><infon key="name_1">surname:Thornton;given-names:JM</infon><infon key="pub-id_doi">10.1006/jmbi.1996.0617</infon><infon key="pub-id_pmid">8947577</infon><infon key="section_type">REF</infon><infon key="source">J Mol Biol</infon><infon key="type">ref</infon><infon key="volume">263</infon><infon key="year">1996</infon><offset>60975</offset><text>Structural families in loops of homologous proteins: automatic classification, modelling and application to antibodies</text></passage><passage><infon key="fpage">228</infon><infon key="lpage">256</infon><infon key="name_0">surname:North;given-names:B</infon><infon key="name_1">surname:Lehmann;given-names:A</infon><infon key="name_2">surname:Dunbrack;given-names:RL</infon><infon key="pub-id_doi">10.1016/j.jmb.2010.10.030</infon><infon key="pub-id_pmid">21035459</infon><infon key="section_type">REF</infon><infon key="source">J Mol Biol</infon><infon key="type">ref</infon><infon key="volume">406</infon><infon key="year">2011</infon><offset>61094</offset><text>A new clustering of antibody CDR loop conformations</text></passage><passage><infon key="fpage">D432</infon><infon key="lpage">D438</infon><infon key="name_0">surname:Adolf-Bryfogle;given-names:J</infon><infon key="name_1">surname:Xu;given-names:Q</infon><infon key="name_2">surname:North;given-names:B</infon><infon key="name_3">surname:Lehmann;given-names:A</infon><infon key="name_4">surname:Dunbrack;given-names:RL</infon><infon key="pub-id_doi">10.1093/nar/gku1106</infon><infon key="pub-id_pmid">25392411</infon><infon key="section_type">REF</infon><infon key="source">Nucleic Acids Res</infon><infon key="type">ref</infon><infon key="volume">43</infon><infon key="year">2015</infon><offset>61146</offset><text>PyIgClassify: a database of antibody CDR structural classifications</text></passage><passage><infon key="fpage">1</infon><infon key="lpage">8</infon><infon key="name_0">surname:Shirai;given-names:H</infon><infon key="name_1">surname:Kidera;given-names:A</infon><infon key="name_2">surname:Nakamura;given-names:H</infon><infon key="pub-id_doi">10.1016/S0014-5793(96)01252-5</infon><infon key="pub-id_pmid">8980108</infon><infon key="section_type">REF</infon><infon key="source">FEBS Lett</infon><infon key="type">ref</infon><infon key="volume">399</infon><infon key="year">1996</infon><offset>61214</offset><text>Structural classification of CDR-H3 in antibodies</text></passage><passage><infon key="fpage">9</infon><infon key="lpage">16</infon><infon key="name_0">surname:Morea;given-names:V</infon><infon key="name_1">surname:Tramontano;given-names:A</infon><infon key="name_2">surname:Rustici;given-names:M</infon><infon key="name_3">surname:Chothia;given-names:C</infon><infon key="name_4">surname:Lesk;given-names:AM</infon><infon key="pub-id_doi">10.1016/S0301-4622(96)02266-1</infon><infon key="pub-id_pmid">9468606</infon><infon key="section_type">REF</infon><infon key="source">Biophys Chem</infon><infon key="type">ref</infon><infon key="volume">68</infon><infon key="year">1997</infon><offset>61264</offset><text>Antibody structure, prediction and redesign</text></passage><passage><infon key="fpage">269</infon><infon key="lpage">294</infon><infon key="name_0">surname:Morea;given-names:V</infon><infon key="name_1">surname:Tramontano;given-names:A</infon><infon key="name_2">surname:Rustici;given-names:M</infon><infon key="name_3">surname:Chothia;given-names:C</infon><infon key="name_4">surname:Lesk;given-names:AM</infon><infon key="pub-id_doi">10.1006/jmbi.1997.1442</infon><infon key="pub-id_pmid">9466909</infon><infon key="section_type">REF</infon><infon key="source">J Mol Biol</infon><infon key="type">ref</infon><infon key="volume">275</infon><infon key="year">1998</infon><offset>61308</offset><text>Conformations of the third hypervariable region in the VH domain of immunoglobulins</text></passage><passage><infon key="fpage">608</infon><infon key="lpage">620</infon><infon key="name_0">surname:Kuroda;given-names:D</infon><infon key="name_1">surname:Shirai;given-names:H</infon><infon key="name_2">surname:Kobori;given-names:M</infon><infon key="name_3">surname:Nakamura;given-names:H</infon><infon key="pub-id_doi">10.1002/prot.22087</infon><infon key="pub-id_pmid">18473362</infon><infon key="section_type">REF</infon><infon key="source">Proteins</infon><infon key="type">ref</infon><infon key="volume">73</infon><infon key="year">2008</infon><offset>61392</offset><text>Structural classification of CDR-H3 revisited: a lesson in antibody modeling</text></passage><passage><infon key="fpage">302</infon><infon key="lpage">311</infon><infon key="name_0">surname:Weitzner;given-names:BD</infon><infon key="name_1">surname:Dunbrack;given-names:RL</infon><infon key="name_2">surname:Gray;given-names:JJ</infon><infon key="pub-id_doi">10.1016/j.str.2014.11.010</infon><infon key="pub-id_pmid">25579815</infon><infon key="section_type">REF</infon><infon key="source">Structure</infon><infon key="type">ref</infon><infon key="volume">23</infon><infon key="year">2015</infon><offset>61469</offset><text>The origin of CDR H3 structural diversity</text></passage><passage><infon key="fpage">3050</infon><infon key="lpage">3066</infon><infon key="name_0">surname:Almagro;given-names:JC</infon><infon key="name_1">surname:Beavers;given-names:MP</infon><infon key="name_2">surname:Hernandez-Guzman;given-names:F</infon><infon key="name_3">surname:Maier;given-names:J</infon><infon key="name_4">surname:Shaulsky;given-names:J</infon><infon key="name_5">surname:Butenhof;given-names:K</infon><infon key="name_6">surname:Labute;given-names:P</infon><infon key="name_7">surname:Thorsteinson;given-names:N</infon><infon key="name_8">surname:Kelly;given-names:K</infon><infon key="name_9">surname:Teplyakov;given-names:A</infon><infon key="pub-id_doi">10.1002/prot.23130</infon><infon key="pub-id_pmid">21935986</infon><infon key="section_type">REF</infon><infon key="source">Proteins</infon><infon key="type">ref</infon><infon key="volume">79</infon><infon key="year">2011</infon><offset>61511</offset><text>Antibody modeling assessment</text></passage><passage><infon key="fpage">1553</infon><infon key="lpage">1562</infon><infon key="name_0">surname:Almagro;given-names:JC</infon><infon key="name_1">surname:Teplyakov;given-names:A</infon><infon key="name_2">surname:Luo;given-names:J</infon><infon key="name_3">surname:Sweet;given-names:R</infon><infon key="name_4">surname:Kodangattil;given-names:S</infon><infon key="name_5">surname:Hernadez-Guzman;given-names:F</infon><infon key="name_6">surname:Gilliland;given-names:GL</infon><infon key="pub-id_doi">10.1002/prot.24567</infon><infon key="pub-id_pmid">24668560</infon><infon key="section_type">REF</infon><infon key="source">Proteins</infon><infon key="type">ref</infon><infon key="volume">82</infon><infon key="year">2014</infon><offset>61540</offset><text>Second Antibody Modeling Assessment (AMA-II)</text></passage><passage><infon key="fpage">1563</infon><infon key="lpage">1582</infon><infon key="name_0">surname:Teplyakov;given-names:A</infon><infon key="name_1">surname:Luo;given-names:J</infon><infon key="name_2">surname:Obmolova;given-names:G</infon><infon key="name_3">surname:Malia;given-names:TJ</infon><infon key="name_4">surname:Sweet;given-names:R</infon><infon key="name_5">surname:Stanfield;given-names:RL</infon><infon key="name_6">surname:Kodangattil;given-names:S</infon><infon key="name_7">surname:Almagro;given-names:JC</infon><infon key="name_8">surname:Gilliland;given-names:GL</infon><infon key="pub-id_doi">10.1002/prot.24554</infon><infon key="pub-id_pmid">24633955</infon><infon key="section_type">REF</infon><infon key="source">Proteins</infon><infon key="type">ref</infon><infon key="volume">82</infon><infon key="year">2014</infon><offset>61585</offset><text>Antibody modeling assessment II. Structures and models</text></passage><passage><infon key="fpage">385</infon><infon key="lpage">396</infon><infon key="name_0">surname:Shi;given-names:L</infon><infon key="name_1">surname:Wheeler;given-names:JC</infon><infon key="name_2">surname:Sweet;given-names:RW</infon><infon key="name_3">surname:Lu;given-names:J</infon><infon key="name_4">surname:Luo;given-names:J</infon><infon key="name_5">surname:Tornetta;given-names:M</infon><infon key="name_6">surname:Whitaker;given-names:B</infon><infon key="name_7">surname:Reddy;given-names:R</infon><infon key="name_8">surname:Brittingham;given-names:R</infon><infon key="name_9">surname:Borozdina;given-names:L</infon><infon key="pub-id_doi">10.1016/j.jmb.2010.01.034</infon><infon key="pub-id_pmid">20114051</infon><infon key="section_type">REF</infon><infon key="source">J Mol Biol</infon><infon key="type">ref</infon><infon key="volume">397</infon><infon key="year">2010</infon><offset>61640</offset><text>De novo selection of high-affinity antibodies from synthetic fab libraries displayed on phage as pIX fusion proteins</text></passage><passage><infon key="fpage">895</infon><infon key="lpage">901</infon><infon key="name_0">surname:de Wildt;given-names:RM</infon><infon key="name_1">surname:Hoet;given-names:RM</infon><infon key="name_2">surname:van Venrooij;given-names:WJ</infon><infon key="name_3">surname:Tomlinson;given-names:IM</infon><infon key="name_4">surname:Winter;given-names:G</infon><infon key="pub-id_doi">10.1006/jmbi.1998.2396</infon><infon key="pub-id_pmid">9887257</infon><infon key="section_type">REF</infon><infon key="source">J Mol Biol</infon><infon key="type">ref</infon><infon key="volume">285</infon><infon key="year">1999</infon><offset>61757</offset><text>Analysis of heavy and light chain pairings indicates that receptor editing shapes the human antibody repertoire</text></passage><passage><infon key="fpage">227</infon><infon key="lpage">233</infon><infon key="name_0">surname:Obmolova;given-names:G</infon><infon key="name_1">surname:Teplyakov;given-names:A</infon><infon key="name_2">surname:Malia;given-names:TJ</infon><infon key="name_3">surname:Grygiel;given-names:TL</infon><infon key="name_4">surname:Sweet;given-names:R</infon><infon key="name_5">surname:Snyder;given-names:LA</infon><infon key="name_6">surname:Gilliland;given-names:GL</infon><infon key="pub-id_doi">10.1016/j.molimm.2012.03.022</infon><infon key="pub-id_pmid">22487721</infon><infon key="section_type">REF</infon><infon key="source">Mol Immunol</infon><infon key="type">ref</infon><infon key="volume">51</infon><infon key="year">2012</infon><offset>61869</offset><text>Structural basis for high selectivity of anti-CCL2 neutralizing antibody CNTO 888</text></passage><passage><infon key="fpage">1107</infon><infon key="lpage">1115</infon><infon key="name_0">surname:Obmolova;given-names:G</infon><infon key="name_1">surname:Malia;given-names:TJ</infon><infon key="name_2">surname:Teplyakov;given-names:A</infon><infon key="name_3">surname:Sweet;given-names:R</infon><infon key="name_4">surname:Gilliland;given-names:GL</infon><infon key="pub-id_doi">10.1107/S2053230X14012552</infon><infon key="pub-id_pmid">25084393</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr</infon><infon key="type">ref</infon><infon key="volume">F70</infon><infon key="year">2014</infon><offset>61951</offset><text>Protein crystallization with microseed matrix screening: application to human germline antibody Fabs</text></passage><passage><infon key="fpage">175</infon><infon key="lpage">182</infon><infon key="name_0">surname:Tramontano;given-names:A</infon><infon key="name_1">surname:Chothia;given-names:C</infon><infon key="name_2">surname:Lesk;given-names:AM</infon><infon key="pub-id_doi">10.1016/S0022-2836(05)80102-0</infon><infon key="pub-id_pmid">2118959</infon><infon key="section_type">REF</infon><infon key="source">J Mol Biol</infon><infon key="type">ref</infon><infon key="volume">215</infon><infon key="year">1990</infon><offset>62052</offset><text>Framework residue-71 is a major determinant of the position and conformation of the 2nd hypervariable region in the VH domains of immunoglobulins</text></passage><passage><infon key="fpage">483</infon><infon key="lpage">498</infon><infon key="name_0">surname:Mas;given-names:MT</infon><infon key="name_1">surname:Smith;given-names:KC</infon><infon key="name_2">surname:Yarmush;given-names:DL</infon><infon key="name_3">surname:Aisaka;given-names:K</infon><infon key="name_4">surname:Fine;given-names:RM</infon><infon key="pub-id_doi">10.1002/prot.340140409</infon><infon key="pub-id_pmid">1438186</infon><infon key="section_type">REF</infon><infon key="source">Proteins</infon><infon key="type">ref</infon><infon key="volume">14</infon><infon key="year">1992</infon><offset>62198</offset><text>Modeling the anti-CEA antibody combining site by homology and conformational search</text></passage><passage><infon key="fpage">83</infon><infon key="lpage">93</infon><infon key="name_0">surname:Stanfield;given-names:RL</infon><infon key="name_1">surname:Takimoto-Kamimura;given-names:M</infon><infon key="name_2">surname:Rini;given-names:JM</infon><infon key="name_3">surname:Profy;given-names:AT</infon><infon key="name_4">surname:Wilson;given-names:IA</infon><infon key="pub-id_doi">10.1016/0969-2126(93)90024-B</infon><infon key="pub-id_pmid">8069628</infon><infon key="section_type">REF</infon><infon key="source">Structure</infon><infon key="type">ref</infon><infon key="volume">1</infon><infon key="year">1993</infon><offset>62282</offset><text>Major antigen-induced domain rearrangements in an antibody</text></passage><passage><infon key="fpage">941</infon><infon key="lpage">953</infon><infon key="name_0">surname:Narayanan;given-names:A</infon><infon key="name_1">surname:Sellers;given-names:BD</infon><infon key="name_2">surname:Jacobson;given-names:MP</infon><infon key="pub-id_doi">10.1016/j.jmb.2009.03.043</infon><infon key="pub-id_pmid">19324053</infon><infon key="section_type">REF</infon><infon key="source">J Mol Biol</infon><infon key="type">ref</infon><infon key="volume">388</infon><infon key="year">2009</infon><offset>62341</offset><text>Energy-based analysis and prediction of the orientation between light- and heavy-chain antibody variable domains</text></passage><passage><infon key="fpage">689</infon><infon key="lpage">697</infon><infon key="name_0">surname:Abhinandan;given-names:KR</infon><infon key="name_1">surname:Martin;given-names:AC</infon><infon key="pub-id_doi">10.1093/protein/gzq043</infon><infon key="pub-id_pmid">20591902</infon><infon key="section_type">REF</infon><infon key="source">Protein Eng Des Sel</infon><infon key="type">ref</infon><infon key="volume">23</infon><infon key="year">2010</infon><offset>62454</offset><text>Analysis and prediction of VH/VL packing in antibodies</text></passage><passage><infon key="fpage">611</infon><infon key="lpage">620</infon><infon key="name_0">surname:Dunbar;given-names:J</infon><infon key="name_1">surname:Fuchs;given-names:A</infon><infon key="name_2">surname:Shi;given-names:J</infon><infon key="name_3">surname:Deane;given-names:CM</infon><infon key="pub-id_doi">10.1093/protein/gzt020</infon><infon key="pub-id_pmid">23708320</infon><infon key="section_type">REF</infon><infon key="source">Protein Eng Des Sel</infon><infon key="type">ref</infon><infon key="volume">26</infon><infon key="year">2013</infon><offset>62509</offset><text>ABangle: characterising the VH-VL orientation in antibodies</text></passage><passage><infon key="fpage">774</infon><infon key="lpage">797</infon><infon key="name_0">surname:Krissinel;given-names:E</infon><infon key="name_1">surname:Henrick;given-names:K</infon><infon key="pub-id_doi">10.1016/j.jmb.2007.05.022</infon><infon key="pub-id_pmid">17681537</infon><infon key="section_type">REF</infon><infon key="source">J Mol Biol</infon><infon key="type">ref</infon><infon key="volume">372</infon><infon key="year">2007</infon><offset>62569</offset><text>Inference of macromolecular assemblies from crystalline state</text></passage><passage><infon key="fpage">946</infon><infon key="lpage">950</infon><infon key="name_0">surname:Lawrence;given-names:MC</infon><infon key="name_1">surname:Colman;given-names:PM</infon><infon key="pub-id_doi">10.1006/jmbi.1993.1648</infon><infon key="pub-id_pmid">8263940</infon><infon key="section_type">REF</infon><infon key="source">J Mol Biol</infon><infon key="type">ref</infon><infon key="volume">234</infon><infon key="year">1993</infon><offset>62631</offset><text>Shape complementarity at protein/protein interfaces</text></passage><passage><infon key="fpage">959</infon><infon key="lpage">965</infon><infon key="name_0">surname:Rini;given-names:JM</infon><infon key="name_1">surname:Schulze-Gahmen;given-names:U</infon><infon key="name_2">surname:Wilson;given-names:IA</infon><infon key="pub-id_doi">10.1126/science.1546293</infon><infon key="pub-id_pmid">1546293</infon><infon key="section_type">REF</infon><infon key="source">Science</infon><infon key="type">ref</infon><infon key="volume">255</infon><infon key="year">1992</infon><offset>62683</offset><text>Structural evidence for induced fit as a mechanism for antibody-antigen recognition</text></passage><passage><infon key="comment">Appendix 1P</infon><infon key="name_0">surname:Lefranc;given-names:MP</infon><infon key="pub-id_doi">10.1002/0471142735.ima01ps40</infon><infon key="pub-id_pmid">18432650</infon><infon key="section_type">REF</infon><infon key="source">Curr Protoc Immunol</infon><infon key="type">ref</infon><infon key="year">2001</infon><offset>62767</offset><text>Nomenclature of the human immunoglobulin genes</text></passage><passage><infon key="name_0">surname:Tomlinson;given-names:IM</infon><infon key="name_1">surname:Williams;given-names:SC</infon><infon key="name_2">surname:Ignatovitch;given-names:O</infon><infon key="name_3">surname:Corbett;given-names:SJ</infon><infon key="name_4">surname:Winter;given-names:G</infon><infon key="section_type">REF</infon><infon key="source">V BASE Sequence Directory, MRC Centre for Protein Engineering</infon><infon key="type">ref</infon><infon key="year">1998</infon><offset>62814</offset></passage><passage><infon key="fpage">182</infon><infon key="lpage">189</infon><infon key="name_0">surname:Zhao;given-names:Y</infon><infon key="name_1">surname:Gutshall;given-names:L</infon><infon key="name_2">surname:Jiang;given-names:H</infon><infon key="name_3">surname:Baker;given-names:A</infon><infon key="name_4">surname:Beil;given-names:E</infon><infon key="name_5">surname:Obmolova;given-names:G</infon><infon key="name_6">surname:Carton;given-names:J</infon><infon key="name_7">surname:Taudte;given-names:S</infon><infon key="name_8">surname:Amegadzie;given-names:B</infon><infon key="pub-id_doi">10.1016/j.pep.2009.04.012</infon><infon key="pub-id_pmid">19442740</infon><infon key="section_type">REF</infon><infon key="source">Protein Expr Purif</infon><infon key="type">ref</infon><infon key="volume">67</infon><infon key="year">2009</infon><offset>62815</offset><text>Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: papain digestion of mAb and transient expression in mammalian cells</text></passage><passage><infon key="fpage">550</infon><infon key="lpage">554</infon><infon key="name_0">surname:D'Arcy;given-names:A</infon><infon key="name_1">surname:Villard;given-names:F</infon><infon key="name_2">surname:Marsh;given-names:M</infon><infon key="pub-id_doi">10.1107/S0907444907007652</infon><infon key="pub-id_pmid">17372361</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr</infon><infon key="type">ref</infon><infon key="volume">D63</infon><infon key="year">2007</infon><offset>62984</offset><text>An automated microseed matrix-screening method for protein crystallization</text></passage><passage><infon key="fpage">927</infon><infon key="lpage">933</infon><infon key="name_0">surname:Obmolova;given-names:G</infon><infon key="name_1">surname:Malia;given-names:TJ</infon><infon key="name_2">surname:Teplyakov;given-names:A</infon><infon key="name_3">surname:Sweet;given-names:R</infon><infon key="name_4">surname:Gilliland;given-names:GL</infon><infon key="pub-id_doi">10.1107/S0907444910026041</infon><infon key="pub-id_pmid">20693692</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr</infon><infon key="type">ref</infon><infon key="volume">D66</infon><infon key="year">2010</infon><offset>63059</offset><text>Promoting crystallization of antibody-antigen complexes via microseed matrix screening</text></passage><passage><infon key="fpage">125</infon><infon key="lpage">132</infon><infon key="name_0">surname:Kabsch;given-names:W</infon><infon key="pub-id_doi">10.1107/S0907444909047337</infon><infon key="pub-id_pmid">20124692</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr</infon><infon key="type">ref</infon><infon key="volume">D66</infon><infon key="year">2010</infon><offset>63146</offset><text>XDS</text></passage><passage><infon key="fpage">658</infon><infon key="lpage">674</infon><infon key="name_0">surname:McCoy;given-names:AJ</infon><infon key="name_1">surname:Grosse-Kunstleve;given-names:RW</infon><infon key="name_2">surname:Adams;given-names:PD</infon><infon key="name_3">surname:Winn;given-names:MD</infon><infon key="name_4">surname:Storoni;given-names:LC</infon><infon key="name_5">surname:Read;given-names:RJ</infon><infon key="pub-id_doi">10.1107/S0021889807021206</infon><infon key="pub-id_pmid">19461840</infon><infon key="section_type">REF</infon><infon key="source">J Appl Crystallogr</infon><infon key="type">ref</infon><infon key="volume">40</infon><infon key="year">2007</infon><offset>63150</offset><text>Phaser crystallographic software</text></passage><passage><infon key="fpage">355</infon><infon key="lpage">367</infon><infon key="name_0">surname:Murshudov;given-names:GN</infon><infon key="name_1">surname:Skubak;given-names:P</infon><infon key="name_2">surname:Lebedev;given-names:AA</infon><infon key="name_3">surname:Pannu;given-names:NS</infon><infon key="name_4">surname:Steiner;given-names:RA</infon><infon key="name_5">surname:Nicholls;given-names:RA</infon><infon key="name_6">surname:Winn;given-names:MD</infon><infon key="name_7">surname:Long;given-names:F</infon><infon key="name_8">surname:Vagin;given-names:AA</infon><infon key="pub-id_doi">10.1107/S0907444911001314</infon><infon key="pub-id_pmid">21460454</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr</infon><infon key="type">ref</infon><infon key="volume">D67</infon><infon key="year">2011</infon><offset>63183</offset><text>REFMAC5 for the refinement of macromolecular crystal structures</text></passage><passage><infon key="fpage">486</infon><infon key="lpage">501</infon><infon key="name_0">surname:Emsley;given-names:P</infon><infon key="name_1">surname:Lohkamp;given-names:B</infon><infon key="name_2">surname:Scott;given-names:WG</infon><infon key="name_3">surname:Cowtan;given-names:K</infon><infon key="pub-id_doi">10.1107/S0907444910007493</infon><infon key="pub-id_pmid">20383002</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr</infon><infon key="type">ref</infon><infon key="volume">D66</infon><infon key="year">2010</infon><offset>63247</offset><text>Features and development of Coot</text></passage><passage><infon key="fpage">235</infon><infon key="lpage">242</infon><infon key="name_0">surname:Winn;given-names:MD</infon><infon key="name_1">surname:Ballard;given-names:CC</infon><infon key="name_2">surname:Cowtan;given-names:KD</infon><infon key="name_3">surname:Dodson;given-names:EJ</infon><infon key="name_4">surname:Emsley;given-names:P</infon><infon key="name_5">surname:Evans;given-names:PR</infon><infon key="name_6">surname:Keegan;given-names:RM</infon><infon key="name_7">surname:Krissinel;given-names:EB</infon><infon key="name_8">surname:Leslie;given-names:AG</infon><infon key="name_9">surname:McCoy;given-names:A</infon><infon key="pub-id_doi">10.1107/S0907444910045749</infon><infon key="pub-id_pmid">21460441</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr</infon><infon key="type">ref</infon><infon key="volume">D67</infon><infon key="year">2011</infon><offset>63280</offset><text>Overview of the CCP4 suite and current developments</text></passage></document></collection>