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protein_structure_NER_independent_val_set

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+ [{"sourceid":"4981400","sourcedb":"","project":"","target":"","text":"Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA The Arabidopsis protein FPA controls flowering time by regulating the alternative 3′-end processing of the FLOWERING LOCUS (FLC) antisense RNA. FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain. The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower. We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 Å resolution. The overall structure is similar to that of the SPOC domain in human SMRT/HDAC1 Associated Repressor Protein (SHARP), although there are also substantial conformational differences between them. Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs. Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain. Introduction Eukaryotic messenger RNAs (mRNAs) are made as precursors through transcription by RNA polymerase II (Pol II), and these primary transcripts undergo extensive processing, including 3′-end cleavage and polyadenylation. In addition, alternative 3′-end cleavage and polyadenylation is an essential and ubiquitous process in eukaryotes. Misregulation of (alternative) 3′-end processing can lead to various genetic defects, cancer and other diseases. There is currently great interest in understanding the molecular mechanisms and functional impacts of alternative 3′-end processing. Recently, the split ends (SPEN) family of proteins was identified as RNA binding proteins that regulate alternative 3′-end cleavage and polyadenylation. They are characterized by possessing N-terminal RNA recognition motifs (RRMs) and a conserved SPEN paralog and ortholog C-terminal (SPOC) domain (Fig 1A). The SPOC domain is believed to mediate protein-protein interactions and has diverse functions among SPEN family proteins, but the molecular mechanism of these functions is not well understood. Sequence conservation of SPOC domains. (A). Domain organization of A. thaliana FPA. (B). Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP. Residues in surface patch 1 are indicated with the orange dots, and those in surface patch 2 with the green dots. The secondary structure elements in the structure of FPA SPOC are labeled. Residues that are strictly conserved among the five proteins are shown in white with a red background, and those that are mostly conserved in red. FPA, a SPEN family protein in Arabidopsis thaliana and other plants, was found to regulate the 3′-end alternative cleavage and polyadenylation of the antisense RNAs of FLOWERING LOCUS (FLC), a flowering repressor gene. FPA promotes the 3′-end processing of class I FLC antisense RNAs, which includes the proximal polyadenylation site. This is associated with histone demethylase activity and down-regulation of FLC transcription. However, the functional mechanism of this complex is still not clear. Although a SPOC domain is found in all the SPEN family proteins, its sequence conservation is rather low. For example, the sequence identity between the SPOC domains of A. thaliana FPA and human SMRT/HDAC1 Associated Repressor Protein (SHARP) is only 19% (Fig 1B). Currently, the SHARP SPOC domain is the only one with structural information. As a first step toward understanding the molecular basis for the regulation of alternative 3′-end processing and flowering by FPA, we have determined the crystal structure of the SPOC domain of A. thaliana FPA at 2.7 Å resolution. The overall structure is similar to that of the SHARP SPOC domain, although there are also substantial conformational differences between them. The structure reveals a surface patch that is conserved among FPA homologs. Results and Discussion Structure of FPA SPOC domain The crystal structure of the SPOC domain of A. thaliana FPA has been determined at 2.7 Å resolution using the selenomethionyl single-wavelength anomalous dispersion method. The expression construct contained residues 433–565 of FPA, but only residues 439–460 and 465–565 are ordered in the crystal. The atomic model has good agreement with the X-ray diffraction data and the expected bond lengths, bond angles and other geometric parameters (Table 1). All the residues are located in the favored regions of the Ramachandran plot (data not shown). The structure has been deposited in the Protein Data Bank, with accession code 5KXF. Summary of crystallographic information. Resolution range (Å)1\t50–2.7 (2.8–2.7)\t \tNumber of observations\t78,008\t \tRmerge (%)\t10.5 (45.3)\t \tI/σI\t24.1 (6.3)\t \tRedundancy\t\t \tCompleteness (%)\t100 (100)\t \tR factor (%)\t19.2 (25.0)\t \tFree R factor (%)\t25.4 (35.4)\t \tRms deviation in bond lengths (Å)\t0.017\t \tRms deviation in bond angles (°)\t1.9\t \t 1The numbers in parentheses are for the highest resolution shell. The crystal structure of the FPA SPOC domain contains a seven-stranded, mostly anti-parallel β-barrel (β1-β7) and three helices (αA-αC) (Fig 2A). Only two of the neighboring strands, β1 and β3, are parallel to each other. Helix αB covers one end of the barrel, while helices αA and αC are located next to each other at one side of the barrel (Fig 2B). The other end of the β-barrel is covered by the loop connecting strands β2 and β3, which contains the disordered 461–464 segment. The center of the barrel is filled with hydrophobic side chains and is not accessible to the solvent. Crystal structure of the SPOC domain of A. thaliana FPA. (A). Schematic drawing of the structure of FPA SPOC domain, colored from blue at the N terminus to red at the C terminus. The view is from the side of the β-barrel. The disordered segment (residues 460–465) is indicated with the dotted line. (B). Structure of the FPA SPOC domain, viewed from the end of the β-barrel, after 90° rotation around the horizontal axis from panel A. All structure figures were produced with PyMOL (www.pymol.org). Comparisons to structural homologs of the SPOC domain Only five structural homologs of the FPA SPOC domain were found in the Protein Data Bank with the DaliLite server, suggesting that the SPOC domain structure is relatively unique. The top hit is the SPOC domain of human SHARP (Fig 3A), with a Z score of 12.3. The other four structural homologs include the β-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D). The next structural homolog has a Z score of 3.0. Structural homologs of the FPA SPOC domain. (A). Overlay of the structures of the FPA SPOC domain (cyan) and the SHARP SPOC domain (gray). The bound position of a doubly-phosphorylated peptide from SMRT is shown in magenta. (B). Overlay of the structures of the FPA SPOC domain (cyan) and the Ku70 β-barrel domain (gray). Ku80 contains a homologous domain (green), which forms a hetero-dimer with that in Ku70. The two domains, and inserted segments on them, mediate the binding of dsDNA (orange). The red rectangle highlights the region of contact between the two β-barrel domains. (C). Overlay of the structures of the FPA SPOC domain (cyan) and the homologous domain in Chp1 (gray). The binding partner of Chp1, Tas3, is shown in green. The red rectangle indicates the region equivalent to the binding site of the SMART phosphopeptide in SHARP SPOC domain, where a loop of Tas3 is also located. (D). Overlay of the structures of the FPA SPOC domain (cyan) and the Med25 ACID (gray). SHARP is a transcriptional co-repressor in the nuclear receptor and Notch/RBP-Jκ signaling pathways. The SPOC domain of SHARP interacts directly with silencing mediator for retinoid and thyroid receptor (SMRT), nuclear receptor co-repressor (N-CoR), HDAC, and other components to represses transcription. While the overall structure of the FPA SPOC domain is similar to that of the SHARP SPOC domain, there are noticeable differences in the positioning of the β-strands and the helices, and most of the loops have substantially different conformations as well (Fig 3A). In addition, the SHARP SPOC domain has three extra helices. One of them covers the other end of the β-barrel, and the other two shield an additional surface of the side of the β-barrel from solvent. A doubly-phosphorylated peptide from SMRT is bound to the side of the barrel, near strands β1 and β3 (Fig 3A). Such a binding mode probably would not be possible in FPA, as the peptide would clash with the β1-β2 loop. The Ku70-Ku80 hetero-dimer is involved in DNA double-strand break repair and the β-barrel domain contributes to DNA binding. In fact, the β-barrel domains of Ku70 and Ku80 form a hetero-dimer, primarily through interactions between the loops connecting the third and fourth strands of the barrel (Fig 3B). The open ends of the two β-barrels face the DNA binding sites, and contact the phosphodiester backbone of the dsDNA. In addition, a long insert connecting strands β2 and β3 in the two domains form an arch-like structure, encircling the dsDNA. Chp1 is a subunit of the RNA-induced initiation of transcriptional gene silencing (RITS) complex. The partner of Chp1, Tas3, is bound between the barrel domain and the second domain of Chp1, and the linker between the two domains is also crucial for this interaction (Fig 3C). It is probably unlikely that the β-barrel itself is sufficient to bind Tas3. Interestingly, a loop in Tas3 contacts strand β3 of the barrel domain, at a location somewhat similar to that of the N-terminal segment of the SMRT peptide in complex with SHARP SPOC domain (Fig 3A). Mediator is a coactivator complex that promotes transcription by Pol II. The Med25 subunit ACID is the target of the potent activator VP16 of the herpes simplex virus. The structure of ACID contains a helix at the C-terminus as well as an extended β1-β2 loop. Nonetheless, the binding site for VP16 has been mapped to roughly the same surface patch, near strands β1 and β3, that is used by the SHARP and Tas3 SPOC domains for binding their partners. A conserved surface patch in the FPA SPOC domain An analysis of the SPOC domain indicates a large surface patch near strands β1, β3, β5 and β6 that is conserved among plant FPA homologs (Fig 4A). This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B). The first surface patch is electropositive in nature (Fig 4C), and residues Arg477 and Tyr515 are also conserved in the SHARP SPOC domain (Fig 1B). In fact, one of the phosphorylated residues of the SMRT peptide interacts with this surface patch (Fig 3A), suggesting that the FPA SPOC domain might also interact with a phosphorylated segment here. In comparison, the second surface patch is more hydrophobic in nature (Fig 4C). A conserved surface patch of FPA SPOC domain. (A). Two views of the molecular surface of FPA SPOC domain colored based on sequence conservation among plant FPA homologs. Purple: most conserved; cyan: least conserved. (B). Residues in the conserved surface patch of FPA SPOC domain. The side chains of the residues are shown in stick models, colored orange in the first sub-patch and green in the second. (C). Molecular surface of FPA SPOC domain colored based on electrostatic potential. Blue: positively charged; red: negatively charged. Testing the requirement of specific conserved amino acids for FPA functions We next examined the potential impact of the conserved surface patch on FPA function in vivo. We mutated two residues, Arg477 and Tyr515, of the surface patch, which are also conserved in the SHARP SPOC domain (Fig 1B) and were found to be functionally important. The mutations were introduced into a transgene designed to express FPA from its native control elements (promoter, introns and 3′ UTR). The resulting transgenes were then stably transformed into an fpa-8 mutant background so that the impact of the mutations on FPA function could be assessed. Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3′-end formation, for example in FPA pre-mRNA (panel B in S1 Fig). We examined independent transgenic lines expressing each R477A and Y515A mutation. In each case, we confirmed that detectable levels of FPA protein expression were restored close to wild-type levels in protein blot analyses using antibodies that specifically recognize FPA (S2 Fig). We then examined the impact of the surface patch mutations on FPA’s function in controlling RNA 3′-end formation by determining whether the mutant proteins functioned in FPA autoregulation and the repression of FLC expression. FPA autoregulates its expression by promoting cleavage and polyadenylation within intron 1 of its own pre-mRNA, resulting in a truncated transcript that does not encode functional protein. We used RNA gel blot analyses to reveal that in each of three independent transgenic lines for each single mutant, rescue of proximally polyadenylated FPA pre-mRNA can be detected (Fig 5A and 5B). We therefore conclude that neither of these mutations disrupted the ability of FPA to promote RNA 3′-end formation in its own transcript. Impact of individual FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA. RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A\n(A), or FPA::FPA Y515A\n(B) using poly(A)+ purified mRNAs. A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs. RNA size (kb) marker (Ambion). TUBULIN was detected as an internal control. Proximally and distally polyadenylated FPA transcripts are marked with arrows. The ratio of distal:proximal polyadenylated forms is given under each lane. (C,D) Impact of individual FPA SPOC domain mutations on FLC transcript levels. qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, 35S::FPA:YFP and FPA::FPA R477A\n(C), FPA::FPA Y515A\n(D) plants. Transcript levels were normalized to the control UBC. Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates. We next examined whether the corresponding mutations disrupted the ability of FPA to control FLC expression. We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D). Since each surface patch mutation appeared to be insufficient to disrupt FPA functions on its own, we combined both mutations into the same transgene. We could again confirm that near wild-type levels of FPA protein were expressed from three independent transgenic lines expressing the FPA R477A;Y515A doubly mutated protein in an fpa-8 mutant background (S3 Fig). We found that FPA R477A;Y515A protein functioned like wild-type FPA to restore FPA pre-mRNA proximal polyadenylation (Fig 6A) and FLC expression to wild-type levels (Fig 6B). Impact of double FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA and FLC expression. (A) RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing FPA::FPA R477A;Y515A using poly(A)+ purified mRNAs. Black arrows indicate the proximally and distally polyadenylated FPA mRNAs. A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs. RNA size (kb) marker (Ambion). TUBULIN was detected as an internal control. The ratio of distal:proximal polyadenylated forms is given under each lane. (B). qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, and FPA::FPA R477A;Y515A plants. Transcript levels were normalized to the control UBC. Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates. Together our findings suggest that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation, or that this combination of mutations is not sufficient to critically disrupt the function of the FPA SPOC domain. Since the corresponding mutations in the SHARP SPOC domain do disrupt its recognition of unphosphorylated SMRT peptides, these observations may reinforce the idea that the features and functions of the FPA SPOC domain differ from those of the only other well-characterized SPOC domain. Materials and Methods Protein expression and purification The SPOC domain (residue 433–565) of A. thaliana FPA was sub-cloned into the pET28a vector (Novagen). The recombinant protein, with an N-terminal hexa-histidine tag, was over-expressed in E. coli BL21 Star (DE3) cells (Novagen), which were induced with 0.4 mM IPTG and allowed to grow at 20°C for 14–18 h. The soluble protein was purified by nickel-charged immobilized-metal affinity chromatography and gel filtration chromatography. The purified protein was concentrated and stored at –80°C in a buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, 10 mM DTT and 5% (v/v) glycerol. The His-tag was not removed for crystallization. The selenomethionine labeled SPOC domain was expressed in E. coli B834(DE3) strain using LeMaster media and purified with the same protocol as the native protein. Protein crystallization Crystals of the native SPOC domain of FPA were grown at 20°C with the sitting-drop vapor diffusion method. The protein solution was at 30 mg/ml concentration, and the reservoir solution contained 0.2 M MgSO4, and 20% (v/v) PEG 3350. Fully-grown crystals were obtained two days after set-up. Crystals of the selenomethionine labeled SPOC domain were grown using the same condition as the native protein. The crystals were cryo-protected in the crystallization solution supplemented with 20% (v/v) glycerol and flash-frozen in liquid nitrogen for data collection at 100K. Data collection and processing A single-wavelength anomalous dispersion (SAD) X-ray diffraction data set on a selenomethionine labeled SPOC domain crystal was collected at the National Synchrotron Light Source (NSLS) beamline X29A using an ADSC Q315r CCD. The diffraction images were processed and scaled with the HKL package. The crystal belongs to space group P65, with unit cell parameters of a = b = 108.2 Å, and c = 34.2 Å. Structure determination and refinement The structure of the SPOC domain was solved by the selenomethionyl SAD method with the program SHELX. The phases were used by program PHENIX for automatic model building. Manual model rebuilding was carried out with Coot. The structure refinement was performed with the program PHENIX, with translation, libration, and screw-rotation (TLS) parameters. The data processing and refinement statistics are summarized in Table 1. The Ramachandran plot showed that 95.8% of the residues are located in the most favored regions, and 4.2% are in additional allowed regions. Generation of constructs with mutated genomic FPA sequence A series of constructs containing a mutated FPA genomic sequence was prepared based on pGreen I 0029 vector. pGreen I 0029 vector with inserted FPA genomic sequence was prepared. In this vector FPA genomic sequence is flanked by 2620bp of the native sequence upstream to the start codon and 1178bp downstream to the stop codon. The vector contains kanamycin resistance genes for both the bacteria and plant hosts. In order to obtain a series of constructs with mutated FPA genomic sequence, FPA sequence in this construct was modified using site-directed mutagenesis. Primers used to prepare required constructs are listed in S1 Table. After the mutagenesis reaction the presence of only the desired mutations was confirmed by sequencing of the whole FPA genomic sequence and flanking regions. Generation of Arabidopsis thaliana transgenic plants All transgenic plants were prepared in fpa-8 mutant background, which is in Col-0 accession. The prepared vectors for Arabidopsis transformations were introduced into electro-competent Agrobacterium tumefaciens cells (C58 CV3101 strain harbouring pSoup vector). The floral dip method was used for plant transformation. Transgenic plants were selected using kanamycin as a selection marker. Presence of the desired mutations in plants was confirmed with specific dCaps markers. Plant growth conditions Wild type Col-0 plants used in this study were obtained from the Nottingham Arabidopsis Stock Centre. Seed of fpa-8 and 35S::FPA:YFP were obtained from Professor Caroline Dean. Plants were grown in pots containing Universal Extra general purpose soil. The glasshouse temperature was maintained at 20°C and the 16 hour daylight was provided by high pressure sodium vapour lamps (Philips Powertone SON-T AGRO 400). In order to grow plants in sterile conditions, seeds were first surface sterilized by a 5 min treatment with sterilizing solution (3% v/v sodium hypochlorite, 0.02% v/v Triton X-100), followed by three washes with 0.02% v/v Triton X-100 and one wash with sterile water. The sterile seeds were sown on MS10 media supplemented with 0.8% w/v agar. MS10 medium was also supplemented with specific antibiotics if required. After sowing, the seeds were stratified at 4°C for two days in order to synchronize their germination. Plants were grown in the tissue culture room at the following conditions: temperature 22°C, 16 hours daylight provided by the Master TL-D 36W/840 (Philips) lamps. Plant protein analysis Total protein samples were prepared using extraction buffer containing: 40 mM Tris-HCl, pH 6.8; 0.1 mM EDTA, pH 8.0; 8 M urea; 1.43 M β-mercaptoethanol, 7% v/v Complete Protease Inhibitors (Roche) and 5 mM PMSF. Equal volumes of samples were separated on 8% SDS-PAGE. Proteins were transferred onto Protran nitrocellulose transfer membrane (Whatman) using wet Criterion blotter system (BioRad). The transfer was performed at room temperature for two hours at a stable voltage of 70 V. Membrane was blocked in 3% (w/v) Milk in TBS for 1h at room temperature followed by overnight incubation with anti-FPA antibody (dilution 1:100 in 3% (w/v) Milk in TBS). After washes the membrane was incubated for 75 min with goat anti-rabbit antibody (Thermo Scientific) (1:3000 dilution in 3% (w/v) Milk in TBS). Protein was detected using SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific). Blots were re-probed following treatment with low pH solution (25mM glycine-HCl, pH 2, 1% (w/v) SDS) followed by blocking for 1h at room temperature in 3% (w/v) Milk in TBS. The membrane was incubated overnight with anti-TUBB2A, tubulin, beta 2A antibody (ARP40177_P050 Aviva systems biology; (dilution 1:1000 in 3% (w/v) Milk in TBS). After washes the membrane was incubated for 75 min with goat anti-rabbit antibody (Thermo Scientific) [1:3000 dilution in 3% (w/v) Milk in TBS]. Signal was detected using SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific). RNA gel blot analysis and RT-qPCR RNA gel blot analysis and RT-qPCR method performed as previously described. Supporting Information References Pre-mRNA processing reaches back to transcription and ahead to translation Molecular mechanisms of eukaryotic pre-mRNA 3' end processing regulation RNA processing and its regulation: global insights into biological networks Structure and function of pre-mRNA 5'-end capping quality control and 3'-end processing Alternative mRNA polyadenylation in eukaryotes: an effective regulator of gene expression Alternative cleavage and polyadenylation: the long and short of it 3' end mRNA processing: molecular mechanisms and implications for health and disease Implications of polyadenylation in health and disease Split end family RNA binding proteins: novel tumor suppressors coupling transcriptional regulation with RNA processing SPOC: a widely distributed domain associated with cancer, apoptosis and transcription The crystal structure of the Split End protein SHARP adds a new layer of complexity to proteins containing RNA recognition motifs FPA, a gene involved in floral induction in Arabidopsis, encodes a protein containing RNA-recognition motifs The spen family protein FPA controls alternative cleavage and polyadenylation of RNA Alternative polyadenylation of antisense RNAs and flowering time control A conserved structural motif reveals the essential transcriptional repression function of Spen proteins and their role in developmental signaling Structural insights into the recruitment of SMRT by the corepressor SHARP under phosphorylative regulation Searching protein structure databases with DaliLite v.3 Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair The Chp1-Tas3 core is a multifunctional platform critical for gene silencing by RITS Structure and VP16 binding of the Mediator Med25 activator interaction domain Structure of the VP16 transactivator target in the Mediator Solution NMR structure of MED25(391–543) comprising the activator-interacting domain (ACID) of human mediator subunit 25 NMR structure of the human Mediator MED25 ACID domain Sharp, an inducible cofactor that integrates nuclear receptor repression and activation SHARP is a novel component of the Notch/RBP-Jkappa signalling pathway ConSurf: an algorithmic tool for the identification of functional regions in proteins by surface mapping of phylogenetic information Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure Processing of X-ray diffraction data collected in oscillation mode Determination of macromolecular structures from anomalous diffraction of synchrotron radiation Substructure solution with SHELXD PHENIX: building a new software for automated crystallographic structure determination Coot: model-building tools for molecular graphics pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation Widespread role for the flowering-time regulators FCA and FPA in RNA-mediated chromatin silencing Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis 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	@@ -0,0 +1,4800 @@

+Structural	O
+insights	O
+and	O
+in	B-experimental_method
+vitro	I-experimental_method
+reconstitution	I-experimental_method
+of	O
+membrane	O
+targeting	O
+and	O
+activation	O
+of	O
+human	B-species
+PI4KB	B-protein
+by	O
+the	O
+ACBD3	B-protein
+protein	O
+Phosphatidylinositol	B-protein
+4	I-protein
+-	I-protein
+kinase	I-protein
+beta	I-protein
+(	O
+PI4KB	B-protein
+)	O
+is	O
+one	O
+of	O
+four	O
+human	B-species
+PI4K	B-protein_type
+enzymes	O
+that	O
+generate	O
+phosphatidylinositol	B-chemical
+4	I-chemical
+-	I-chemical
+phosphate	I-chemical
+(	O
+PI4P	B-chemical
+),	O
+a	O
+minor	O
+but	O
+essential	O
+regulatory	O
+lipid	O
+found	O
+in	O
+all	O
+eukaryotic	B-taxonomy_domain
+cells	O
+.	O
+To	O
+convert	O
+their	O
+lipid	O
+substrates	O
+,	O
+PI4Ks	B-protein_type
+must	O
+be	O
+recruited	O
+to	O
+the	O
+correct	O
+membrane	O
+compartment	O
+.	O
+PI4KB	B-protein
+is	O
+critical	O
+for	O
+the	O
+maintenance	O
+of	O
+the	O
+Golgi	O
+and	O
+trans	O
+Golgi	O
+network	O
+(	O
+TGN	O
+)	O
+PI4P	B-chemical
+pools	O
+,	O
+however	O
+,	O
+the	O
+actual	O
+targeting	O
+mechanism	O
+of	O
+PI4KB	B-protein
+to	O
+the	O
+Golgi	O
+and	O
+TGN	O
+membranes	O
+is	O
+unknown	O
+.	O
+Here	O
+,	O
+we	O
+present	O
+an	O
+NMR	B-experimental_method
+structure	B-evidence
+of	O
+the	O
+complex	O
+of	O
+PI4KB	B-protein
+and	O
+its	O
+interacting	O
+partner	O
+,	O
+Golgi	B-protein_type
+adaptor	I-protein_type
+protein	I-protein_type
+acyl	B-protein
+-	I-protein
+coenzyme	I-protein
+A	I-protein
+binding	I-protein
+domain	I-protein
+containing	I-protein
+protein	I-protein
+3	I-protein
+(	O
+ACBD3	B-protein
+).	O
+We	O
+show	O
+that	O
+ACBD3	B-protein
+is	O
+capable	O
+of	O
+recruiting	O
+PI4KB	B-protein
+to	O
+membranes	O
+both	O
+in	O
+vitro	O
+and	O
+in	O
+vivo	O
+,	O
+and	O
+that	O
+membrane	O
+recruitment	O
+of	O
+PI4KB	B-protein
+by	O
+ACBD3	B-protein
+increases	O
+its	O
+enzymatic	B-evidence
+activity	I-evidence
+and	O
+that	O
+the	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+complex	O
+formation	O
+is	O
+essential	O
+for	O
+proper	O
+function	O
+of	O
+the	O
+Golgi	O
+.	O
+Phosphatidylinositol	B-protein
+4	I-protein
+-	I-protein
+kinase	I-protein
+beta	I-protein
+(	O
+PI4KB	B-protein
+,	O
+also	O
+known	O
+as	O
+PI4K	B-protein
+IIIβ	I-protein
+)	O
+is	O
+a	O
+soluble	O
+cytosolic	O
+protein	O
+yet	O
+its	O
+function	O
+is	O
+to	O
+phosphorylate	O
+membrane	O
+lipids	O
+.	O
+It	O
+is	O
+one	O
+of	O
+four	O
+human	B-species
+PI4K	B-protein_type
+enzymes	O
+that	O
+phosphorylate	O
+phosphatidylinositol	B-chemical
+(	O
+PI	B-chemical
+)	O
+to	O
+generate	O
+phosphatidylinositol	B-chemical
+4	I-chemical
+-	I-chemical
+phosphate	I-chemical
+(	O
+PI4P	B-chemical
+).	O
+PI4P	B-chemical
+is	O
+an	O
+essential	O
+lipid	O
+found	O
+in	O
+various	O
+membrane	O
+compartments	O
+including	O
+the	O
+Golgi	O
+and	O
+trans	O
+-	O
+Golgi	O
+network	O
+(	O
+TGN	O
+),	O
+the	O
+plasma	O
+membrane	O
+and	O
+the	O
+endocytic	O
+compartments	O
+.	O
+In	O
+these	O
+locations	O
+,	O
+PI4P	B-chemical
+plays	O
+an	O
+important	O
+role	O
+in	O
+cell	O
+signaling	O
+and	O
+lipid	O
+transport	O
+,	O
+and	O
+serves	O
+as	O
+a	O
+precursor	O
+for	O
+higher	O
+phosphoinositides	B-chemical
+or	O
+as	O
+a	O
+docking	O
+site	O
+for	O
+clathrin	B-protein_type
+adaptor	O
+or	O
+lipid	O
+transfer	O
+proteins	O
+.	O
+A	O
+wide	O
+range	O
+of	O
+positive	B-taxonomy_domain
+-	I-taxonomy_domain
+sense	I-taxonomy_domain
+single	I-taxonomy_domain
+-	I-taxonomy_domain
+stranded	I-taxonomy_domain
+RNA	I-taxonomy_domain
+viruses	I-taxonomy_domain
+(+	O
+RNA	B-taxonomy_domain
+viruses	I-taxonomy_domain
+),	O
+including	O
+many	O
+that	O
+are	O
+important	O
+human	B-species
+pathogens	O
+,	O
+hijack	O
+human	B-species
+PI4KA	B-protein
+or	O
+PI4KB	B-protein
+enzymes	O
+to	O
+generate	O
+specific	O
+PI4P	B-chemical
+-	O
+enriched	O
+organelles	O
+called	O
+membranous	O
+webs	O
+or	O
+replication	O
+factories	O
+.	O
+These	O
+structures	B-evidence
+are	O
+essential	O
+for	O
+effective	O
+viral	B-taxonomy_domain
+replication	O
+.	O
+Recently	O
+,	O
+highly	O
+specific	O
+PI4KB	B-protein
+inhibitors	O
+were	O
+developed	O
+as	O
+potential	O
+antivirals	O
+.	O
+PI4K	B-protein_type
+kinases	B-protein_type
+must	O
+be	O
+recruited	O
+to	O
+the	O
+correct	O
+membrane	O
+type	O
+to	O
+fulfill	O
+their	O
+enzymatic	O
+functions	O
+.	O
+Type	B-protein_type
+II	I-protein_type
+PI4Ks	I-protein_type
+(	O
+PI4K2A	B-protein
+and	O
+PI4K2B	B-protein
+)	O
+are	O
+heavily	B-protein_state
+palmitoylated	I-protein_state
+and	O
+thus	O
+behave	O
+as	O
+membrane	B-protein
+proteins	I-protein
+.	O
+In	O
+contrast	O
+,	O
+type	B-protein_type
+III	I-protein_type
+PI4Ks	I-protein_type
+(	O
+PI4KA	B-protein
+and	O
+PI4KB	B-protein
+)	O
+are	O
+soluble	O
+cytosolic	O
+proteins	O
+that	O
+are	O
+recruited	O
+to	O
+appropriate	O
+membranes	O
+indirectly	O
+via	O
+protein	O
+-	O
+protein	O
+interactions	O
+.	O
+The	O
+recruitment	O
+of	O
+PI4KA	B-protein
+to	O
+the	O
+plasma	O
+membrane	O
+by	O
+EFR3	B-protein
+and	O
+TTC7	B-protein
+is	O
+relatively	O
+well	O
+understood	O
+even	O
+at	O
+the	O
+structural	O
+level	O
+,	O
+but	O
+,	O
+the	O
+actual	O
+molecular	O
+mechanism	O
+of	O
+PI4KB	B-protein
+recruitment	O
+to	O
+the	O
+Golgi	O
+is	O
+still	O
+poorly	O
+understood	O
+.	O
+Acyl	B-protein
+-	I-protein
+coenzyme	I-protein
+A	I-protein
+binding	I-protein
+domain	I-protein
+containing	I-protein
+protein	I-protein
+3	I-protein
+(	O
+ACBD3	B-protein
+,	O
+also	O
+known	O
+as	O
+GCP60	B-protein
+and	O
+PAP7	B-protein
+)	O
+is	O
+a	O
+Golgi	O
+resident	O
+protein	O
+.	O
+Its	O
+membrane	O
+localization	O
+is	O
+mediated	O
+by	O
+the	O
+interaction	O
+with	O
+the	O
+Golgi	O
+integral	O
+protein	O
+golgin	B-protein
+B1	I-protein
+/	O
+giantin	B-protein
+.	O
+ACBD3	B-protein
+functions	O
+as	O
+an	O
+adaptor	O
+protein	O
+and	O
+signaling	O
+hub	O
+across	O
+cellular	O
+signaling	O
+pathways	O
+.	O
+ACBD3	B-protein
+can	O
+interact	O
+with	O
+a	O
+number	O
+of	O
+proteins	O
+including	O
+golgin	B-protein
+A3	I-protein
+/	O
+golgin	B-protein
+-	I-protein
+160	I-protein
+to	O
+regulate	O
+apoptosis	O
+,	O
+Numb	B-protein_type
+proteins	I-protein_type
+to	O
+control	O
+asymmetric	O
+cell	O
+division	O
+and	O
+neuronal	O
+differentiation	O
+,	O
+metal	B-protein_type
+transporter	I-protein_type
+DMT1	B-protein
+and	O
+monomeric	B-oligomeric_state
+G	B-protein_type
+protein	I-protein_type
+Dexras1	B-protein
+to	O
+maintain	O
+iron	B-chemical
+homeostasis	O
+,	O
+and	O
+the	O
+lipid	B-protein_type
+kinase	I-protein_type
+PI4KB	B-protein
+to	O
+regulate	O
+lipid	O
+homeostasis	O
+.	O
+ACBD3	B-protein
+has	O
+been	O
+also	O
+implicated	O
+in	O
+the	O
+pathology	O
+of	O
+neurodegenerative	O
+diseases	O
+such	O
+as	O
+Huntington	O
+’	O
+s	O
+disease	O
+due	O
+to	O
+its	O
+interactions	O
+with	O
+a	O
+polyglutamine	B-structure_element
+repeat	I-structure_element
+-	O
+containing	O
+mutant	B-protein_state
+huntingtin	B-protein
+and	O
+the	O
+striatal	O
+-	O
+selective	O
+monomeric	B-oligomeric_state
+G	B-protein_type
+protein	I-protein_type
+Rhes	B-protein
+/	O
+Dexras2	B-protein
+.	O
+ACBD3	B-protein
+is	O
+a	O
+binding	O
+partner	O
+of	O
+viral	B-taxonomy_domain
+non	B-protein_type
+-	I-protein_type
+structural	I-protein_type
+3A	I-protein_type
+proteins	I-protein_type
+and	O
+a	O
+host	O
+factor	O
+of	O
+several	O
+picornaviruses	B-taxonomy_domain
+including	O
+poliovirus	B-taxonomy_domain
+,	O
+coxsackievirus	B-taxonomy_domain
+B3	I-taxonomy_domain
+,	O
+and	O
+Aichi	B-taxonomy_domain
+virus	I-taxonomy_domain
+.	O
+We	O
+present	O
+a	O
+biochemical	B-experimental_method
+and	I-experimental_method
+structural	I-experimental_method
+characterization	I-experimental_method
+of	O
+the	O
+molecular	O
+complex	O
+composed	O
+of	O
+the	O
+ACBD3	B-protein
+protein	O
+and	O
+the	O
+PI4KB	B-protein
+enzyme	O
+.	O
+We	O
+show	O
+that	O
+ACBD3	B-protein
+can	O
+recruit	O
+PI4KB	B-protein
+to	O
+model	O
+membranes	O
+as	O
+well	O
+as	O
+redirect	O
+PI4KB	B-protein
+to	O
+cellular	O
+membranes	O
+where	O
+it	O
+is	O
+not	O
+naturally	O
+found	O
+.	O
+Our	O
+data	O
+also	O
+show	O
+that	O
+ACBD3	B-protein
+regulates	O
+the	O
+enzymatic	B-evidence
+activity	I-evidence
+of	O
+PI4KB	B-protein
+kinase	B-protein_type
+through	O
+membrane	O
+recruitment	O
+rather	O
+than	O
+allostery	O
+.	O
+ACBD3	B-protein
+and	O
+PI4KB	B-protein
+interact	O
+with	O
+1	O
+:	O
+1	O
+stoichiometry	O
+with	O
+submicromolar	O
+affinity	O
+In	O
+order	O
+to	O
+verify	O
+the	O
+interactions	O
+between	O
+ACBD3	B-protein
+and	O
+PI4KB	B-protein
+we	O
+expressed	B-experimental_method
+and	I-experimental_method
+purified	I-experimental_method
+both	O
+proteins	O
+.	O
+To	O
+increase	O
+yields	O
+of	O
+bacterial	B-experimental_method
+expression	I-experimental_method
+the	O
+intrinsically	B-structure_element
+disordered	I-structure_element
+region	I-structure_element
+of	O
+PI4KB	B-protein
+(	O
+residues	O
+423	B-residue_range
+–	I-residue_range
+522	I-residue_range
+)	O
+was	O
+removed	B-experimental_method
+(	O
+Fig	O
+.	O
+1A	O
+).	O
+This	O
+internal	O
+deletion	B-experimental_method
+does	O
+not	O
+significantly	O
+affect	O
+the	O
+kinase	B-protein_type
+activity	O
+(	O
+SI	O
+Fig	O
+.	O
+1A	O
+)	O
+or	O
+interaction	O
+with	O
+ACBD3	B-protein
+(	O
+SI	O
+Fig	O
+.	O
+1B	O
+,	O
+C	O
+).	O
+In	O
+an	O
+in	B-experimental_method
+vitro	I-experimental_method
+binding	I-experimental_method
+assay	I-experimental_method
+,	O
+ACBD3	B-protein
+co	B-experimental_method
+-	I-experimental_method
+purified	I-experimental_method
+with	I-experimental_method
+the	I-experimental_method
+NiNTA	I-experimental_method
+-	I-experimental_method
+immobilized	I-experimental_method
+N	O
+-	O
+terminal	O
+His6GB1	B-protein_state
+-	I-protein_state
+tagged	I-protein_state
+PI4KB	B-protein
+(	O
+Fig	O
+.	O
+1B	O
+,	O
+left	O
+panel	O
+),	O
+suggesting	O
+a	O
+direct	O
+interaction	O
+.	O
+Using	O
+a	O
+mammalian	B-experimental_method
+two	I-experimental_method
+-	I-experimental_method
+hybrid	I-experimental_method
+assay	I-experimental_method
+Greninger	O
+and	O
+colleagues	O
+localized	B-evidence
+this	O
+interaction	O
+to	O
+the	O
+Q	B-structure_element
+domain	I-structure_element
+of	O
+ACBD3	B-protein
+(	O
+named	O
+according	O
+to	O
+its	O
+high	O
+content	O
+of	O
+glutamine	B-residue_name
+residues	O
+)	O
+and	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+PI4KB	B-protein
+preceding	O
+its	O
+helical	B-structure_element
+domain	I-structure_element
+.	O
+We	O
+expressed	B-experimental_method
+the	O
+Q	B-structure_element
+domain	I-structure_element
+of	O
+ACBD3	B-protein
+(	O
+residues	O
+241	B-residue_range
+–	I-residue_range
+308	I-residue_range
+)	O
+and	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+PI4KB	B-protein
+(	O
+residues	O
+1	B-residue_range
+–	I-residue_range
+68	I-residue_range
+)	O
+in	O
+E	B-species
+.	I-species
+coli	I-species
+and	O
+using	O
+purified	O
+recombinant	O
+proteins	O
+,	O
+we	O
+confirmed	O
+that	O
+these	O
+two	O
+domains	O
+are	O
+sufficient	O
+to	O
+maintain	O
+the	O
+interaction	O
+(	O
+Fig	O
+.	O
+1B	O
+,	O
+middle	O
+and	O
+right	O
+panel	O
+).	O
+Because	O
+it	O
+has	O
+been	O
+reported	O
+that	O
+ACBD3	B-protein
+can	O
+dimerize	B-oligomeric_state
+in	O
+a	O
+mammalian	B-experimental_method
+two	I-experimental_method
+-	I-experimental_method
+hybrid	I-experimental_method
+assay	I-experimental_method
+,	O
+we	O
+were	O
+interested	O
+in	O
+determining	O
+the	O
+stoichiometry	O
+of	O
+the	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+protein	O
+complex	O
+.	O
+The	O
+sedimentation	B-evidence
+coefficients	I-evidence
+of	O
+ACBD3	B-protein
+and	O
+PI4KB	B-protein
+alone	B-protein_state
+,	O
+or	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+complex	O
+were	O
+determined	O
+by	O
+analytical	B-experimental_method
+ultracentrifugation	I-experimental_method
+and	O
+found	O
+to	O
+be	O
+3	O
+.	O
+1	O
+S	O
+,	O
+4	O
+.	O
+1	O
+S	O
+,	O
+and	O
+5	O
+.	O
+1	O
+S	O
+.	O
+These	O
+values	O
+correspond	O
+to	O
+molecular	B-evidence
+weights	I-evidence
+of	O
+approximately	O
+55	O
+kDa	O
+,	O
+80	O
+kDa	O
+,	O
+and	O
+130	O
+kDa	O
+,	O
+respectively	O
+.	O
+This	O
+result	O
+suggests	O
+that	O
+both	O
+proteins	O
+are	O
+monomeric	B-oligomeric_state
+and	O
+the	O
+stoichiometry	O
+of	O
+the	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+protein	O
+complex	O
+is	O
+1	O
+:	O
+1	O
+(	O
+Fig	O
+.	O
+1C	O
+,	O
+left	O
+panel	O
+).	O
+Similar	O
+results	O
+were	O
+obtained	O
+for	O
+the	O
+complex	O
+of	O
+the	O
+Q	B-structure_element
+domain	I-structure_element
+of	O
+ACBD3	B-protein
+and	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+PI4KB	B-protein
+(	O
+Fig	O
+.	O
+1C	O
+,	O
+right	O
+panel	O
+).	O
+We	O
+also	O
+determined	O
+the	O
+strength	O
+of	O
+the	O
+interaction	O
+between	O
+recombinant	O
+full	B-protein_state
+length	I-protein_state
+ACBD3	B-protein
+and	O
+PI4KB	B-protein
+using	O
+surface	B-experimental_method
+plasmon	I-experimental_method
+resonance	I-experimental_method
+(	O
+SPR	B-experimental_method
+).	O
+SPR	B-experimental_method
+measurements	O
+revealed	O
+a	O
+strong	O
+interaction	O
+with	O
+a	O
+Kd	B-evidence
+value	O
+of	O
+320	O
++/−	O
+130	O
+nM	O
+(	O
+Fig	O
+.	O
+1D	O
+,	O
+SI	O
+Fig	O
+.	O
+1D	O
+).	O
+We	O
+concluded	O
+that	O
+ACBD3	B-protein
+and	O
+PI4KB	B-protein
+interact	O
+directly	O
+through	O
+the	O
+Q	B-structure_element
+domain	I-structure_element
+of	O
+ACBD3	B-protein
+and	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+PI4KB	B-protein
+forming	O
+a	O
+1	O
+:	O
+1	O
+complex	O
+with	O
+a	O
+dissociation	B-evidence
+constant	I-evidence
+in	O
+the	O
+submicromolar	O
+range	O
+.	O
+Structural	B-experimental_method
+analysis	I-experimental_method
+of	O
+the	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+complex	O
+Full	B-protein_state
+length	I-protein_state
+ACBD3	B-protein
+and	O
+PI4KB	B-protein
+both	O
+contain	O
+large	O
+intrinsically	B-structure_element
+disordered	I-structure_element
+regions	I-structure_element
+that	O
+impede	O
+crystallization	O
+.	O
+We	O
+used	O
+hydrogen	B-experimental_method
+-	I-experimental_method
+deuterium	I-experimental_method
+exchange	I-experimental_method
+mass	I-experimental_method
+spectrometry	I-experimental_method
+(	O
+HDX	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+)	O
+analysis	O
+of	O
+the	O
+complex	O
+to	O
+determine	O
+which	O
+parts	O
+of	O
+the	O
+complex	O
+are	O
+well	B-protein_state
+folded	I-protein_state
+(	O
+SI	O
+Fig	O
+.	O
+2	O
+).	O
+However	O
+,	O
+we	O
+were	O
+unable	O
+to	O
+obtain	O
+crystals	B-evidence
+even	O
+when	O
+using	O
+significantly	O
+truncated	B-protein_state
+constructs	O
+that	O
+included	O
+only	O
+the	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+and	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+PI4KB	B-protein
+.	O
+For	O
+this	O
+reason	O
+,	O
+we	O
+produced	O
+an	O
+isotopically	B-protein_state
+labeled	I-protein_state
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+and	O
+isotopically	B-protein_state
+labeled	I-protein_state
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+:	O
+PI4KB	B-protein
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+protein	O
+complex	O
+and	O
+used	O
+NMR	B-experimental_method
+spectroscopy	I-experimental_method
+for	O
+structural	O
+characterization	O
+.	O
+As	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+protein	O
+complex	O
+was	O
+prepared	O
+by	O
+co	B-experimental_method
+-	I-experimental_method
+expression	I-experimental_method
+of	O
+both	O
+proteins	O
+,	O
+the	O
+samples	O
+consisted	O
+of	O
+an	O
+equimolar	O
+mixture	O
+of	O
+two	O
+uniformly	O
+15N	B-chemical
+/	O
+13C	B-chemical
+labelled	B-protein_state
+molecules	O
+.	O
+Comprehensive	O
+backbone	O
+and	O
+side	O
+-	O
+chain	O
+resonance	O
+assignments	O
+for	O
+the	O
+free	B-protein_state
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+and	O
+the	O
+complex	O
+,	O
+as	O
+illustrated	O
+by	O
+the	O
+2D	B-experimental_method
+15N	I-experimental_method
+/	I-experimental_method
+1H	I-experimental_method
+HSQC	I-experimental_method
+spectra	B-evidence
+(	O
+SI	O
+Figs	O
+3	O
+and	O
+4	O
+),	O
+were	O
+obtained	O
+using	O
+a	O
+standard	O
+combination	O
+of	O
+triple	B-experimental_method
+-	I-experimental_method
+resonance	I-experimental_method
+experiments	I-experimental_method
+,	O
+as	O
+described	O
+previously	O
+.	O
+Backbone	O
+amide	O
+signals	O
+(	O
+15N	B-chemical
+and	O
+1H	B-chemical
+)	O
+for	O
+the	O
+free	B-protein_state
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+were	O
+nearly	O
+completely	O
+assigned	O
+apart	O
+from	O
+the	O
+first	O
+four	O
+N	O
+-	O
+terminal	O
+residues	O
+(	O
+Met1	B-residue_range
+-	I-residue_range
+Lys4	I-residue_range
+)	O
+and	O
+Gln44	B-residue_name_number
+.	O
+Over	O
+93	O
+%	O
+of	O
+non	O
+-	O
+exchangeable	O
+side	O
+-	O
+chain	O
+signals	O
+were	O
+assigned	O
+for	O
+the	O
+free	B-protein_state
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+.	O
+Apart	O
+from	O
+the	O
+four	O
+N	O
+-	O
+terminal	O
+residues	O
+,	O
+the	O
+side	O
+-	O
+chain	O
+assignments	O
+were	O
+missing	O
+for	O
+Gln	B-residue_name
+(	O
+Hg3	O
+),	O
+Gln	B-residue_name
+(	O
+Ha	O
+/	O
+Hb	O
+/	O
+Hg	O
+),	O
+Gln44	B-residue_name_number
+(	O
+Ha	O
+/	O
+Hb	O
+/	O
+Hg	O
+)	O
+and	O
+Gln48	B-residue_name_number
+(	O
+Hg	O
+)	O
+mainly	O
+due	O
+to	O
+extensive	O
+overlaps	O
+within	O
+the	O
+spectral	O
+regions	O
+populated	O
+by	O
+highly	O
+abundant	O
+glutamine	B-residue_name
+side	O
+-	O
+chain	O
+resonances	O
+.	O
+The	O
+protein	O
+complex	O
+yielded	O
+relatively	O
+well	O
+resolved	O
+spectra	B-evidence
+(	O
+SI	O
+Fig	O
+.	O
+4	O
+)	O
+that	O
+resulted	O
+in	O
+assignment	O
+of	O
+backbone	O
+amide	O
+signals	O
+for	O
+all	O
+residues	O
+apart	O
+from	O
+Gln	B-residue_name
+(	O
+ACBD3	B-protein
+)	O
+and	O
+Ala2	B-residue_name_number
+(	O
+PI4KB	B-protein
+).	O
+The	O
+essentially	O
+complete	O
+15N	B-chemical
+,	O
+13C	B-chemical
+and	O
+1H	B-chemical
+resonance	O
+assignments	O
+allowed	O
+automated	O
+assignment	O
+of	O
+the	O
+NOEs	B-evidence
+identified	O
+in	O
+the	O
+3D	B-experimental_method
+15N	I-experimental_method
+/	I-experimental_method
+1H	I-experimental_method
+NOESY	I-experimental_method
+-	I-experimental_method
+HSQC	I-experimental_method
+and	O
+13C	B-experimental_method
+/	I-experimental_method
+1H	I-experimental_method
+HMQC	I-experimental_method
+-	I-experimental_method
+NOESY	I-experimental_method
+spectra	B-evidence
+that	O
+were	O
+subsequently	O
+used	O
+in	O
+structural	B-experimental_method
+calculation	I-experimental_method
+.	O
+Structural	B-evidence
+statistics	I-evidence
+for	O
+the	O
+final	O
+water	O
+-	O
+refined	O
+sets	O
+of	O
+structures	B-evidence
+are	O
+shown	O
+in	O
+SI	O
+Table	O
+1	O
+.	O
+This	O
+structure	B-evidence
+revealed	O
+that	O
+the	O
+Q	B-structure_element
+domain	I-structure_element
+forms	O
+a	O
+two	B-structure_element
+helix	I-structure_element
+hairpin	I-structure_element
+.	O
+The	O
+first	O
+helix	B-structure_element
+bends	O
+sharply	O
+over	O
+the	O
+second	O
+helix	B-structure_element
+and	O
+creates	O
+a	O
+fold	O
+resembling	O
+a	O
+three	B-structure_element
+helix	I-structure_element
+bundle	I-structure_element
+that	O
+serves	O
+as	O
+a	O
+nest	O
+for	O
+one	O
+helix	B-structure_element
+of	O
+the	O
+PI4KB	B-protein
+N	O
+-	O
+terminus	O
+(	O
+residues	O
+44	B-residue_range
+–	I-residue_range
+64	I-residue_range
+,	O
+from	O
+this	O
+point	O
+on	O
+referred	O
+to	O
+as	O
+the	O
+kinase	B-structure_element
+helix	I-structure_element
+)	O
+(	O
+Fig	O
+.	O
+2A	O
+).	O
+Preceding	O
+the	O
+kinase	B-structure_element
+helix	I-structure_element
+are	O
+three	O
+ordered	O
+residues	O
+(	O
+Val42	B-residue_name_number
+,	O
+Ile43	B-residue_name_number
+,	O
+and	O
+Asp44	B-residue_name_number
+)	O
+that	O
+also	O
+contribute	O
+to	O
+the	O
+interaction	O
+(	O
+Fig	O
+.	O
+2B	O
+).	O
+The	O
+remaining	O
+part	O
+of	O
+the	O
+PI4KB	B-protein
+N	O
+-	O
+termini	O
+,	O
+however	O
+,	O
+is	O
+disordered	O
+(	O
+SI	O
+Fig	O
+.	O
+5	O
+).	O
+Almost	O
+all	O
+of	O
+the	O
+PI4KB	B-complex_assembly
+:	I-complex_assembly
+ACBD3	I-complex_assembly
+interactions	B-bond_interaction
+are	I-bond_interaction
+hydrophobic	I-bond_interaction
+with	O
+the	O
+exception	O
+of	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+between	O
+the	O
+side	O
+chains	O
+of	O
+ACBD3	B-protein
+Tyr261	B-residue_name_number
+and	O
+PI4KB	B-protein
+His63	B-residue_name_number
+,	O
+and	O
+between	O
+the	O
+sidechain	O
+of	O
+ACBD3	B-protein
+Tyr288	B-residue_name_number
+and	O
+the	O
+PI4KB	B-protein
+backbone	O
+(	O
+Asp44	B-residue_name_number
+)	O
+(	O
+Fig	O
+.	O
+2B	O
+).	O
+Interestingly	O
+,	O
+we	O
+noted	O
+that	O
+the	O
+PI4KB	B-protein
+helix	B-structure_element
+is	O
+amphipathic	B-protein_state
+and	O
+its	O
+hydrophobic	B-site
+surface	I-site
+leans	O
+on	O
+the	O
+Q	B-structure_element
+domain	I-structure_element
+(	O
+Fig	O
+.	O
+2C	O
+).	O
+To	O
+corroborate	O
+the	O
+structural	B-evidence
+data	I-evidence
+,	O
+we	O
+introduced	B-experimental_method
+a	O
+number	O
+of	O
+point	B-experimental_method
+mutations	I-experimental_method
+and	O
+validated	O
+their	O
+effect	O
+on	O
+complex	O
+formation	O
+using	O
+an	O
+in	B-experimental_method
+vitro	I-experimental_method
+pull	I-experimental_method
+-	I-experimental_method
+down	I-experimental_method
+assay	I-experimental_method
+(	O
+Fig	O
+.	O
+2D	O
+).	O
+Wild	B-protein_state
+type	I-protein_state
+ACBD3	B-protein
+protein	O
+co	B-experimental_method
+-	I-experimental_method
+purified	I-experimental_method
+together	O
+with	O
+the	O
+NiNTA	O
+-	O
+immobilized	O
+His6	B-protein_state
+-	I-protein_state
+tagged	I-protein_state
+wild	B-protein_state
+type	I-protein_state
+PI4KB	B-protein
+as	O
+well	O
+as	O
+with	O
+the	O
+PI4KB	B-protein
+V42A	B-mutant
+and	O
+V47A	B-mutant
+mutants	B-protein_state
+,	O
+but	O
+not	O
+with	O
+mutants	B-protein_state
+within	O
+the	O
+imminent	O
+binding	B-site
+interface	I-site
+(	O
+I43A	B-mutant
+,	O
+V55A	B-mutant
+,	O
+L56A	B-mutant
+).	O
+As	O
+predicted	O
+,	O
+wild	B-protein_state
+type	I-protein_state
+PI4KB	B-protein
+interacted	O
+with	O
+the	O
+ACBD3	B-protein
+Y266A	B-mutant
+mutant	B-protein_state
+and	O
+slightly	O
+with	O
+the	O
+Y285A	B-mutant
+mutant	B-protein_state
+,	O
+but	O
+not	O
+with	O
+the	O
+F258A	B-mutant
+,	O
+H284A	B-mutant
+,	O
+and	O
+Y288A	B-mutant
+mutants	B-protein_state
+(	O
+Fig	O
+.	O
+2D	O
+).	O
+ACBD3	B-protein
+efficiently	O
+recruits	O
+the	O
+PI4KB	B-protein
+enzyme	O
+to	O
+membranes	O
+We	O
+next	O
+sought	O
+to	O
+determine	O
+if	O
+the	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+interaction	O
+drives	O
+membrane	O
+localization	O
+of	O
+the	O
+PI4KB	B-protein
+enzyme	O
+.	O
+To	O
+do	O
+this	O
+,	O
+we	O
+first	O
+established	O
+an	O
+in	B-experimental_method
+vitro	I-experimental_method
+membrane	I-experimental_method
+recruitment	I-experimental_method
+system	I-experimental_method
+using	O
+Giant	B-experimental_method
+Unilamellar	I-experimental_method
+Vesicles	I-experimental_method
+(	O
+GUVs	B-experimental_method
+)	O
+containing	O
+the	O
+PI4KB	B-protein
+substrate	O
+–	O
+the	O
+PI	B-chemical
+lipid	O
+.	O
+We	O
+observed	O
+that	O
+PI4KB	B-protein
+kinase	B-protein_type
+was	O
+not	O
+membrane	O
+localized	B-evidence
+when	O
+added	O
+to	O
+the	O
+GUVs	B-experimental_method
+at	O
+600	O
+nM	O
+concentration	O
+,	O
+whereas	O
+non	O
+-	O
+covalent	O
+tethering	O
+of	O
+ACBD3	B-protein
+to	O
+the	O
+surface	O
+of	O
+the	O
+GUVs	B-experimental_method
+,	O
+using	O
+the	O
+His6	O
+tag	O
+on	O
+ACBD3	B-protein
+and	O
+the	O
+DGS	B-chemical
+-	I-chemical
+NTA	I-chemical
+(	I-chemical
+Ni	I-chemical
+)	I-chemical
+lipid	I-chemical
+,	O
+led	O
+to	O
+efficient	O
+PI4KB	B-protein
+membrane	O
+localization	O
+(	O
+Fig	O
+.	O
+3A	O
+).	O
+We	O
+hypothesized	O
+that	O
+if	O
+ACBD3	B-protein
+is	O
+one	O
+of	O
+the	O
+main	O
+Golgi	O
+localization	B-evidence
+signals	I-evidence
+for	O
+PI4KB	B-protein
+,	O
+overexpression	B-experimental_method
+of	O
+the	O
+Q	B-structure_element
+domain	I-structure_element
+should	O
+decrease	O
+the	O
+amount	O
+of	O
+the	O
+endogenous	O
+kinase	B-protein_type
+on	O
+the	O
+Golgi	O
+.	O
+Indeed	O
+,	O
+we	O
+observed	O
+loss	O
+for	O
+endogenous	O
+PI4KB	B-protein
+signal	O
+on	O
+the	O
+Golgi	O
+in	O
+cells	O
+overexpressing	B-experimental_method
+the	O
+GFP	B-experimental_method
+–	O
+Q	B-structure_element
+domain	I-structure_element
+construct	O
+(	O
+Fig	O
+.	O
+3B	O
+upper	O
+panel	O
+).	O
+We	O
+attribute	O
+the	O
+loss	O
+of	O
+signal	B-evidence
+to	O
+the	O
+immunostaining	O
+protocol	O
+-	O
+the	O
+kinase	B-protein_type
+that	O
+is	O
+not	O
+bound	O
+to	O
+Golgi	O
+is	O
+lost	O
+during	O
+the	O
+permeabilization	O
+step	O
+and	O
+hence	O
+the	O
+“	O
+disappearance	O
+”	O
+of	O
+the	O
+signal	B-evidence
+because	O
+overexpression	B-experimental_method
+of	O
+GFP	B-experimental_method
+alone	O
+or	O
+a	O
+non	B-protein_state
+-	I-protein_state
+binding	I-protein_state
+Q	B-structure_element
+domain	I-structure_element
+mutant	B-protein_state
+has	O
+no	O
+effect	O
+on	O
+the	O
+localization	B-evidence
+of	O
+the	O
+endogenous	O
+PI4KB	B-protein
+(	O
+Fig	O
+.	O
+3B	O
+).	O
+Given	O
+this	O
+result	O
+,	O
+overexpression	B-experimental_method
+of	O
+the	O
+Q	B-structure_element
+domain	I-structure_element
+should	O
+also	O
+interfere	O
+with	O
+the	O
+PI4KB	B-protein
+dependent	O
+Golgi	O
+functions	O
+.	O
+Ceramide	B-chemical
+transport	O
+and	O
+accumulation	O
+in	O
+Golgi	O
+is	O
+a	O
+well	O
+-	O
+known	O
+PI4KB	B-protein
+dependent	O
+process	O
+.	O
+We	O
+have	O
+used	O
+fluorescently	B-protein_state
+labeled	I-protein_state
+ceramide	B-chemical
+and	O
+analyzed	O
+its	O
+trafficking	O
+in	O
+non	O
+-	O
+transfected	O
+cells	O
+and	O
+cell	O
+overexpressing	B-experimental_method
+the	O
+Q	B-structure_element
+domain	I-structure_element
+.	O
+As	O
+expected	O
+,	O
+the	O
+Golgi	O
+accumulation	O
+of	O
+ceramide	B-chemical
+was	O
+not	O
+observed	O
+in	O
+cells	O
+expressing	B-experimental_method
+the	O
+wt	B-protein_state
+Q	B-structure_element
+domain	I-structure_element
+while	O
+cells	O
+expressing	O
+RFP	B-experimental_method
+or	O
+the	O
+mutant	B-protein_state
+Q	B-structure_element
+domain	I-structure_element
+accumulated	O
+ceramide	B-chemical
+normally	O
+(	O
+Fig	O
+.	O
+3C	O
+)	O
+suggesting	O
+that	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+complex	O
+formation	O
+is	O
+crucial	O
+for	O
+the	O
+normal	O
+function	O
+of	O
+Golgi	O
+.	O
+We	O
+further	O
+analyzed	O
+the	O
+function	O
+of	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+interaction	O
+in	O
+membrane	O
+recruitment	O
+of	O
+PI4KB	B-protein
+in	O
+living	O
+cells	O
+using	O
+fluorescently	B-protein_state
+tagged	I-protein_state
+proteins	O
+.	O
+We	O
+used	O
+the	O
+rapamycin	B-chemical
+-	O
+inducible	O
+heteromerization	O
+of	O
+FKBP12	B-protein
+(	O
+FK506	B-protein
+binding	I-protein
+protein	I-protein
+12	I-protein
+)	O
+and	O
+FRB	B-structure_element
+(	O
+fragment	B-structure_element
+of	O
+mTOR	B-protein
+that	O
+binds	O
+rapamycin	B-chemical
+)	O
+system	O
+.	O
+We	O
+fused	B-experimental_method
+the	O
+FRB	B-structure_element
+to	O
+residues	O
+34	B-residue_range
+–	I-residue_range
+63	I-residue_range
+of	O
+the	O
+mitochondrial	B-structure_element
+localization	I-structure_element
+signal	I-structure_element
+from	O
+mitochondrial	B-protein
+A	I-protein
+-	I-protein
+kinase	I-protein
+anchor	I-protein
+protein	I-protein
+1	I-protein
+(	O
+AKAP1	B-protein
+)	O
+and	O
+CFP	B-experimental_method
+.	O
+The	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+was	O
+then	O
+fused	B-experimental_method
+to	I-experimental_method
+FKBP12	B-protein
+and	O
+mRFP	B-experimental_method
+(	O
+Fig	O
+.	O
+3D	O
+).	O
+We	O
+analyzed	O
+localization	B-evidence
+of	O
+the	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+and	O
+GFP	B-experimental_method
+–	O
+PI4KB	B-protein
+before	O
+and	O
+after	O
+the	O
+addition	O
+of	O
+rapamycin	B-chemical
+.	O
+As	O
+a	O
+control	O
+we	O
+used	O
+H284A	B-mutant
+mutant	B-protein_state
+of	O
+the	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+that	O
+does	O
+not	O
+significantly	O
+bind	O
+PI4KB	B-protein
+kinase	B-protein_type
+.	O
+In	O
+every	O
+case	O
+the	O
+ACDB3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+was	O
+rapidly	O
+(	O
+within	O
+5	O
+minutes	O
+)	O
+recruited	O
+to	O
+the	O
+mitochondrial	O
+membrane	O
+upon	O
+addition	O
+of	O
+rapamycin	B-chemical
+,	O
+but	O
+only	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+protein	O
+effectively	O
+directed	O
+the	O
+kinase	B-protein_type
+to	O
+the	O
+mitochondria	O
+(	O
+Fig	O
+.	O
+3E	O
+,	O
+Movie	O
+1	O
+and	O
+2	O
+).	O
+Notably	O
+,	O
+we	O
+observed	O
+that	O
+when	O
+the	O
+GFP	B-experimental_method
+-	O
+PI4KB	B-protein
+kinase	B-protein_type
+is	O
+co	B-experimental_method
+-	I-experimental_method
+expressed	I-experimental_method
+with	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+ACDB3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+it	O
+loses	O
+its	O
+typical	O
+Golgi	O
+localization	B-evidence
+(	O
+Fig	O
+.	O
+3E	O
+upper	O
+panel	O
+).	O
+However	O
+,	O
+PI4KB	B-protein
+retains	O
+it	O
+Golgi	O
+localization	B-evidence
+when	O
+co	B-experimental_method
+-	I-experimental_method
+expressed	I-experimental_method
+with	O
+the	O
+non	B-protein_state
+-	I-protein_state
+interacting	I-protein_state
+Q	B-structure_element
+domain	I-structure_element
+mutant	B-protein_state
+(	O
+Fig	O
+.	O
+3E	O
+lower	O
+panel	O
+).	O
+ACBD3	B-protein
+increases	O
+PI4KB	B-protein
+enzymatic	B-evidence
+activity	I-evidence
+by	O
+recruiting	O
+PI4KB	B-protein
+to	O
+close	O
+vicinity	O
+of	O
+its	O
+substrate	O
+To	O
+test	O
+whether	O
+ACBD3	B-protein
+can	O
+stimulate	O
+PI4KB	B-protein
+kinase	B-protein_type
+enzymatic	B-evidence
+activity	I-evidence
+we	O
+performed	O
+a	O
+standard	O
+luminescent	B-experimental_method
+kinase	I-experimental_method
+assay	I-experimental_method
+using	O
+PI	B-chemical
+-	O
+containing	O
+micelles	O
+as	O
+the	O
+substrate	O
+.	O
+We	O
+observed	O
+no	O
+effect	O
+on	O
+the	O
+kinase	B-protein_type
+activity	O
+of	O
+PI4KB	B-protein
+(	O
+Fig	O
+.	O
+4A	O
+)	O
+suggesting	O
+that	O
+ACBD3	B-protein
+does	O
+not	O
+directly	O
+affect	O
+the	O
+enzyme	O
+(	O
+e	O
+.	O
+g	O
+.	O
+induction	O
+of	O
+a	O
+conformation	O
+change	O
+).	O
+However	O
+,	O
+in	O
+vivo	O
+ACBD3	B-protein
+is	O
+located	O
+at	O
+the	O
+Golgi	O
+membranes	O
+,	O
+whereas	O
+in	O
+this	O
+experiment	O
+,	O
+ACBD3	B-protein
+was	O
+located	O
+in	O
+the	O
+solution	O
+and	O
+PI	B-chemical
+is	O
+provided	O
+as	O
+micelles	O
+.	O
+For	O
+this	O
+,	O
+we	O
+again	O
+turned	O
+to	O
+the	O
+GUV	B-experimental_method
+system	O
+with	O
+ACBD3	B-protein
+localized	B-evidence
+to	O
+the	O
+GUV	B-experimental_method
+membrane	O
+.	O
+The	O
+GUVs	B-experimental_method
+contained	O
+10	O
+%	O
+PI	B-chemical
+to	O
+serve	O
+as	O
+a	O
+substrate	O
+for	O
+PI4KB	B-protein
+kinase	B-protein_type
+.	O
+The	O
+buffer	O
+also	O
+contained	O
+CFP	B-experimental_method
+-	O
+SidC	B-protein
+,	O
+which	O
+binds	O
+to	O
+PI4P	B-chemical
+with	O
+nanomolar	O
+affinity	O
+.	O
+This	O
+enabled	O
+visualization	O
+of	O
+the	O
+kinase	B-protein_type
+reaction	O
+using	O
+a	O
+confocal	B-experimental_method
+microscope	I-experimental_method
+.	O
+We	O
+compared	O
+the	O
+efficiency	O
+of	O
+the	O
+phosphorylation	B-ptm
+reaction	O
+of	O
+the	O
+kinase	B-protein_type
+alone	B-protein_state
+with	O
+that	O
+of	O
+kinase	B-protein_type
+recruited	O
+to	O
+the	O
+surface	O
+of	O
+the	O
+GUVs	B-experimental_method
+by	O
+ACBD3	B-protein
+.	O
+Reaction	O
+was	O
+also	O
+performed	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+ATP	B-chemical
+as	O
+a	O
+negative	O
+control	O
+(	O
+Fig	O
+.	O
+4B	O
+).	O
+These	O
+experiments	O
+showed	O
+that	O
+PI4KB	B-protein
+enzymatic	B-evidence
+activity	I-evidence
+increases	O
+when	O
+ACBD3	B-protein
+is	O
+membrane	O
+localized	O
+(	O
+Fig	O
+.	O
+4C	O
+,	O
+SI	O
+Fig	O
+.	O
+6	O
+).	O
+Membrane	O
+recruitment	O
+of	O
+PI4KB	B-protein
+enzyme	O
+is	O
+crucial	O
+to	O
+ensure	O
+its	O
+proper	O
+function	O
+at	O
+the	O
+Golgi	O
+and	O
+TGN	O
+.	O
+However	O
+,	O
+the	O
+molecular	O
+mechanism	O
+and	O
+structural	O
+basis	O
+for	O
+PI4KB	B-protein
+interaction	O
+with	O
+the	O
+membrane	O
+is	O
+poorly	O
+understood	O
+.	O
+In	O
+principle	O
+,	O
+any	O
+of	O
+the	O
+binding	O
+partners	O
+of	O
+PI4KB	B-protein
+could	O
+play	O
+a	O
+role	O
+in	O
+membrane	O
+recruitment	O
+.	O
+To	O
+date	O
+,	O
+several	O
+PI4KB	B-protein
+interacting	O
+proteins	O
+have	O
+been	O
+reported	O
+,	O
+including	O
+the	O
+small	B-protein_type
+GTPases	I-protein_type
+Rab11	B-protein
+and	O
+Arf1	B-protein
+,	O
+the	O
+Golgi	O
+resident	O
+acyl	B-protein
+-	I-protein
+CoA	I-protein
+binding	I-protein
+domain	I-protein
+containing	I-protein
+3	I-protein
+(	O
+ACBD3	B-protein
+)	O
+protein	O
+,	O
+neuronal	B-protein
+calcium	I-protein
+sensor	I-protein
+-	I-protein
+1	I-protein
+(	O
+NCS	B-protein
+-	I-protein
+1	I-protein
+also	O
+known	O
+as	O
+frequenin	B-protein
+in	O
+yeast	B-taxonomy_domain
+)	O
+and	O
+the	O
+14	B-protein_type
+-	I-protein_type
+3	I-protein_type
+-	I-protein_type
+3	I-protein_type
+proteins	I-protein_type
+.	O
+The	O
+monomeric	B-oligomeric_state
+G	B-protein_type
+protein	I-protein_type
+Rab11	B-protein
+binds	O
+mammalian	B-taxonomy_domain
+PI4KB	B-protein
+through	O
+the	O
+helical	B-structure_element
+domain	I-structure_element
+of	O
+the	O
+kinase	B-protein_type
+.	O
+Although	O
+Rab11	B-protein
+does	O
+not	O
+appear	O
+to	O
+be	O
+required	O
+for	O
+recruitment	O
+of	O
+PI4KB	B-protein
+to	O
+the	O
+Golgi	O
+,	O
+PI4KB	B-protein
+is	O
+required	O
+for	O
+Golgi	O
+recruitment	O
+of	O
+Rab11	B-protein
+.	O
+Arf1	B-protein
+,	O
+the	O
+other	O
+small	B-protein_type
+GTP	I-protein_type
+binding	I-protein_type
+protein	I-protein_type
+,	O
+is	O
+known	O
+to	O
+influence	O
+the	O
+activity	O
+and	O
+localization	O
+of	O
+PI4KB	B-protein
+,	O
+but	O
+it	O
+does	O
+not	O
+appear	O
+to	O
+interact	O
+directly	O
+with	O
+PI4KB	B-protein
+(	O
+our	O
+unpublished	O
+data	O
+).	O
+The	O
+yeast	B-taxonomy_domain
+homologue	O
+of	O
+NCS1	B-protein
+called	O
+frequenin	B-protein
+has	O
+been	O
+shown	O
+to	O
+interact	O
+with	O
+Pik1p	B-protein
+,	O
+the	O
+yeast	B-taxonomy_domain
+orthologue	O
+of	O
+PI4KB	B-protein
+and	O
+regulate	O
+its	O
+activity	O
+and	O
+perhaps	O
+its	O
+membrane	O
+association	O
+,	O
+but	O
+the	O
+role	O
+of	O
+NCS	B-protein
+-	I-protein
+1	I-protein
+in	O
+PI4KB	B-protein
+recruitment	O
+in	O
+mammalian	B-taxonomy_domain
+cells	O
+is	O
+unclear	O
+.	O
+NCS	B-protein
+-	I-protein
+1	I-protein
+is	O
+an	O
+N	O
+-	O
+terminally	O
+myristoylated	B-protein_state
+protein	O
+that	O
+participates	O
+in	O
+exocytosis	O
+.	O
+It	O
+is	O
+expressed	O
+only	O
+in	O
+certain	O
+cell	O
+types	O
+,	O
+suggesting	O
+that	O
+if	O
+it	O
+contributes	O
+to	O
+PI4KB	B-protein
+membrane	O
+recruitment	O
+,	O
+it	O
+does	O
+so	O
+in	O
+a	O
+tissues	O
+specific	O
+manner	O
+.	O
+The	O
+interaction	O
+of	O
+PI4KB	B-protein
+with	O
+14	B-protein_type
+-	I-protein_type
+3	I-protein_type
+-	I-protein_type
+3	I-protein_type
+proteins	I-protein_type
+,	O
+promoted	O
+by	O
+phosphorylation	B-ptm
+of	O
+PI4KB	B-protein
+by	O
+protein	B-protein
+kinase	I-protein
+D	I-protein
+,	O
+influences	O
+the	O
+activity	O
+of	O
+PI4KB	B-protein
+by	O
+stabilizing	O
+its	O
+active	B-protein_state
+conformation	O
+.	O
+However	O
+,	O
+14	B-protein_type
+-	I-protein_type
+3	I-protein_type
+-	I-protein_type
+3	I-protein_type
+proteins	I-protein_type
+do	O
+not	O
+appear	O
+to	O
+interfere	O
+with	O
+membrane	O
+recruitment	O
+of	O
+this	O
+kinase	B-protein_type
+.	O
+ACBD3	B-protein
+is	O
+a	O
+Golgi	O
+resident	O
+protein	O
+,	O
+conserved	B-protein_state
+among	O
+vertebrates	B-taxonomy_domain
+(	O
+SI	O
+Fig	O
+.	O
+7	O
+),	O
+that	O
+interacts	O
+directly	O
+with	O
+PI4KB	B-protein
+(	O
+see	O
+also	O
+SI	O
+Fig	O
+.	O
+8	O
+and	O
+SI	O
+Discussion	O
+),	O
+and	O
+whose	O
+genetic	O
+inactivation	O
+interferes	O
+with	O
+the	O
+Golgi	O
+localization	O
+of	O
+the	O
+kinase	B-protein_type
+.	O
+For	O
+these	O
+reasons	O
+we	O
+focused	O
+on	O
+the	O
+interaction	O
+of	O
+the	O
+PI4KB	B-protein
+enzyme	O
+with	O
+the	O
+Golgi	O
+resident	O
+ACBD3	B-protein
+protein	O
+in	O
+this	O
+study	O
+.	O
+Here	O
+we	O
+present	O
+the	O
+mechanism	O
+for	O
+membrane	O
+recruitment	O
+of	O
+PI4KB	B-protein
+by	O
+the	O
+Golgi	O
+resident	O
+ACBD3	B-protein
+protein	O
+.	O
+We	O
+show	O
+that	O
+these	O
+proteins	O
+interact	O
+directly	O
+with	O
+a	O
+Kd	B-evidence
+value	O
+in	O
+the	O
+submicromolar	O
+range	O
+.	O
+The	O
+interaction	O
+is	O
+sufficient	O
+to	O
+recruit	O
+PI4KB	B-protein
+to	O
+model	O
+membranes	O
+in	O
+vitro	O
+as	O
+well	O
+as	O
+to	O
+the	O
+mitochondria	O
+where	O
+PI4KB	B-protein
+is	O
+never	O
+naturally	O
+found	O
+.	O
+To	O
+understand	O
+this	O
+process	O
+at	O
+the	O
+atomic	O
+level	O
+we	O
+solved	B-experimental_method
+the	O
+solution	B-evidence
+structure	I-evidence
+of	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+sub	O
+complex	O
+(	O
+Fig	O
+.	O
+1A	O
+)	O
+and	O
+found	O
+that	O
+the	O
+PI4KB	B-protein
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+contains	O
+a	O
+short	B-structure_element
+amphipatic	I-structure_element
+helix	I-structure_element
+(	O
+residues	O
+44	B-residue_range
+–	I-residue_range
+64	I-residue_range
+)	O
+that	O
+binds	O
+the	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+.	O
+The	O
+Q	B-structure_element
+domain	I-structure_element
+adopts	O
+a	O
+helical	B-structure_element
+hairpin	I-structure_element
+fold	I-structure_element
+that	O
+is	O
+further	O
+stabilized	O
+upon	O
+binding	O
+the	O
+kinase	B-structure_element
+helix	I-structure_element
+(	O
+Fig	O
+.	O
+2A	O
+).	O
+Our	O
+data	O
+strongly	O
+suggest	O
+that	O
+formation	O
+of	O
+the	O
+complex	O
+does	O
+not	O
+directly	O
+influence	O
+the	O
+catalytic	O
+abilities	O
+of	O
+the	O
+kinase	B-protein_type
+but	O
+experiments	O
+with	O
+model	O
+membranes	O
+revealed	O
+that	O
+ACBD3	B-protein
+enhances	O
+catalytic	O
+activity	O
+of	O
+the	O
+kinase	B-protein_type
+by	O
+a	O
+recruitment	O
+based	O
+mechanism	O
+;	O
+it	O
+recruits	O
+the	O
+kinase	B-protein_type
+to	O
+the	O
+membrane	O
+and	O
+thus	O
+increases	O
+the	O
+local	O
+concentration	O
+of	O
+the	O
+substrate	O
+in	O
+the	O
+vicinity	O
+of	O
+the	O
+kinase	B-protein_type
+.	O
+Based	O
+on	O
+our	O
+and	O
+previously	O
+published	O
+structures	B-evidence
+we	O
+built	O
+a	O
+pseudoatomic	B-evidence
+model	I-evidence
+of	O
+PI4KB	B-protein
+multi	O
+-	O
+protein	O
+assembly	O
+on	O
+the	O
+membrane	O
+(	O
+Fig	O
+.	O
+5	O
+)	O
+that	O
+illustrates	O
+how	O
+the	O
+enzyme	O
+is	O
+recruited	O
+and	O
+positioned	O
+towards	O
+its	O
+lipidic	O
+substrate	O
+and	O
+how	O
+it	O
+in	O
+turn	O
+recruits	O
+Rab11	B-protein
+.	O
++	B-taxonomy_domain
+RNA	I-taxonomy_domain
+viruses	I-taxonomy_domain
+replicate	O
+at	O
+specific	O
+PI4P	B-chemical
+-	O
+enriched	O
+membranous	O
+compartments	O
+.	O
+These	O
+are	O
+called	O
+replication	O
+factories	O
+(	O
+because	O
+they	O
+enhance	O
+viral	B-taxonomy_domain
+replication	O
+)	O
+or	O
+membranous	O
+webs	O
+(	O
+because	O
+of	O
+their	O
+appearance	O
+under	O
+the	O
+electron	O
+microscope	O
+).	O
+To	O
+generate	O
+replication	O
+factories	O
+,	O
+viruses	B-taxonomy_domain
+hijack	O
+several	O
+host	O
+factors	O
+including	O
+the	O
+PI4K	B-protein_type
+kinases	B-protein_type
+to	O
+secure	O
+high	O
+content	O
+of	O
+the	O
+PI4P	B-chemical
+lipid	B-chemical
+.	O
+Non	B-protein_type
+-	I-protein_type
+structural	I-protein_type
+3A	I-protein_type
+proteins	I-protein_type
+from	O
+many	O
+picornaviruses	B-taxonomy_domain
+from	O
+the	O
+Enterovirus	B-taxonomy_domain
+(	O
+e	O
+.	O
+g	O
+.	O
+poliovirus	B-species
+,	O
+coxsackievirus	B-species
+-	I-species
+B3	I-species
+,	O
+rhinovirus	B-species
+-	I-species
+14	I-species
+)	O
+and	O
+Kobuvirus	B-taxonomy_domain
+(	O
+e	O
+.	O
+g	O
+.	O
+Aichi	B-species
+virus	I-species
+-	I-species
+1	I-species
+)	O
+genera	O
+directly	O
+interact	O
+with	O
+ACBD3	B-protein
+.	O
+Our	O
+data	O
+suggest	O
+that	O
+they	O
+could	O
+do	O
+this	O
+via	O
+3A	B-complex_assembly
+:	I-complex_assembly
+ACBD3	I-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+complex	O
+formation	O
+.	O
+The	O
+structure	B-evidence
+of	O
+the	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+and	O
+the	O
+kinase	B-structure_element
+helix	I-structure_element
+described	O
+here	O
+provides	O
+a	O
+novel	O
+opportunity	O
+for	O
+further	O
+research	O
+on	O
+the	O
+role	O
+of	O
+ACBD3	B-protein
+,	O
+PI4KB	B-protein
+,	O
+and	O
+the	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+interaction	O
+in	O
+picornaviral	B-taxonomy_domain
+replication	O
+.	O
+This	O
+could	O
+eventually	O
+have	O
+implications	O
+for	O
+therapeutic	O
+intervention	O
+to	O
+combat	O
+picornaviruses	B-taxonomy_domain
+-	O
+mediated	O
+diseases	O
+ranging	O
+from	O
+polio	O
+to	O
+the	O
+common	O
+cold	O
+.	O
+Biochemical	B-experimental_method
+characterization	I-experimental_method
+of	O
+the	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+complex	O
+.	O
+(	O
+A	O
+)	O
+Schematic	O
+representation	O
+of	O
+the	O
+ACBD3	B-protein
+and	O
+PI4KB	B-protein
+constructs	O
+used	O
+for	O
+the	O
+experiments	O
+.	O
+ACBD3	B-protein
+contains	O
+the	O
+acyl	B-structure_element
+-	I-structure_element
+CoA	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+(	O
+ACBD	B-structure_element
+),	O
+charged	B-structure_element
+amino	I-structure_element
+acids	I-structure_element
+region	I-structure_element
+(	O
+CAR	B-structure_element
+),	O
+glutamine	B-structure_element
+rich	I-structure_element
+region	I-structure_element
+(	O
+Q	B-structure_element
+),	O
+and	O
+Golgi	B-structure_element
+dynamics	I-structure_element
+domain	I-structure_element
+(	O
+GOLD	B-structure_element
+).	O
+PI4KB	B-protein
+is	O
+composed	O
+of	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+,	O
+helical	B-structure_element
+domain	I-structure_element
+,	O
+and	O
+kinase	B-structure_element
+domain	I-structure_element
+which	O
+can	O
+be	O
+divided	O
+into	O
+N	B-structure_element
+-	I-structure_element
+and	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+lobes	I-structure_element
+.	O
+(	O
+B	O
+)	O
+In	B-experimental_method
+vitro	I-experimental_method
+pull	I-experimental_method
+-	I-experimental_method
+down	I-experimental_method
+assay	I-experimental_method
+.	O
+Pull	B-experimental_method
+-	I-experimental_method
+down	I-experimental_method
+assays	I-experimental_method
+were	O
+performed	O
+using	O
+NiNTA	O
+-	O
+immobilized	O
+N	O
+-	O
+terminal	O
+His6GB1	B-protein_state
+-	I-protein_state
+tagged	I-protein_state
+proteins	O
+as	O
+indicated	O
+and	O
+untagged	B-protein_state
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+PI4KB	B-protein
+or	O
+ACBD3	B-protein
+.	O
+The	O
+inputs	O
+and	O
+bound	O
+proteins	O
+were	O
+analyzed	O
+on	O
+SDS	B-experimental_method
+gels	I-experimental_method
+stained	O
+with	O
+Coomassie	O
+Blue	O
+.	O
+Please	O
+,	O
+see	O
+SI	O
+Fig	O
+.	O
+9	O
+for	O
+original	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+gels	O
+.	O
+(	O
+C	O
+)	O
+Analytical	B-experimental_method
+Ultracentrifugation	I-experimental_method
+.	O
+AUC	B-experimental_method
+analysis	O
+of	O
+the	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+complex	O
+at	O
+the	O
+concentration	O
+of	O
+5	O
+μM	O
+(	O
+both	O
+proteins	O
+,	O
+left	O
+panel	O
+)	O
+and	O
+ACBD3	B-complex_assembly
+Q	I-complex_assembly
+domain	I-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+N	I-complex_assembly
+terminal	I-complex_assembly
+region	I-complex_assembly
+complex	O
+at	O
+the	O
+concentration	O
+of	O
+35	O
+μM	O
+(	O
+both	O
+proteins	O
+,	O
+right	O
+panel	O
+).	O
+(	O
+D	O
+)	O
+Surface	B-experimental_method
+plasmon	I-experimental_method
+resonance	I-experimental_method
+.	O
+SPR	B-experimental_method
+analysis	O
+of	O
+the	O
+PI4KB	B-protein
+binding	O
+to	O
+immobilized	O
+ACBD3	B-protein
+.	O
+Sensorgrams	B-evidence
+for	O
+four	O
+concentrations	O
+of	O
+PI4KB	B-protein
+are	O
+shown	O
+.	O
+Structural	B-experimental_method
+analysis	I-experimental_method
+of	O
+the	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+complex	O
+.	O
+(	O
+A	O
+)	O
+Overall	O
+structure	B-evidence
+of	O
+the	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+by	O
+itself	O
+and	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+the	O
+PI4KB	B-protein
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+.	O
+Superposition	B-experimental_method
+of	O
+the	O
+30	O
+converged	O
+structures	B-evidence
+obtained	O
+for	O
+the	O
+Q	B-structure_element
+domain	I-structure_element
+(	O
+top	O
+)	O
+and	O
+the	O
+45	O
+converged	O
+structures	B-evidence
+obtained	O
+for	O
+the	O
+complex	O
+(	O
+bottom	O
+),	O
+with	O
+only	O
+the	O
+folded	B-protein_state
+part	O
+of	O
+PI4KB	B-protein
+shown	O
+(	O
+see	O
+SI	O
+Fig	O
+.	O
+2	O
+for	O
+the	O
+complete	O
+view	O
+).	O
+(	O
+B	O
+)	O
+Detailed	O
+view	O
+of	O
+the	O
+complex	O
+.	O
+The	O
+interaction	O
+is	O
+facilitated	O
+by	O
+only	O
+two	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+(	O
+ACBD3	B-protein
+Tyr261	B-residue_name_number
+:	O
+PI4KB	B-protein
+His63	B-residue_name_number
+and	O
+ACBD3	B-protein
+Tyr288	B-residue_name_number
+:	O
+PI4KB	B-protein
+Asp44	B-residue_name_number
+),	O
+while	O
+the	O
+hydrophobic	B-site
+surface	I-site
+of	O
+the	O
+kinase	B-structure_element
+helix	I-structure_element
+nests	O
+in	O
+the	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+.	O
+ACBD3	B-protein
+is	O
+shown	O
+in	O
+magenta	O
+and	O
+PI4KB	B-protein
+in	O
+orange	O
+.	O
+(	O
+C	O
+)	O
+Top	O
+view	O
+of	O
+the	O
+kinase	B-structure_element
+helix	I-structure_element
+.	O
+The	O
+kinase	B-structure_element
+helix	I-structure_element
+is	O
+amphipathic	B-protein_state
+and	O
+its	O
+hydrophobic	B-site
+surface	I-site
+overlaps	O
+with	O
+the	O
+ACBD3	B-protein
+binding	B-site
+surface	I-site
+(	O
+shown	O
+in	O
+magenta	O
+).	O
+Strong	O
+and	O
+weak	O
+hydrophobes	O
+are	O
+in	O
+green	O
+and	O
+cyan	O
+respectively	O
+,	O
+basic	O
+residues	O
+in	O
+blue	O
+,	O
+acidic	O
+residues	O
+in	O
+red	O
+and	O
+nonpolar	O
+hydrophilic	O
+residues	O
+in	O
+orange	O
+.	O
+(	O
+D	O
+)	O
+Pull	B-experimental_method
+-	I-experimental_method
+down	I-experimental_method
+assay	I-experimental_method
+with	O
+a	O
+NiNTA	O
+-	O
+immobilized	O
+N	O
+-	O
+terminally	O
+His6GB1	B-protein_state
+-	I-protein_state
+tagged	I-protein_state
+PI4KB	B-protein
+kinase	B-protein_type
+and	O
+untagged	B-protein_state
+ACBD3	B-protein
+protein	O
+.	O
+Wild	B-protein_state
+type	I-protein_state
+proteins	O
+and	O
+selected	O
+point	O
+mutants	B-protein_state
+of	O
+both	O
+PI4KB	B-protein
+and	O
+ACBD3	B-protein
+were	O
+used	O
+.	O
+Please	O
+,	O
+see	O
+SI	O
+Fig	O
+.	O
+9	O
+for	O
+original	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+gels	O
+.	O
+ACBD3	B-protein
+is	O
+sufficient	O
+to	O
+recruit	O
+the	O
+PI4KB	B-protein
+kinase	B-protein_type
+to	O
+membranes	O
+.	O
+(	O
+A	O
+)	O
+GUVs	B-experimental_method
+recruitment	I-experimental_method
+assay	I-experimental_method
+.	O
+Top	O
+–	O
+Virtually	O
+no	O
+membrane	O
+bound	O
+kinase	B-protein_type
+was	O
+observed	O
+when	O
+600	O
+nM	O
+PI4KB	B-protein
+was	O
+added	O
+to	O
+the	O
+GUVs	B-experimental_method
+.	O
+Bottom	O
+–	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+600	O
+nM	O
+GUV	B-protein_state
+tethered	I-protein_state
+ACBD3	B-protein
+a	O
+significant	O
+signal	O
+of	O
+the	O
+kinase	B-protein_type
+is	O
+detected	O
+on	O
+the	O
+surface	O
+of	O
+GUVs	B-experimental_method
+.	O
+(	O
+B	O
+)	O
+Golgi	B-experimental_method
+displacement	I-experimental_method
+experiment	I-experimental_method
+.	O
+Upper	O
+panel	O
+:	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+fused	O
+to	O
+GFP	B-experimental_method
+was	O
+overexpressed	B-experimental_method
+and	O
+the	O
+endogenous	O
+PI4KB	B-protein
+was	O
+immunostained	B-experimental_method
+.	O
+Middle	O
+panel	O
+:	O
+The	O
+same	O
+experiment	O
+performed	O
+with	O
+GFP	B-experimental_method
+alone	O
+.	O
+Lower	O
+panel	O
+:	O
+The	O
+same	O
+experiment	O
+performed	O
+with	O
+mutant	B-protein_state
+Q	B-structure_element
+domain	I-structure_element
+(	O
+F258A	B-mutant
+,	O
+H284A	B-mutant
+,	O
+Y288A	B-mutant
+)	O
+that	O
+does	O
+not	O
+bind	O
+the	O
+PI4KB	B-protein
+.	O
+(	O
+C	O
+)	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+overexpression	B-experimental_method
+inhibits	O
+ceramide	B-chemical
+transport	O
+to	O
+Golgi	O
+–	O
+COS	O
+-	O
+7	O
+cells	O
+transfected	O
+with	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+-	O
+FKBP	B-protein
+-	O
+mRFP	B-experimental_method
+were	O
+loaded	O
+with	O
+0	O
+.	O
+05	O
+μM	O
+Bodipy	B-chemical
+FL	I-chemical
+-	I-chemical
+Ceramide	I-chemical
+for	O
+20	O
+min	O
+,	O
+then	O
+washed	O
+and	O
+depicted	O
+after	O
+20	O
+min	O
+.	O
+Middle	O
+panel	O
+–	O
+The	O
+same	O
+experiment	O
+performed	O
+with	O
+mRFP	B-experimental_method
+-	O
+FKBP	B-protein
+alone	O
+.	O
+Lower	O
+panel	O
+–	O
+The	O
+same	O
+experiment	O
+performed	O
+with	O
+mutant	B-protein_state
+Q	B-structure_element
+domain	I-structure_element
+(	O
+F258A	B-mutant
+,	O
+H284A	B-mutant
+,	O
+Y288A	B-mutant
+)	O
+that	O
+does	O
+not	O
+bind	O
+the	O
+PI4KB	B-protein
+.	O
+(	O
+D	O
+)	O
+Scheme	O
+of	O
+the	O
+mitochondria	B-experimental_method
+recruitment	I-experimental_method
+experiment	I-experimental_method
+.	O
+–	O
+The	O
+AKAP1	B-protein
+-	O
+FRB	B-structure_element
+-	O
+CFP	B-experimental_method
+construct	O
+is	O
+localized	B-evidence
+at	O
+the	O
+outer	O
+mitochondrial	O
+membrane	O
+,	O
+while	O
+the	O
+GFP	B-experimental_method
+-	O
+PI4KB	B-protein
+and	O
+Q	B-structure_element
+domain	I-structure_element
+-	O
+FKBP	B-protein
+-	O
+mRFP	B-experimental_method
+constructs	O
+are	O
+localized	B-evidence
+in	O
+the	O
+cytoplasm	O
+where	O
+they	O
+can	O
+form	O
+a	O
+complex	O
+.	O
+Upon	O
+addition	O
+of	O
+rapamycin	B-chemical
+the	O
+Q	B-structure_element
+domain	I-structure_element
+-	O
+FKBP	B-protein
+-	O
+mRFP	B-experimental_method
+construct	O
+translocates	O
+to	O
+the	O
+mitochondria	O
+and	O
+takes	O
+GFP	B-experimental_method
+-	O
+PI4KB	B-protein
+with	O
+it	O
+.	O
+(	O
+E	O
+)	O
+Mitochondria	B-experimental_method
+recruitment	I-experimental_method
+experiment	I-experimental_method
+.	O
+Left	O
+–	O
+cells	O
+transfected	O
+with	O
+AKAP1	B-protein
+-	O
+FRB	B-structure_element
+-	O
+CFP	B-experimental_method
+,	O
+GFP	B-experimental_method
+-	O
+PI4KB	B-protein
+and	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+Q	B-structure_element
+domain	I-structure_element
+-	O
+FKBP	B-protein
+-	O
+mRFP	B-experimental_method
+constructs	O
+before	O
+and	O
+five	O
+minutes	O
+after	O
+addition	O
+of	O
+rapamycin	B-chemical
+.	O
+Right	O
+–	O
+The	O
+same	O
+experiment	O
+performed	O
+using	O
+the	O
+H264A	B-mutant
+Q	B-structure_element
+domain	I-structure_element
+mutant	B-protein_state
+.	O
+ACBD3	B-protein
+indirectly	O
+increases	O
+the	O
+activity	O
+of	O
+PI4KB	B-protein
+.	O
+(	O
+A	O
+)	O
+Micelles	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+kinase	I-experimental_method
+assay	I-experimental_method
+–	O
+PI	B-chemical
+in	O
+TX100	O
+micelles	O
+was	O
+used	O
+in	O
+a	O
+luminescent	B-experimental_method
+kinase	I-experimental_method
+assay	I-experimental_method
+and	O
+the	O
+production	O
+of	O
+PI4P	B-chemical
+was	O
+measured	O
+.	O
+Bar	O
+graph	O
+presents	O
+the	O
+mean	O
+values	O
+of	O
+PI4P	B-chemical
+generated	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+the	O
+proteins	O
+as	O
+indicated	O
+,	O
+normalized	O
+to	O
+the	O
+amount	O
+of	O
+PI4P	B-chemical
+generated	O
+by	O
+PI4KB	B-protein
+alone	O
+.	O
+Error	O
+bars	O
+are	O
+standard	B-evidence
+errors	I-evidence
+of	I-evidence
+the	I-evidence
+mean	I-evidence
+(	O
+SEM	B-evidence
+)	O
+based	O
+on	O
+three	O
+independent	O
+experiments	O
+.	O
+(	O
+B	O
+)	O
+GUV	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+phosphorylation	I-experimental_method
+assay	I-experimental_method
+–	O
+GUVs	B-experimental_method
+containing	O
+10	O
+%	O
+PI	B-chemical
+were	O
+used	O
+as	O
+a	O
+substrate	O
+and	O
+the	O
+production	O
+of	O
+PI4P	B-chemical
+was	O
+measured	O
+using	O
+the	O
+CFP	B-experimental_method
+-	I-experimental_method
+SidC	I-experimental_method
+biosensor	I-experimental_method
+.	O
+(	O
+C	O
+)–	O
+Quantification	O
+of	O
+the	O
+GUV	B-experimental_method
+phosphorylation	I-experimental_method
+assay	I-experimental_method
+–	O
+Mean	B-evidence
+membrane	I-evidence
+fluorescence	I-evidence
+intensity	I-evidence
+of	O
+the	O
+PI4P	B-chemical
+reporter	O
+(	O
+SidC	B-protein
+-	O
+label	O
+)	O
+under	O
+different	O
+protein	O
+/	O
+ATP	B-chemical
+conditions	O
+.	O
+The	O
+mean	B-evidence
+membrane	I-evidence
+intensity	I-evidence
+value	O
+is	O
+relative	O
+to	O
+the	O
+background	O
+signal	O
+and	O
+the	O
+difference	O
+between	O
+the	O
+membrane	O
+and	O
+background	O
+signal	O
+in	O
+the	O
+reference	O
+system	O
+lacking	O
+ATP	B-chemical
+.	O
+The	O
+error	O
+bars	O
+stand	O
+for	O
+SEM	B-evidence
+based	O
+on	O
+three	O
+independent	O
+experiments	O
+(	O
+also	O
+SI	O
+Fig	O
+.	O
+6	O
+).	O
+Pseudoatomic	B-evidence
+model	I-evidence
+of	O
+the	O
+PI4KB	B-protein
+multiprotein	O
+complex	O
+assembly	O
+.	O
+PI4KB	B-protein
+in	O
+orange	O
+,	O
+Rab11	B-protein
+in	O
+purple	O
+,	O
+ACBD3	B-protein
+in	O
+blue	O
+.	O
+The	O
+model	O
+is	O
+based	O
+on	O
+our	O
+NMR	B-experimental_method
+structure	B-evidence
+and	O
+a	O
+previously	O
+published	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+PI4KB	B-complex_assembly
+:	I-complex_assembly
+Rab11	I-complex_assembly
+complex	O
+(	O
+PDB	O
+code	O
+4D0L	O
+),	O
+ACBD	B-structure_element
+and	O
+GOLD	B-structure_element
+domain	O
+were	O
+homology	B-experimental_method
+modeled	I-experimental_method
+based	O
+on	O
+high	O
+sequence	O
+identity	O
+structures	B-evidence
+produced	O
+by	O
+the	O
+Phyre2	B-experimental_method
+web	O
+server	O
+.	O
+The	O
+GOLD	B-structure_element
+domain	O
+is	O
+tethered	O
+to	O
+the	O
+membrane	O
+by	O
+GolginB1	B-protein
+(	O
+also	O
+known	O
+as	O
+Giantin	B-protein
+)	O
+which	O
+is	O
+not	O
+shown	O
+for	O
+clarity	O
+.	O
+Intrinsically	B-structure_element
+disordered	I-structure_element
+linkers	I-structure_element
+are	O
+modeled	O
+in	O
+an	O
+arbitrary	O
+but	O
+physically	O
+plausible	O
+conformation	O
+.	O

annotation_IOB/PMC4817029.tsv ADDED Viewed

	@@ -0,0 +1,6158 @@

+Molecular	O
+characterization	O
+of	O
+a	O
+family	B-protein_type
+5	I-protein_type
+glycoside	I-protein_type
+hydrolase	I-protein_type
+suggests	O
+an	O
+induced	O
+-	O
+fit	O
+enzymatic	O
+mechanism	O
+Glycoside	B-protein_type
+hydrolases	I-protein_type
+(	O
+GHs	B-protein_type
+)	O
+play	O
+fundamental	O
+roles	O
+in	O
+the	O
+decomposition	O
+of	O
+lignocellulosic	O
+biomaterials	O
+.	O
+Here	O
+,	O
+we	O
+report	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+structure	B-evidence
+of	O
+a	O
+cellulase	B-protein_type
+from	O
+Bacillus	B-species
+licheniformis	I-species
+(	O
+BlCel5B	B-protein
+),	O
+a	O
+member	O
+of	O
+the	O
+GH5	B-protein_type
+subfamily	I-protein_type
+4	I-protein_type
+that	O
+is	O
+entirely	O
+dependent	O
+on	O
+its	O
+two	O
+ancillary	B-structure_element
+modules	I-structure_element
+(	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+and	O
+CBM46	B-structure_element
+)	O
+for	O
+catalytic	O
+activity	O
+.	O
+Using	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+,	O
+small	B-experimental_method
+-	I-experimental_method
+angle	I-experimental_method
+X	I-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+scattering	I-experimental_method
+and	O
+molecular	B-experimental_method
+dynamics	I-experimental_method
+simulations	I-experimental_method
+,	O
+we	O
+propose	O
+that	O
+the	O
+C	O
+-	O
+terminal	O
+CBM46	B-structure_element
+caps	O
+the	O
+distal	O
+N	O
+-	O
+terminal	O
+catalytic	B-structure_element
+domain	I-structure_element
+(	O
+CD	B-structure_element
+)	O
+to	O
+establish	O
+a	O
+fully	B-protein_state
+functional	I-protein_state
+active	B-site
+site	I-site
+via	O
+a	O
+combination	O
+of	O
+large	O
+-	O
+scale	O
+multidomain	O
+conformational	O
+selection	O
+and	O
+induced	O
+-	O
+fit	O
+mechanisms	O
+.	O
+The	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+is	O
+pivoting	O
+the	O
+packing	O
+and	O
+unpacking	O
+motions	O
+of	O
+CBM46	B-structure_element
+relative	O
+to	O
+CD	B-structure_element
+in	O
+the	O
+assembly	O
+of	O
+the	O
+binding	B-site
+subsite	I-site
+.	O
+This	O
+is	O
+the	O
+first	O
+example	O
+of	O
+a	O
+multidomain	O
+GH	B-protein_type
+relying	O
+on	O
+large	O
+amplitude	O
+motions	O
+of	O
+the	O
+CBM46	B-structure_element
+for	O
+assembly	O
+of	O
+the	O
+catalytically	B-protein_state
+competent	I-protein_state
+form	O
+of	O
+the	O
+enzyme	O
+.	O
+Plant	B-taxonomy_domain
+biomass	O
+-	O
+the	O
+most	O
+abundant	O
+source	O
+of	O
+carbohydrates	B-chemical
+on	O
+Earth	O
+-	O
+is	O
+primarily	O
+composed	O
+of	O
+cellulose	B-chemical
+microfibrils	O
+surrounded	O
+by	O
+a	O
+hydrated	O
+heteropolymeric	O
+matrix	O
+of	O
+hemicellulose	B-chemical
+and	O
+lignin	B-chemical
+.	O
+Plant	B-taxonomy_domain
+biomass	O
+may	O
+be	O
+subjected	O
+to	O
+thermo	O
+-	O
+chemical	O
+pretreatments	O
+and	O
+enzymatic	O
+reactions	O
+to	O
+produce	O
+soluble	O
+fermentable	O
+sugars	B-chemical
+.	O
+The	O
+canonical	O
+model	O
+of	O
+hydrolytic	O
+degradation	O
+of	O
+cellulose	B-chemical
+requires	O
+at	O
+least	O
+three	O
+classes	O
+of	O
+enzymes	O
+.	O
+Cellobiohydrolases	B-protein_type
+(	O
+CBHs	B-protein_type
+)	O
+processively	O
+cleave	O
+the	O
+glycosidic	O
+bonds	O
+at	O
+the	O
+reducing	O
+and	O
+non	O
+-	O
+reducing	O
+ends	O
+of	O
+cellulose	B-chemical
+chains	O
+in	O
+crystalline	O
+regions	O
+to	O
+produce	O
+cellobiose	B-chemical
+.	O
+Endoglucanases	B-protein_type
+(	O
+EGs	B-protein_type
+)	O
+introduce	O
+random	O
+cuts	O
+in	O
+the	O
+amorphous	O
+regions	O
+of	O
+cellulose	B-chemical
+and	O
+create	O
+new	O
+chain	O
+extremities	O
+for	O
+CBH	B-protein_type
+attack	O
+;	O
+thus	O
+,	O
+these	O
+enzymes	O
+act	O
+synergistically	O
+.	O
+The	O
+released	O
+cellobiose	B-chemical
+molecules	O
+are	O
+then	O
+enzymatically	O
+converted	O
+into	O
+glucose	B-chemical
+by	O
+β	B-protein_type
+-	I-protein_type
+glucosidases	I-protein_type
+.	O
+The	O
+molecular	O
+architecture	O
+of	O
+glycoside	B-protein_type
+hydrolases	I-protein_type
+(	O
+GHs	B-protein_type
+)	O
+frequently	O
+consists	O
+of	O
+a	O
+catalytic	B-structure_element
+domain	I-structure_element
+(	O
+CD	B-structure_element
+),	O
+where	O
+hydrolysis	O
+occurs	O
+,	O
+and	O
+one	O
+or	O
+more	O
+ancillary	B-structure_element
+modules	I-structure_element
+(	O
+AMs	B-structure_element
+),	O
+which	O
+are	O
+usually	O
+connected	O
+by	O
+less	B-protein_state
+structured	I-protein_state
+linkers	B-structure_element
+.	O
+The	O
+most	O
+common	O
+type	O
+of	O
+AMs	B-structure_element
+are	O
+carbohydrate	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+modules	I-structure_element
+(	O
+CBMs	B-structure_element
+),	O
+which	O
+are	O
+able	O
+to	O
+recognize	O
+and	O
+bind	O
+specific	O
+carbohydrate	B-chemical
+chains	O
+.	O
+Generally	O
+distinct	O
+and	O
+independent	O
+structural	O
+domains	O
+,	O
+the	O
+CBMs	B-structure_element
+facilitate	O
+carbohydrate	B-chemical
+hydrolysis	O
+by	O
+increasing	O
+the	O
+local	O
+concentration	O
+of	O
+enzymes	O
+at	O
+the	O
+surface	O
+of	O
+insoluble	O
+substrates	O
+,	O
+thereby	O
+targeting	O
+the	O
+CD	B-structure_element
+component	O
+to	O
+its	O
+cognate	O
+ligands	O
+.	O
+CBMs	B-structure_element
+might	O
+also	O
+disrupt	O
+the	O
+crystalline	O
+structure	O
+of	O
+cellulose	B-chemical
+microfibrils	O
+,	O
+although	O
+the	O
+underlying	O
+mechanism	O
+remains	O
+poorly	O
+understood	O
+.	O
+Thus	O
+,	O
+CBMs	B-structure_element
+enhance	O
+the	O
+accessibility	O
+of	O
+CDs	B-structure_element
+to	O
+carbohydrate	B-chemical
+chains	O
+to	O
+improve	O
+enzymatic	O
+activity	O
+,	O
+making	O
+them	O
+important	O
+candidates	O
+for	O
+the	O
+development	O
+of	O
+effective	O
+biomass	O
+-	O
+degrading	O
+enzymes	O
+in	O
+industrial	O
+settings	O
+.	O
+Although	O
+there	O
+are	O
+examples	O
+of	O
+active	B-protein_state
+GHs	B-protein_type
+that	O
+lack	B-protein_state
+AMs	B-structure_element
+,	O
+the	O
+majority	O
+of	O
+the	O
+enzymes	O
+depend	O
+on	O
+AMs	B-structure_element
+for	O
+activity	O
+.	O
+In	O
+several	O
+cases	O
+,	O
+CBMs	B-structure_element
+were	O
+shown	O
+to	O
+extend	O
+and	O
+complement	O
+the	O
+CD	B-structure_element
+substrate	B-site
+-	I-site
+binding	I-site
+site	I-site
+in	O
+multimodular	O
+carbohydrate	B-protein_type
+-	I-protein_type
+active	I-protein_type
+enzymes	I-protein_type
+,	O
+such	O
+as	O
+endo	B-protein_type
+/	I-protein_type
+exocellulase	I-protein_type
+E4	B-protein
+from	O
+Thermobifida	B-species
+fusca	I-species
+,	O
+chitinase	B-protein
+B	I-protein
+from	O
+Serratia	B-species
+marcescens	I-species
+,	O
+a	O
+starch	B-protein_type
+phosphatase	I-protein_type
+from	O
+Arabidopsis	B-species
+thaliana	I-species
+and	O
+a	O
+GH5	B-protein_type
+subfamily	I-protein_type
+4	I-protein_type
+(	O
+GH5_4	B-protein_type
+)	O
+endoglucanase	B-protein_type
+from	O
+Bacillus	B-species
+halodurans	I-species
+(	O
+BhCel5B	B-protein
+).	O
+A	O
+pioneer	O
+work	O
+of	O
+Sakon	O
+et	O
+al	O
+.	O
+revealed	O
+that	O
+rigid	O
+structural	O
+extension	O
+of	O
+the	O
+GH9	B-protein_type
+CD	B-structure_element
+by	O
+a	O
+type	B-structure_element
+C	I-structure_element
+CBM3	I-structure_element
+imprints	O
+a	O
+processive	O
+mode	O
+of	O
+action	O
+to	O
+this	O
+endoglucanase	B-protein_type
+.	O
+Further	O
+publications	O
+showed	O
+that	O
+CBM	B-structure_element
+-	O
+based	O
+structural	O
+extensions	O
+of	O
+the	O
+active	B-site
+site	I-site
+are	O
+important	O
+for	O
+substrate	O
+engagement	O
+and	O
+recognition	O
+.	O
+Recently	O
+,	O
+Venditto	O
+et	O
+al	O
+.	O
+reported	O
+the	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+structure	I-evidence
+of	O
+the	O
+tri	B-structure_element
+-	I-structure_element
+modular	I-structure_element
+GH5_4	B-protein_type
+endoglucanase	B-protein_type
+from	O
+Bacillus	B-species
+halodurans	I-species
+(	O
+31	O
+%	O
+sequence	O
+identity	O
+to	O
+BlCel5B	B-protein
+),	O
+with	O
+the	O
+CBM46	B-structure_element
+extension	O
+of	O
+the	O
+active	B-site
+site	I-site
+appended	O
+to	O
+the	O
+CD	B-structure_element
+via	O
+an	O
+immunoglobulin	B-structure_element
+(	I-structure_element
+Ig	I-structure_element
+)-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+.	O
+Removal	B-experimental_method
+of	I-experimental_method
+the	O
+CBM46	B-structure_element
+caused	O
+a	O
+~	O
+60	O
+-	O
+fold	O
+reduction	O
+of	O
+the	O
+activity	O
+of	O
+the	O
+enzyme	O
+against	O
+β	B-chemical
+-	I-chemical
+glucans	I-chemical
+,	O
+but	O
+showed	O
+little	O
+or	O
+no	O
+effect	O
+against	O
+xyloglucan	B-chemical
+hydrolysis	O
+.	O
+Moreover	O
+,	O
+the	O
+CBM46	B-structure_element
+mediated	O
+a	O
+significant	O
+increase	O
+in	O
+the	O
+BhCel5B	B-protein
+activity	O
+in	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+settings	O
+.	O
+Modeling	B-experimental_method
+of	O
+cellotriose	B-chemical
+in	O
+the	O
+negative	B-site
+subsites	I-site
+of	O
+the	O
+active	B-site
+site	I-site
+of	O
+BhCel5B	B-protein
+demonstrated	O
+the	O
+structural	B-protein_state
+conservation	I-protein_state
+of	O
+the	O
+-	B-residue_number
+1	I-residue_number
+position	O
+,	O
+but	O
+provided	O
+little	O
+information	O
+about	O
+direct	O
+interactions	O
+between	O
+CBM46	B-structure_element
+and	O
+the	O
+substrate	O
+.	O
+It	O
+was	O
+speculated	O
+that	O
+β	O
+-	O
+1	O
+,	O
+3	O
+kink	O
+of	O
+the	O
+β	B-chemical
+-	I-chemical
+glucan	I-chemical
+might	O
+allow	O
+the	O
+ligand	O
+to	O
+reach	O
+for	O
+the	O
+CBM46	B-structure_element
+,	O
+whereas	O
+pure	O
+β	O
+-	O
+1	O
+,	O
+4	O
+linkages	O
+in	O
+the	O
+backbone	O
+of	O
+xyloglucan	B-chemical
+chains	O
+would	O
+restrict	O
+binding	O
+to	O
+the	O
+CD	B-structure_element
+,	O
+thus	O
+explaining	O
+the	O
+lack	O
+of	O
+influence	O
+of	O
+the	O
+CBM46	B-structure_element
+on	O
+the	O
+enzymatic	O
+activity	O
+of	O
+BhCel5B	B-protein
+against	O
+xyloglucans	B-chemical
+in	O
+solution	O
+.	O
+It	O
+was	O
+also	O
+argued	O
+that	O
+the	O
+CBM46	B-structure_element
+could	O
+potentialize	O
+the	O
+activity	O
+by	O
+driving	O
+BhCel5B	B-protein
+towards	O
+xyloglucan	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+regions	I-structure_element
+in	O
+the	O
+context	O
+of	O
+the	O
+plant	B-taxonomy_domain
+cell	O
+walls	O
+,	O
+but	O
+no	O
+large	O
+-	O
+scale	O
+conformational	O
+adjustments	O
+of	O
+the	O
+AMs	B-structure_element
+have	O
+been	O
+shown	O
+to	O
+occur	O
+or	O
+suggested	O
+to	O
+take	O
+part	O
+in	O
+the	O
+enzymatic	O
+activity	O
+.	O
+Although	O
+initially	O
+introduced	O
+as	O
+contradictory	O
+theories	O
+,	O
+these	O
+two	O
+limiting	O
+cases	O
+can	O
+be	O
+unified	O
+considering	O
+the	O
+flux	O
+description	O
+concept	O
+or	O
+the	O
+extended	B-protein_state
+conformational	O
+selection	O
+model	O
+.	O
+While	O
+local	O
+ligand	O
+-	O
+induced	O
+conformational	O
+adjustments	O
+have	O
+been	O
+reported	O
+for	O
+carbohydrate	B-protein_type
+-	I-protein_type
+active	I-protein_type
+enzymes	I-protein_type
+,	O
+cognate	O
+ligands	O
+recognition	O
+and	O
+hydrolysis	O
+mediated	O
+by	O
+a	O
+large	O
+-	O
+scale	O
+conformational	O
+mobility	O
+of	O
+distinct	O
+domains	O
+in	O
+multidomain	O
+settings	O
+is	O
+uncommon	O
+for	O
+endoglucanases	B-protein_type
+.	O
+Here	O
+,	O
+we	O
+report	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+a	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+GH5_4	B-protein_type
+enzyme	O
+from	O
+Bacillus	B-species
+licheniformis	I-species
+(	O
+BlCel5B	B-protein
+)	O
+that	O
+exhibits	O
+two	O
+AMs	B-structure_element
+(	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+and	O
+CBM46	B-structure_element
+)	O
+appended	O
+to	O
+the	O
+CD	B-structure_element
+.	O
+We	O
+structurally	B-experimental_method
+and	I-experimental_method
+functionally	I-experimental_method
+characterize	I-experimental_method
+the	O
+enzyme	O
+using	O
+a	O
+combination	O
+of	O
+protein	B-experimental_method
+crystallography	I-experimental_method
+,	O
+small	B-experimental_method
+-	I-experimental_method
+angle	I-experimental_method
+X	I-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+scattering	I-experimental_method
+(	O
+SAXS	B-experimental_method
+),	O
+molecular	B-experimental_method
+dynamics	I-experimental_method
+computer	I-experimental_method
+simulations	I-experimental_method
+and	O
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutagenesis	I-experimental_method
+,	O
+and	O
+show	O
+that	O
+the	O
+AMs	B-structure_element
+and	O
+their	O
+conformational	O
+mobility	O
+are	O
+essential	O
+for	O
+the	O
+enzymatic	O
+activity	O
+of	O
+BlCel5B	B-protein
+.	O
+We	O
+find	O
+that	O
+the	O
+large	O
+-	O
+scale	O
+conformational	O
+adjustments	O
+of	O
+the	O
+distal	O
+CBM46	B-structure_element
+mediated	O
+by	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+hinge	I-structure_element
+domain	I-structure_element
+are	O
+crucial	O
+in	O
+active	B-site
+-	I-site
+site	I-site
+assembly	O
+for	O
+optimal	O
+substrate	O
+binding	O
+and	O
+hydrolysis	O
+.	O
+We	O
+propose	O
+that	O
+the	O
+BlCel5B	B-protein
+conformational	O
+selection	O
+/	O
+induced	O
+-	O
+fit	O
+mechanism	O
+of	O
+hydrolysis	O
+represents	O
+a	O
+novel	O
+paradigm	O
+that	O
+applies	O
+to	O
+several	O
+GH5_4	B-protein_type
+members	O
+and	O
+,	O
+possibly	O
+,	O
+to	O
+a	O
+number	O
+of	O
+other	O
+multidomain	O
+GHs	B-protein_type
+.	O
+BlCel5B	B-protein
+Crystal	B-evidence
+Structure	I-evidence
+BlCel5B	B-protein
+crystals	B-evidence
+in	O
+the	O
+substrate	B-protein_state
+-	I-protein_state
+free	I-protein_state
+form	O
+and	O
+complexed	B-protein_state
+with	I-protein_state
+cellopentaose	B-chemical
+(	O
+C5	B-chemical
+)	O
+were	O
+obtained	O
+and	O
+diffracted	O
+to	O
+1	O
+.	O
+7	O
+Å	O
+and	O
+1	O
+.	O
+75	O
+Å	O
+resolutions	O
+,	O
+respectively	O
+(	O
+Supplementary	O
+Table	O
+1	O
+).	O
+The	O
+substrate	B-protein_state
+-	I-protein_state
+free	I-protein_state
+and	O
+complexed	B-protein_state
+structures	B-evidence
+exhibited	O
+no	O
+substantial	O
+conformational	O
+differences	O
+(	O
+with	O
+the	O
+exception	O
+of	O
+the	O
+substrate	O
+).	O
+Because	O
+of	O
+minor	O
+variations	O
+in	O
+the	O
+loops	B-structure_element
+located	O
+distal	O
+to	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+site	I-site
+,	O
+a	O
+root	B-evidence
+mean	I-evidence
+squared	I-evidence
+deviation	I-evidence
+(	O
+rmsd	B-evidence
+)	O
+of	O
+0	O
+.	O
+33	O
+Å	O
+between	O
+the	O
+complexed	B-protein_state
+and	O
+substrate	B-protein_state
+-	I-protein_state
+free	I-protein_state
+structures	B-evidence
+was	O
+observed	O
+.	O
+A	O
+single	O
+protein	O
+chain	O
+occupies	O
+the	O
+asymmetric	O
+unit	O
+,	O
+and	O
+most	O
+of	O
+the	O
+residues	O
+were	O
+built	O
+,	O
+with	O
+the	O
+exception	O
+of	O
+the	O
+first	B-residue_range
+17	I-residue_range
+residues	I-residue_range
+and	O
+those	O
+in	O
+the	O
+loop	B-structure_element
+between	O
+L398	B-residue_name_number
+and	O
+P405	B-residue_name_number
+due	O
+to	O
+weak	O
+electron	B-evidence
+density	I-evidence
+.	O
+The	O
+BlCel5B	B-protein
+structure	B-evidence
+comprises	O
+three	O
+distinct	O
+domains	O
+:	O
+an	O
+N	O
+-	O
+terminal	O
+CD	B-structure_element
+(	O
+residues	O
+18	B-residue_range
+to	I-residue_range
+330	I-residue_range
+),	O
+an	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+(	O
+residues	O
+335	B-residue_range
+to	I-residue_range
+428	I-residue_range
+)	O
+and	O
+a	O
+family	B-structure_element
+46	I-structure_element
+CBM	I-structure_element
+(	O
+residues	O
+432	B-residue_range
+to	I-residue_range
+533	I-residue_range
+)	O
+(	O
+Fig	O
+.	O
+1A	O
+,	O
+B	O
+).	O
+Similarly	O
+to	O
+other	O
+members	O
+of	O
+the	O
+GH5	B-protein_type
+family	O
+,	O
+the	O
+CD	B-structure_element
+of	O
+BlCel5B	B-protein
+has	O
+a	O
+typical	O
+TIM	B-structure_element
+barrel	I-structure_element
+fold	I-structure_element
+with	O
+eight	O
+inner	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+and	O
+eight	O
+outer	O
+α	B-structure_element
+helices	I-structure_element
+that	O
+are	O
+interconnected	O
+by	O
+loops	B-structure_element
+and	O
+three	O
+short	O
+α	B-structure_element
+helices	I-structure_element
+.	O
+Very	O
+short	O
+linkers	B-structure_element
+,	O
+D429	B-structure_element
+-	I-structure_element
+D430	I-structure_element
+-	I-structure_element
+P431	I-structure_element
+and	O
+V331	B-structure_element
+-	I-structure_element
+P332	I-structure_element
+-	I-structure_element
+N333	I-structure_element
+-	I-structure_element
+A334	I-structure_element
+,	O
+connect	O
+the	O
+CBM46	B-structure_element
+to	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+and	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+to	O
+the	O
+CD	B-structure_element
+,	O
+respectively	O
+.	O
+Both	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+and	O
+CBM46	B-structure_element
+have	O
+a	O
+β	B-structure_element
+-	I-structure_element
+sandwich	I-structure_element
+fold	I-structure_element
+composed	O
+of	O
+two	O
+β	B-structure_element
+-	I-structure_element
+sheets	I-structure_element
+of	O
+four	O
+and	O
+three	O
+antiparallel	B-structure_element
+β	I-structure_element
+-	I-structure_element
+strands	I-structure_element
+interconnected	O
+by	O
+loops	B-structure_element
+and	O
+a	O
+short	O
+α	B-structure_element
+helix	I-structure_element
+between	O
+strands	B-structure_element
+β3	B-structure_element
+and	O
+β4	B-structure_element
+(	O
+Fig	O
+.	O
+1C	O
+).	O
+A	O
+structural	B-experimental_method
+comparison	I-experimental_method
+between	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+and	O
+the	O
+CBM46	B-structure_element
+using	O
+the	O
+Dali	B-experimental_method
+server	I-experimental_method
+yielded	O
+an	O
+rmsd	B-evidence
+of	O
+2	O
+.	O
+3	O
+Å	O
+and	O
+a	O
+Z	B-evidence
+-	I-evidence
+score	I-evidence
+of	O
+10	O
+.	O
+2	O
+.	O
+A	O
+structure	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+search	I-experimental_method
+performed	O
+using	O
+the	O
+same	O
+server	O
+showed	O
+that	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+is	O
+similar	O
+to	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+from	O
+a	O
+recently	O
+solved	B-experimental_method
+crystal	B-evidence
+structure	I-evidence
+of	O
+a	O
+tri	B-structure_element
+-	I-structure_element
+modular	I-structure_element
+GH5_4	B-protein_type
+enzyme	O
+from	O
+Bacillus	B-species
+halodurans	I-species
+,	O
+BhCel5B	B-protein
+,	O
+with	O
+rmsd	B-evidence
+=	O
+1	O
+.	O
+3	O
+Å	O
+and	O
+Z	B-evidence
+-	I-evidence
+score	I-evidence
+=	O
+15	O
+.	O
+3	O
+.	O
+The	O
+CBM46	B-structure_element
+from	O
+BhCel5B	B-protein
+is	O
+the	O
+most	O
+structurally	O
+similar	O
+to	O
+BlCel5B	B-protein
+CBM46	B-structure_element
+,	O
+with	O
+rmsd	B-evidence
+=	O
+1	O
+.	O
+6	O
+Å	O
+and	O
+Z	B-evidence
+-	I-evidence
+score	I-evidence
+=	O
+12	O
+.	O
+4	O
+.	O
+The	O
+sequence	O
+identity	O
+relative	O
+to	O
+BhCel5B	B-protein
+,	O
+however	O
+,	O
+is	O
+low	O
+(	O
+28	O
+%	O
+for	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+and	O
+25	O
+%	O
+for	O
+CBM46	B-structure_element
+).	O
+The	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+,	O
+adjacent	O
+to	O
+the	O
+CD	B-structure_element
+,	O
+contains	O
+only	O
+one	O
+tyrosine	B-residue_name
+(	O
+Y367	B-residue_name_number
+)	O
+exposed	O
+to	O
+solvent	O
+and	O
+no	O
+tryptophan	B-residue_name
+residues	O
+.	O
+Because	O
+aromatic	O
+residues	O
+play	O
+a	O
+major	O
+role	O
+in	O
+glucose	B-chemical
+recognition	O
+,	O
+this	O
+observation	O
+suggests	O
+that	O
+substrate	O
+binding	O
+may	O
+not	O
+be	O
+the	O
+primary	O
+function	O
+of	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+.	O
+In	O
+contrast	O
+,	O
+the	O
+CBM46	B-structure_element
+has	O
+three	O
+tryptophan	B-residue_name
+residues	O
+,	O
+two	O
+of	O
+which	O
+face	O
+the	O
+CD	B-structure_element
+substrate	B-site
+binding	I-site
+site	I-site
+(	O
+Fig	O
+.	O
+1A	O
+),	O
+indicating	O
+that	O
+it	O
+may	O
+be	O
+actively	O
+engaged	O
+in	O
+the	O
+carbohydrate	B-chemical
+binding	O
+.	O
+Electron	B-evidence
+density	I-evidence
+maps	I-evidence
+clearly	O
+reveal	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+a	O
+cellotetraose	B-chemical
+(	O
+C4	B-chemical
+)	O
+and	O
+not	O
+a	O
+soaked	O
+cellopentaose	B-chemical
+(	O
+C5	B-chemical
+)	O
+in	O
+the	O
+CD	B-structure_element
+negative	B-site
+substrate	I-site
+-	I-site
+binding	I-site
+subsites	I-site
+(	O
+Fig	O
+.	O
+1D	O
+),	O
+indicating	O
+that	O
+BlCel5B	B-protein
+is	O
+catalytically	B-protein_state
+active	I-protein_state
+in	O
+the	O
+crystal	O
+state	O
+and	O
+able	O
+to	O
+cleave	O
+a	O
+C5	B-chemical
+molecule	O
+.	O
+The	O
+lack	B-evidence
+of	I-evidence
+electron	I-evidence
+density	I-evidence
+verifies	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+the	O
+fifth	B-residue_number
+glucose	B-chemical
+moiety	O
+from	O
+the	O
+soaked	O
+C5	B-chemical
+,	O
+and	O
+a	O
+closer	O
+inspection	O
+of	O
+the	O
+structure	B-evidence
+confirmed	O
+that	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+a	O
+fifth	B-residue_number
+glucose	B-chemical
+unit	O
+would	O
+be	O
+sterically	O
+hindered	O
+by	O
+the	O
+catalytic	B-site
+residues	I-site
+on	O
+the	O
+reducing	O
+end	O
+and	O
+by	O
+residue	O
+R234	B-residue_name_number
+of	O
+a	O
+symmetry	O
+-	O
+related	O
+enzyme	O
+molecule	O
+on	O
+the	O
+non	O
+-	O
+reducing	O
+end	O
+.	O
+The	O
+ability	O
+of	O
+BlCel5B	B-protein
+to	O
+cleave	O
+C5	B-chemical
+into	O
+glucose	B-chemical
+and	O
+C4	B-chemical
+molecules	O
+in	O
+solution	O
+was	O
+demonstrated	O
+by	O
+enzymatic	B-experimental_method
+product	I-experimental_method
+profile	I-experimental_method
+mass	I-experimental_method
+spectrometry	I-experimental_method
+analysis	O
+(	O
+Fig	O
+.	O
+2A	O
+).	O
+The	O
+C4	B-chemical
+oligomer	O
+in	O
+the	O
+BlCel5B	B-protein
+binding	B-site
+site	I-site
+is	O
+coordinated	B-bond_interaction
+by	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+to	O
+residues	O
+N36	B-residue_name_number
+,	O
+H113	B-residue_name_number
+,	O
+H114	B-residue_name_number
+,	O
+N158	B-residue_name_number
+,	O
+W301	B-residue_name_number
+,	O
+and	O
+N303	B-residue_name_number
+and	O
+by	O
+a	O
+CH	B-bond_interaction
+-	I-bond_interaction
+π	I-bond_interaction
+interaction	I-bond_interaction
+with	O
+residue	O
+W47	B-residue_name_number
+(	O
+Fig	O
+.	O
+1D	O
+).	O
+These	O
+residues	O
+belong	O
+to	O
+the	O
+CD	B-structure_element
+and	O
+are	O
+conserved	B-protein_state
+in	O
+the	O
+GH5	B-protein_type
+family	O
+.	O
+BlCel5B	B-protein
+enzymatic	O
+activity	O
+BlCel5B	B-protein
+exhibits	O
+optimum	O
+activity	O
+toward	O
+carboxymethylcellulose	B-chemical
+(	O
+CMC	B-chemical
+;	O
+8	O
+.	O
+7	O
+U	O
+/	O
+mg	O
+)	O
+at	O
+a	O
+pH	O
+of	O
+4	O
+.	O
+0	O
+and	O
+55	O
+°	O
+C	O
+and	O
+retains	O
+approximately	O
+half	O
+of	O
+its	O
+maximum	O
+activity	O
+at	O
+80	O
+°	O
+C	O
+,	O
+demonstrating	O
+considerable	O
+thermal	O
+stability	O
+(	O
+Fig	O
+.	O
+2B	O
+,	O
+C	O
+).	O
+BlCel5B	B-protein
+is	O
+also	O
+active	B-protein_state
+on	O
+β	B-chemical
+-	I-chemical
+glucan	I-chemical
+(	O
+34	O
+U	O
+/	O
+mg	O
+),	O
+lichenan	B-chemical
+(	O
+17	O
+.	O
+8	O
+U	O
+/	O
+mg	O
+)	O
+and	O
+xyloglucan	B-chemical
+(	O
+15	O
+.	O
+7	O
+U	O
+/	O
+mg	O
+)	O
+substrates	O
+(	O
+Table	O
+1	O
+),	O
+whereas	O
+no	O
+activity	O
+was	O
+detected	O
+on	O
+galactomannan	B-chemical
+,	O
+rye	B-taxonomy_domain
+arabinoxylan	B-chemical
+,	O
+1	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+β	I-chemical
+-	I-chemical
+mannan	I-chemical
+or	O
+the	O
+insoluble	O
+substrate	O
+Azo	B-chemical
+-	I-chemical
+Avicel	I-chemical
+.	O
+Kinetic	O
+parameters	O
+were	O
+calculated	O
+assuming	O
+Michaelis	B-experimental_method
+-	I-experimental_method
+Menten	I-experimental_method
+behavior	I-experimental_method
+with	O
+CMC	B-chemical
+as	O
+substrate	O
+:	O
+KM	B-evidence
+=	O
+1	O
+.	O
+78	O
+g	O
+L	O
+−	O
+1	O
+and	O
+Vmax	B-evidence
+=	O
+1	O
+.	O
+41	O
+×	O
+10	O
+−	O
+4	O
+g	O
+s	O
+−	O
+1	O
+mg	O
+protein	O
+−	O
+1	O
+(	O
+Fig	O
+.	O
+2D	O
+).	O
+Although	O
+BlCel5B	B-protein
+is	O
+not	O
+a	O
+highly	O
+active	B-protein_state
+enzyme	O
+against	O
+one	O
+specific	O
+substrate	O
+as	O
+compared	O
+to	O
+others	O
+GH5_4	B-protein_type
+,	O
+it	O
+has	O
+the	O
+advantage	O
+of	O
+being	O
+active	B-protein_state
+against	O
+different	O
+substrates	O
+with	O
+β	O
+-	O
+1	O
+,	O
+3	O
+and	O
+/	O
+or	O
+β	O
+-	O
+1	O
+,	O
+4	O
+glycosidic	O
+linkages	O
+.	O
+To	O
+understand	O
+the	O
+importance	O
+of	O
+the	O
+ancillary	B-structure_element
+modules	I-structure_element
+for	O
+BlCel5B	B-protein
+activity	O
+,	O
+enzymatic	B-experimental_method
+assays	I-experimental_method
+were	O
+carried	O
+out	O
+using	O
+four	O
+enzyme	O
+mutants	B-protein_state
+:	O
+a	O
+CBM46	B-structure_element
+deletion	B-experimental_method
+(	O
+ΔCBM46	B-mutant
+)	O
+and	O
+an	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
++	O
+CBM46	B-structure_element
+deletion	B-experimental_method
+(	O
+ΔIg	B-mutant
+-	I-mutant
+CBM46	I-mutant
+)	O
+as	O
+well	O
+as	O
+point	B-experimental_method
+mutations	I-experimental_method
+of	O
+the	O
+CBM46	B-structure_element
+inner	O
+surface	O
+residues	O
+W479A	B-mutant
+and	O
+W481A	B-mutant
+.	O
+These	O
+mutants	B-protein_state
+were	O
+expressed	B-experimental_method
+and	I-experimental_method
+purified	I-experimental_method
+as	O
+described	O
+for	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+enzyme	O
+.	O
+Strikingly	O
+,	O
+neither	O
+of	O
+the	O
+deletion	B-protein_state
+variants	I-protein_state
+exhibited	O
+detectable	O
+activity	O
+toward	O
+any	O
+of	O
+the	O
+substrates	O
+tested	O
+using	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+BlCel5B	B-protein
+(	O
+Table	O
+1	O
+),	O
+demonstrating	O
+that	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+and	O
+the	O
+CBM46	B-structure_element
+are	O
+essential	O
+for	O
+BlCel5B	B-protein
+activity	O
+.	O
+Thermal	B-experimental_method
+shift	I-experimental_method
+assays	I-experimental_method
+were	O
+conducted	O
+to	O
+confirm	O
+structural	O
+stability	O
+of	O
+the	O
+mutants	B-protein_state
+(	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+).	O
+All	O
+of	O
+the	O
+constructs	O
+showed	O
+similar	O
+melting	B-evidence
+temperatures	I-evidence
+:	O
+62	O
+°	O
+C	O
+for	O
+BlCel5B	B-protein
+,	O
+58	O
+°	O
+C	O
+for	O
+BlCel5BΔCBM46	B-mutant
+,	O
+56	O
+°	O
+C	O
+for	O
+BlCel5BΔIg	B-mutant
+-	I-mutant
+CBM46	I-mutant
+,	O
+65	O
+°	O
+C	O
+for	O
+BlCel5BW479A	B-mutant
+and	O
+59	O
+°	O
+C	O
+for	O
+BlCel5BW479A	B-mutant
+,	O
+thus	O
+confirming	O
+their	O
+proper	O
+overall	O
+fold	O
+.	O
+We	O
+also	O
+examined	O
+the	O
+function	O
+of	O
+the	O
+CBM46	B-structure_element
+inner	O
+surface	B-site
+residues	O
+W479	B-residue_name_number
+and	O
+W481	B-residue_name_number
+(	O
+Fig	O
+.	O
+1A	O
+)	O
+in	O
+BlCel5B	B-protein
+activity	O
+by	O
+performing	O
+enzymatic	B-experimental_method
+assays	I-experimental_method
+with	O
+W479A	B-mutant
+and	O
+W481A	B-mutant
+mutants	B-protein_state
+.	O
+Both	O
+mutations	B-experimental_method
+reduced	O
+enzymatic	O
+activity	O
+toward	O
+all	O
+tested	O
+substrates	O
+(	O
+Table	O
+1	O
+),	O
+with	O
+W481A	B-mutant
+having	O
+a	O
+stronger	O
+effect	O
+than	O
+W479A	B-mutant
+(~	O
+64	O
+%	O
+vs	O
+.	O
+79	O
+%	O
+activity	O
+relative	O
+to	O
+wt	B-protein_state
+BlCel5B	B-protein
+using	O
+β	B-chemical
+-	I-chemical
+glucan	I-chemical
+and	O
+~	O
+10	O
+%	O
+vs	O
+.	O
+50	O
+%	O
+using	O
+CMC	B-chemical
+).	O
+This	O
+indicates	O
+that	O
+CBM46	B-structure_element
+must	O
+interact	O
+with	O
+the	O
+substrate	O
+via	O
+residues	O
+W479	B-residue_name_number
+and	O
+W481	B-residue_name_number
+.	O
+However	O
+,	O
+since	O
+the	O
+BlCel5B	B-protein
+crystal	B-evidence
+structure	I-evidence
+exhibits	O
+no	O
+close	B-protein_state
+contact	O
+between	O
+these	O
+residues	O
+and	O
+the	O
+substrate	O
+,	O
+these	O
+results	O
+suggest	O
+the	O
+existence	O
+of	O
+large	O
+-	O
+amplitude	O
+interdomain	O
+motions	O
+that	O
+may	O
+enable	O
+direct	O
+interactions	O
+between	O
+CBM46	B-structure_element
+and	O
+the	O
+carbohydrate	B-chemical
+.	O
+BlCelB5	B-protein
+dynamics	O
+and	O
+binding	B-site
+-	I-site
+site	I-site
+architecture	O
+Molecular	B-experimental_method
+dynamics	I-experimental_method
+(	O
+MD	B-experimental_method
+)	O
+simulations	B-experimental_method
+were	O
+performed	O
+to	O
+investigate	O
+the	O
+conformational	O
+mobility	O
+of	O
+BlCel5B	B-protein
+.	O
+In	O
+the	O
+simulations	B-experimental_method
+of	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+for	O
+BlCel5B	B-protein
+bound	B-protein_state
+to	I-protein_state
+C4	B-chemical
+,	O
+the	O
+substrate	O
+dissociates	O
+from	O
+the	O
+protein	O
+within	O
+the	O
+first	O
+100	O
+ns	O
+of	O
+the	O
+simulation	B-experimental_method
+time	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2A	O
+).	O
+This	O
+observation	O
+suggests	O
+that	O
+cellotetraose	B-chemical
+does	O
+not	O
+exhibit	O
+detectable	O
+affinity	O
+for	O
+this	O
+specific	O
+BlCel5B	B-protein
+conformation	O
+in	O
+solution	O
+,	O
+as	O
+one	O
+might	O
+otherwise	O
+expect	O
+for	O
+a	O
+reaction	O
+product	O
+.	O
+No	O
+changes	O
+beyond	O
+local	O
+fluctuations	O
+were	O
+observed	O
+in	O
+any	O
+of	O
+the	O
+three	O
+BlCel5B	B-protein
+domains	O
+within	O
+the	O
+time	O
+scale	O
+of	O
+these	O
+runs	O
+(	O
+400	O
+ns	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+2B	O
+).	O
+However	O
+,	O
+the	O
+CBM46	B-structure_element
+and	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+domains	I-structure_element
+did	O
+exhibit	O
+rigid	O
+body	O
+-	O
+like	O
+motions	O
+relative	O
+to	O
+the	O
+CD	B-structure_element
+,	O
+with	O
+rmsd	B-evidence
+values	O
+around	O
+2	O
+.	O
+3	O
+Å	O
+and	O
+1	O
+.	O
+8	O
+Å	O
+,	O
+respectively	O
+,	O
+suggesting	O
+that	O
+BlCel5B	B-protein
+may	O
+execute	O
+large	O
+-	O
+amplitude	O
+interdomain	O
+motions	O
+over	O
+longer	O
+time	O
+scales	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2B	O
+,	O
+C	O
+).	O
+Accordingly	O
+,	O
+simulations	B-experimental_method
+were	O
+then	O
+performed	O
+using	O
+accelerated	B-experimental_method
+molecular	I-experimental_method
+dynamics	I-experimental_method
+(	O
+aMD	B-experimental_method
+)	O
+techniques	O
+to	O
+probe	O
+BlCel5B	B-protein
+interdomain	O
+motions	O
+.	O
+aMD	B-experimental_method
+enhances	O
+conformational	O
+sampling	O
+by	O
+raising	O
+the	O
+basins	O
+of	O
+the	O
+dihedral	B-evidence
+potential	I-evidence
+energy	I-evidence
+surface	I-evidence
+without	O
+affecting	O
+the	O
+general	O
+form	O
+of	O
+the	O
+atomistic	O
+potential	O
+,	O
+thereby	O
+increasing	O
+transition	O
+rates	O
+between	O
+different	O
+local	O
+minima	O
+.	O
+aMD	B-experimental_method
+trajectories	B-evidence
+corresponding	O
+to	O
+more	O
+than	O
+1	O
+.	O
+0	O
+μs	O
+of	O
+conventional	O
+MD	B-experimental_method
+runs	O
+were	O
+generated	O
+.	O
+During	O
+these	O
+simulations	B-experimental_method
+,	O
+we	O
+observed	O
+occlusive	O
+conformations	O
+between	O
+CBM46	B-structure_element
+and	O
+CD	B-structure_element
+that	O
+resulted	O
+in	O
+a	O
+rearrangement	O
+of	O
+the	O
+enzyme	O
+’	O
+s	O
+architecture	O
+around	O
+the	O
+active	B-site
+site	I-site
+(	O
+Video	O
+S1	O
+).	O
+Figure	O
+3A	O
+shows	O
+BlCel5B	B-protein
+in	O
+the	O
+crystallographic	B-experimental_method
+conformation	O
+(	O
+red	O
+)	O
+and	O
+in	O
+a	O
+selected	O
+configuration	O
+obtained	O
+with	O
+aMD	B-experimental_method
+(	O
+blue	O
+)	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+the	O
+substrate	O
+.	O
+Interdomain	O
+motions	O
+were	O
+gauged	O
+by	O
+the	O
+time	O
+evolution	O
+of	O
+the	O
+distance	B-evidence
+between	O
+the	O
+α	O
+carbons	O
+of	O
+residues	O
+I120	B-residue_name_number
+and	O
+E477	B-residue_name_number
+(	O
+represented	O
+as	O
+spheres	O
+in	O
+Fig	O
+.	O
+3A	O
+),	O
+belonging	O
+to	O
+the	O
+CD	B-structure_element
+and	O
+CBM46	B-structure_element
+,	O
+respectively	O
+.	O
+Figure	O
+3C	O
+shows	O
+that	O
+the	O
+I120	B-residue_name_number
+-	O
+E477	B-residue_name_number
+distance	B-evidence
+(	O
+red	O
+curve	O
+)	O
+gradually	O
+decreases	O
+from	O
+~	O
+35	O
+Å	O
+to	O
+~	O
+7	O
+Å	O
+within	O
+the	O
+first	O
+half	O
+of	O
+the	O
+1	O
+.	O
+0	O
+μs	O
+aMD	B-experimental_method
+trajectory	B-evidence
+,	O
+indicating	O
+a	O
+transition	O
+between	O
+the	O
+semi	B-protein_state
+-	I-protein_state
+open	I-protein_state
+(	O
+crystallographic	B-experimental_method
+)	O
+and	O
+occluded	B-protein_state
+(	O
+aMD	B-experimental_method
+sampled	O
+)	O
+configurations	O
+.	O
+During	O
+the	O
+second	O
+half	O
+of	O
+the	O
+aMD	B-experimental_method
+simulation	I-experimental_method
+,	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+enzyme	O
+remained	O
+in	O
+the	O
+closed	B-protein_state
+conformation	O
+,	O
+with	O
+the	O
+CBM46	B-structure_element
+covering	O
+the	O
+carbohydrate	B-site
+-	I-site
+binding	I-site
+site	I-site
+.	O
+These	O
+results	O
+suggest	O
+that	O
+BlCel5B	B-protein
+undergoes	O
+large	O
+-	O
+scale	O
+interdomain	O
+movements	O
+that	O
+enable	O
+interactions	O
+between	O
+CBM46	B-structure_element
+and	O
+the	O
+substrate	O
+bound	B-protein_state
+to	I-protein_state
+the	O
+CD	B-structure_element
+.	O
+To	O
+study	O
+the	O
+interactions	O
+of	O
+BlCel5B	B-protein
+with	O
+a	O
+non	O
+-	O
+hydrolyzed	O
+glucan	B-chemical
+chain	O
+,	O
+we	O
+built	O
+a	O
+model	O
+structure	B-evidence
+with	O
+a	O
+cellooctaose	B-chemical
+(	O
+C8	B-chemical
+)	O
+chain	O
+spanning	O
+the	O
+entire	O
+positive	B-site
+(+	I-site
+1	I-site
+to	I-site
++	I-site
+4	I-site
+)	I-site
+and	O
+negative	B-site
+(−	I-site
+4	I-site
+to	I-site
+−	I-site
+1	I-site
+)	I-site
+subsites	B-site
+of	O
+the	O
+enzyme	O
+.	O
+Starting	O
+from	O
+the	O
+crystallographic	O
+BlCel5B	B-protein
+conformation	O
+,	O
+the	O
+C8	B-chemical
+molecule	O
+deviated	O
+significantly	O
+from	O
+the	O
+active	B-site
+site	I-site
+and	O
+assumed	O
+a	O
+non	O
+-	O
+productive	O
+binding	O
+mode	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2D	O
+).	O
+This	O
+observation	O
+suggests	O
+that	O
+the	O
+open	B-protein_state
+conformation	O
+of	O
+BlCel5B	B-protein
+is	O
+not	O
+able	O
+to	O
+hold	O
+the	O
+substrate	O
+in	O
+a	O
+position	O
+suitable	O
+for	O
+hydrolysis	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2E	O
+).	O
+However	O
+,	O
+after	O
+subjecting	O
+the	O
+BlCel5B	B-complex_assembly
+-	I-complex_assembly
+C8	I-complex_assembly
+complex	O
+to	O
+a	O
+0	O
+.	O
+5	O
+μs	O
+aMD	B-experimental_method
+simulation	I-experimental_method
+with	O
+harmonic	O
+restraints	O
+on	O
+the	O
+C8	B-chemical
+chain	O
+to	O
+prevent	O
+it	O
+from	O
+deviating	O
+from	O
+the	O
+productive	O
+binding	O
+mode	O
+,	O
+the	O
+CBM46	B-structure_element
+readily	O
+closed	B-protein_state
+over	O
+the	O
+CD	B-structure_element
+and	O
+trapped	O
+the	O
+C8	B-chemical
+chain	O
+in	O
+position	O
+for	O
+hydrolysis	O
+(	O
+Fig	O
+.	O
+3B	O
+).	O
+In	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+the	O
+substrate	O
+,	O
+CBM46	B-structure_element
+adopts	O
+a	O
+final	O
+conformation	O
+intermediate	O
+between	O
+the	O
+crystallographic	B-evidence
+structure	I-evidence
+and	O
+that	O
+observed	O
+in	O
+the	O
+substrate	B-protein_state
+-	I-protein_state
+free	I-protein_state
+BlCel5B	B-protein
+aMD	B-experimental_method
+simulations	I-experimental_method
+;	O
+this	O
+is	O
+illustrated	O
+by	O
+the	O
+I120	B-residue_name_number
+-	O
+E477	B-residue_name_number
+distance	B-evidence
+,	O
+which	O
+stabilizes	O
+near	O
+20	O
+Å	O
+in	O
+the	O
+closed	B-protein_state
+configuration	O
+that	O
+traps	O
+the	O
+C8	B-chemical
+molecule	O
+(	O
+in	O
+contrast	O
+to	O
+~	O
+7	O
+Å	O
+for	O
+substrate	B-protein_state
+-	I-protein_state
+free	I-protein_state
+BlCel5B	B-protein
+)	O
+(	O
+Fig	O
+.	O
+3C	O
+).	O
+This	O
+BlCel5B	B-complex_assembly
+-	I-complex_assembly
+C8	I-complex_assembly
+configuration	O
+remains	O
+stable	O
+over	O
+an	O
+additional	O
+500	O
+ns	O
+of	O
+conventional	O
+MD	B-experimental_method
+simulation	I-experimental_method
+with	O
+no	O
+restraints	O
+(	O
+Fig	O
+.	O
+3C	O
+cyan	O
+line	O
+,	O
+Supplementary	O
+Fig	O
+.	O
+2E	O
+,	O
+F	O
+).	O
+A	O
+closer	O
+inspection	O
+of	O
+the	O
+productive	O
+binding	O
+mode	O
+obtained	O
+from	O
+these	O
+extensive	O
+simulations	B-experimental_method
+reveals	O
+that	O
+the	O
+CBM46	B-structure_element
+tryptophan	B-residue_name
+residues	O
+W479	B-residue_name_number
+and	O
+W481	B-residue_name_number
+(	O
+along	O
+with	O
+CD	B-structure_element
+tryptophan	B-residue_name
+residues	O
+)	O
+play	O
+important	O
+roles	O
+in	O
+carbohydrate	B-chemical
+recognition	O
+and	O
+orientation	O
+by	O
+creating	O
+a	O
+tunnel	B-site
+-	O
+like	O
+topology	O
+along	O
+the	O
+BlCel5B	B-protein
+binding	B-site
+cleft	I-site
+,	O
+as	O
+depicted	O
+in	O
+Fig	O
+.	O
+3D	O
+.	O
+Together	O
+,	O
+these	O
+results	O
+indicate	O
+that	O
+CBM46	B-structure_element
+is	O
+a	O
+key	O
+component	O
+of	O
+the	O
+catalytic	B-protein_state
+active	I-protein_state
+complex	O
+,	O
+providing	O
+an	O
+explanation	O
+as	O
+to	O
+why	O
+CBM46	B-structure_element
+is	O
+essential	O
+for	O
+the	O
+enzymatic	O
+activity	O
+of	O
+BlCel5B	B-protein
+.	O
+To	O
+enable	O
+substantially	O
+longer	O
+time	O
+scales	O
+compared	O
+to	O
+atomistic	B-experimental_method
+simulations	I-experimental_method
+,	O
+we	O
+further	O
+explored	O
+the	O
+dynamics	O
+of	O
+BlCel5B	B-protein
+using	O
+coarse	B-experimental_method
+-	I-experimental_method
+grained	I-experimental_method
+MD	I-experimental_method
+(	O
+CG	B-experimental_method
+-	I-experimental_method
+MD	I-experimental_method
+)	O
+simulations	B-experimental_method
+.	O
+We	O
+performed	O
+three	O
+independent	O
+~	O
+120	O
+μs	O
+CG	B-experimental_method
+-	I-experimental_method
+MD	I-experimental_method
+simulations	I-experimental_method
+,	O
+for	O
+a	O
+total	O
+of	O
+approximately	O
+360	O
+μs	O
+of	O
+sampling	O
+.	O
+The	O
+distance	B-evidence
+between	O
+the	O
+α	O
+carbons	O
+of	O
+two	O
+residues	O
+centrally	O
+positioned	O
+in	O
+the	O
+CD	B-structure_element
+and	O
+CBM46	B-structure_element
+(	O
+Fig	O
+.	O
+4A	O
+)	O
+was	O
+monitored	O
+,	O
+and	O
+the	O
+results	O
+shown	O
+in	O
+Fig	O
+.	O
+4B	O
+indicate	O
+that	O
+the	O
+wide	O
+-	O
+amplitude	O
+events	O
+described	O
+above	O
+frequently	O
+appear	O
+in	O
+this	O
+time	O
+scale	O
+.	O
+The	O
+computed	B-evidence
+distance	I-evidence
+distribution	I-evidence
+depicted	O
+in	O
+Fig	O
+.	O
+4C	O
+indicates	O
+three	O
+main	O
+conformational	O
+states	O
+ranging	O
+from	O
+(	O
+I	O
+)	O
+closed	B-protein_state
+conformations	O
+similar	O
+to	O
+those	O
+encountered	O
+in	O
+the	O
+substrate	B-protein_state
+-	I-protein_state
+free	I-protein_state
+aMD	B-experimental_method
+simulations	I-experimental_method
+,	O
+in	O
+which	O
+CBM46	B-structure_element
+interacts	O
+with	O
+the	O
+CD	B-structure_element
+to	O
+shape	O
+the	O
+substrate	B-site
+binding	I-site
+site	I-site
+,	O
+to	O
+(	O
+II	O
+)	O
+semi	B-protein_state
+-	I-protein_state
+open	I-protein_state
+conformations	O
+similar	O
+to	O
+the	O
+crystallographic	B-evidence
+structure	I-evidence
+,	O
+and	O
+(	O
+III	O
+)	O
+extended	B-protein_state
+BlCel5B	B-protein
+conformations	O
+in	O
+which	O
+the	O
+CD	B-structure_element
+and	O
+CBM46	B-structure_element
+are	O
+even	O
+further	O
+apart	O
+than	O
+in	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+.	O
+BlCel5B	B-protein
+conformers	O
+fit	O
+the	O
+SAXS	B-experimental_method
+envelope	B-evidence
+SAXS	B-experimental_method
+experiments	O
+were	O
+conducted	O
+to	O
+assess	O
+BlCel5B	B-protein
+conformational	O
+states	O
+in	O
+solution	O
+,	O
+and	O
+the	O
+results	O
+revealed	O
+the	O
+enzyme	O
+in	O
+its	O
+monomeric	B-oligomeric_state
+form	O
+,	O
+with	O
+average	O
+values	O
+of	O
+Rg	B-evidence
+=	O
+27	O
+.	O
+17	O
+Å	O
+and	O
+Dmax	B-evidence
+=	O
+87	O
+.	O
+59	O
+Å	O
+(	O
+Supplementary	O
+Table	O
+2	O
+).	O
+The	O
+ab	B-experimental_method
+initio	I-experimental_method
+dummy	I-experimental_method
+atom	I-experimental_method
+model	I-experimental_method
+(	O
+DAM	B-experimental_method
+)	O
+demonstrated	O
+that	O
+the	O
+SAXS	B-experimental_method
+-	O
+derived	O
+BlCel5B	B-protein
+molecular	O
+envelope	B-evidence
+could	O
+not	O
+be	O
+single	O
+-	O
+handedly	O
+filled	O
+by	O
+any	O
+of	O
+the	O
+main	O
+conformational	O
+states	O
+encountered	O
+in	O
+the	O
+simulations	B-experimental_method
+(	O
+Fig	O
+.	O
+4D	O
+).	O
+It	O
+is	O
+known	O
+that	O
+a	O
+Kratky	B-evidence
+plot	I-evidence
+exhibits	O
+a	O
+peak	O
+with	O
+an	O
+elevated	O
+baseline	O
+at	O
+high	O
+q	O
+for	O
+a	O
+monodisperse	O
+system	O
+composed	O
+of	O
+multi	O
+-	O
+domain	O
+particles	O
+with	O
+flexible	O
+extensions	O
+.	O
+Indeed	O
+,	O
+an	O
+elevation	O
+of	O
+the	O
+baseline	O
+toward	O
+a	O
+hyperbolic	O
+-	O
+like	O
+curve	O
+was	O
+observed	O
+for	O
+BlCel5B	B-protein
+,	O
+indicating	O
+a	O
+considerable	O
+degree	O
+of	O
+molecular	O
+mobility	O
+in	O
+solution	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3	O
+).	O
+Thus	O
+,	O
+the	O
+conformational	O
+heterogeneity	O
+of	O
+the	O
+enzyme	O
+can	O
+be	O
+decomposed	O
+in	O
+structural	O
+terms	O
+as	O
+a	O
+combination	O
+of	O
+conformational	O
+states	O
+identified	O
+in	O
+our	O
+crystallographic	B-experimental_method
+and	I-experimental_method
+MD	I-experimental_method
+studies	I-experimental_method
+.	O
+We	O
+found	O
+that	O
+the	O
+SAXS	B-experimental_method
+envelope	B-evidence
+can	O
+be	O
+well	O
+represented	O
+by	O
+considering	O
+the	O
+superimposition	B-experimental_method
+of	O
+three	O
+different	O
+representative	O
+molecular	O
+conformations	O
+of	O
+BlCel5B	B-protein
+(	O
+Fig	O
+.	O
+4E	O
+):	O
+a	O
+closed	B-protein_state
+or	O
+CBM46	B-structure_element
+/	O
+CD	B-structure_element
+-	O
+occluded	B-protein_state
+conformation	O
+extracted	O
+from	O
+the	O
+simulations	B-experimental_method
+with	O
+a	O
+relative	O
+weight	O
+of	O
+26	O
+%,	O
+a	O
+semi	B-protein_state
+-	I-protein_state
+open	I-protein_state
+conformation	O
+represented	O
+by	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+corresponding	O
+to	O
+40	O
+%,	O
+and	O
+an	O
+extended	B-protein_state
+conformation	O
+based	O
+on	O
+simulations	B-experimental_method
+that	O
+is	O
+responsible	O
+for	O
+34	O
+%	O
+of	O
+the	O
+SAXS	B-experimental_method
+envelope	B-evidence
+.	O
+The	O
+resulting	O
+average	B-evidence
+scattering	I-evidence
+curve	I-evidence
+from	O
+this	O
+model	O
+fits	O
+the	O
+experimental	O
+protein	O
+scattering	B-evidence
+intensity	I-evidence
+,	O
+with	O
+χ	B-evidence
+=	O
+1	O
+.	O
+89	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3	O
+).	O
+GH5_4	B-protein_type
+phylogenetic	B-experimental_method
+analysis	I-experimental_method
+After	O
+the	O
+exclusion	O
+of	O
+partial	O
+sequences	O
+and	O
+the	O
+suppression	O
+of	O
+highly	O
+identical	O
+members	O
+(	O
+higher	O
+than	O
+90	O
+%	O
+identity	O
+),	O
+144	O
+sequences	O
+containing	O
+between	O
+277	B-residue_range
+and	I-residue_range
+400	I-residue_range
+residues	O
+were	O
+aligned	B-experimental_method
+and	O
+used	O
+to	O
+construct	O
+a	O
+phylogenetic	B-evidence
+tree	I-evidence
+(	O
+Supplementary	O
+Fig	O
+.	O
+4A	O
+).	O
+According	O
+to	O
+PFAM	O
+database	O
+conserved	O
+domain	O
+classification	O
+,	O
+128	O
+GH5	B-protein_type
+enzymes	O
+have	O
+an	O
+architecture	O
+consisting	O
+of	O
+an	O
+N	O
+-	O
+terminal	O
+catalytic	B-structure_element
+module	I-structure_element
+,	O
+a	O
+CBM_X2	B-structure_element
+module	O
+and	O
+an	O
+unknown	O
+module	O
+of	O
+approximately	O
+100	O
+residues	O
+at	O
+the	O
+C	O
+-	O
+terminus	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4B	O
+).	O
+Of	O
+these	O
+,	O
+12	O
+enzymes	O
+have	O
+an	O
+additional	O
+CBM1	B-structure_element
+,	O
+and	O
+5	O
+have	O
+a	O
+CBM2	B-structure_element
+at	O
+the	O
+N	O
+-	O
+terminal	O
+region	O
+.	O
+Based	O
+on	O
+this	O
+PFAM	O
+architecture	O
+and	O
+CAZy	O
+subfamily	O
+classification	O
+,	O
+all	O
+the	O
+144	O
+enzymes	O
+(	O
+including	O
+BlCel5B	B-protein
+)	O
+belong	O
+to	O
+the	O
+GH5_4	B-protein_type
+subfamily	O
+and	O
+group	O
+together	O
+in	O
+the	O
+same	O
+branch	O
+of	O
+the	O
+phylogenetic	B-evidence
+tree	I-evidence
+,	O
+evidencing	O
+a	O
+common	O
+ancestor	O
+.	O
+These	O
+results	O
+support	O
+the	O
+hypothesis	O
+that	O
+the	O
+enzymes	O
+may	O
+employ	O
+the	O
+same	O
+mechanism	O
+by	O
+which	O
+ligand	O
+binding	O
+is	O
+mediated	O
+by	O
+an	O
+extensive	O
+conformational	O
+breathing	O
+of	O
+the	O
+enzyme	O
+that	O
+involves	O
+the	O
+large	O
+-	O
+scale	O
+movement	O
+of	O
+CBM46	B-structure_element
+around	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+(	O
+CBM_X2	B-structure_element
+)	O
+as	O
+a	O
+structural	B-structure_element
+hinge	I-structure_element
+.	O
+Here	O
+,	O
+we	O
+elucidate	O
+the	O
+trimodular	B-protein_state
+molecular	O
+architecture	O
+of	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+BlCel5B	B-protein
+,	O
+a	O
+member	O
+of	O
+the	O
+GH5_4	B-protein_type
+subfamily	O
+,	O
+for	O
+which	O
+large	O
+-	O
+scale	O
+conformational	O
+dynamics	O
+appears	O
+to	O
+play	O
+a	O
+central	O
+role	O
+in	O
+its	O
+enzymatic	O
+activity	O
+.	O
+Full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+BlCel5B	B-protein
+is	O
+active	B-protein_state
+on	O
+both	O
+cellulosic	B-chemical
+and	O
+hemicellulosic	B-chemical
+substrates	O
+and	O
+auxiliary	O
+modules	O
+are	O
+crucial	O
+for	O
+its	O
+activity	O
+.	O
+Most	O
+carbohydrate	B-protein_type
+-	I-protein_type
+active	I-protein_type
+enzymes	I-protein_type
+are	O
+modular	O
+and	O
+consist	O
+of	O
+a	O
+catalytic	B-structure_element
+domain	I-structure_element
+appended	O
+to	O
+one	O
+or	O
+more	O
+separate	O
+AMs	B-structure_element
+.	O
+AMs	B-structure_element
+,	O
+such	O
+as	O
+CBMs	B-structure_element
+,	O
+typically	O
+recognize	O
+carbohydrates	B-chemical
+and	O
+target	O
+their	O
+cognate	O
+catalytic	B-structure_element
+domains	I-structure_element
+toward	O
+the	O
+substrate	O
+.	O
+Because	O
+the	O
+structural	B-experimental_method
+analysis	I-experimental_method
+of	O
+the	O
+protein	O
+is	O
+challenging	O
+if	O
+the	O
+linkers	B-structure_element
+connecting	O
+the	O
+structural	O
+subunits	O
+of	O
+the	O
+enzyme	O
+are	O
+long	O
+and	O
+flexible	O
+,	O
+the	O
+standard	O
+approach	O
+is	O
+to	O
+study	O
+the	O
+domains	O
+separately	O
+.	O
+In	O
+this	O
+work	O
+,	O
+a	O
+combination	O
+of	O
+protein	B-experimental_method
+crystallography	I-experimental_method
+,	O
+computational	B-experimental_method
+molecular	I-experimental_method
+dynamics	I-experimental_method
+,	O
+and	O
+SAXS	B-experimental_method
+analyses	O
+enabled	O
+the	O
+identification	O
+of	O
+a	O
+new	O
+conformational	O
+selection	O
+-	O
+based	O
+molecular	O
+mechanism	O
+that	O
+involves	O
+GH5	B-protein_type
+catalytic	B-structure_element
+domain	I-structure_element
+and	O
+two	O
+AMs	B-structure_element
+in	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+BlCel5B	B-protein
+.	O
+We	O
+observed	O
+that	O
+the	O
+BlCel5B	B-protein
+distal	O
+CBM46	B-structure_element
+is	O
+directly	O
+involved	O
+in	O
+shaping	O
+the	O
+local	O
+architecture	O
+of	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+site	I-site
+.	O
+Although	O
+the	O
+CD	B-structure_element
+alone	B-protein_state
+appears	O
+unable	O
+to	O
+bind	O
+the	O
+substrate	O
+for	O
+catalysis	O
+,	O
+the	O
+AMs	B-structure_element
+exhibit	O
+open	B-protein_state
+-	O
+close	B-protein_state
+motions	O
+that	O
+allow	O
+the	O
+substrate	O
+to	O
+be	O
+captured	O
+in	O
+a	O
+suitable	O
+position	O
+for	O
+hydrolysis	O
+.	O
+Here	O
+,	O
+we	O
+advocate	O
+that	O
+large	O
+-	O
+amplitude	O
+motions	O
+of	O
+AMs	B-structure_element
+are	O
+crucial	O
+for	O
+assembling	O
+the	O
+enzyme	O
+into	O
+its	O
+active	B-protein_state
+conformation	O
+,	O
+highlighting	O
+a	O
+new	O
+function	O
+of	O
+CBMs	B-structure_element
+.	O
+This	O
+mechanism	O
+of	O
+substrate	O
+binding	O
+closely	O
+resembles	O
+the	O
+extended	B-protein_state
+conformational	O
+selection	O
+model	O
+,	O
+with	O
+the	O
+induced	O
+-	O
+fit	O
+mechanism	O
+of	O
+reaction	O
+as	O
+its	O
+limiting	O
+case	O
+.	O
+To	O
+the	O
+best	O
+of	O
+our	O
+knowledge	O
+,	O
+this	O
+enzymatic	O
+mechanism	O
+has	O
+not	O
+been	O
+proposed	O
+previously	O
+for	O
+any	O
+GH	B-protein_type
+.	O
+The	O
+CD	B-site
+binding	I-site
+site	I-site
+of	O
+BlCel5B	B-protein
+is	O
+open	O
+and	O
+relatively	O
+flat	O
+and	O
+is	O
+thus	O
+barely	O
+able	O
+to	O
+properly	O
+hold	O
+the	O
+substrate	O
+in	O
+position	O
+for	O
+catalysis	O
+without	O
+assistance	O
+from	O
+the	O
+CBM46	B-structure_element
+.	O
+In	O
+contrast	O
+,	O
+other	O
+GH5s	B-protein_type
+belonging	O
+to	O
+subfamily	O
+4	O
+listed	O
+in	O
+the	O
+Protein	O
+Data	O
+Bank	O
+exhibit	O
+a	O
+deep	O
+binding	B-site
+cleft	I-site
+or	O
+tunnel	B-site
+that	O
+can	O
+effectively	O
+entrap	O
+the	O
+substrate	O
+for	O
+catalysis	O
+(	O
+Fig	O
+.	O
+5	O
+).	O
+Due	O
+to	O
+the	O
+marked	O
+interdomain	O
+conformational	O
+rearrangement	O
+observed	O
+in	O
+our	O
+simulations	B-experimental_method
+,	O
+the	O
+CBM46	B-structure_element
+generates	O
+a	O
+confined	O
+binding	B-site
+site	I-site
+in	O
+BlCel5B	B-protein
+that	O
+resembles	O
+the	O
+binding	B-site
+site	I-site
+architecture	O
+of	O
+the	O
+other	O
+GH5	B-protein_type
+enzymes	O
+that	O
+lack	B-protein_state
+AMs	B-structure_element
+.	O
+Thus	O
+,	O
+BlCel5B	B-protein
+appears	O
+to	O
+have	O
+adopted	O
+a	O
+strategy	O
+of	O
+CBM46	B-structure_element
+-	O
+mediated	O
+interactions	O
+for	O
+proper	O
+functioning	O
+.	O
+Although	O
+the	O
+homologous	O
+BhCel5B	B-protein
+has	O
+the	O
+same	O
+domain	O
+architecture	O
+of	O
+BlCel5B	B-protein
+and	O
+belongs	O
+to	O
+the	O
+same	O
+subfamily	O
+(	O
+a	O
+comparison	O
+of	O
+the	O
+sequence	O
+and	O
+structure	B-evidence
+of	O
+BlCel5B	B-protein
+and	O
+BhCel5B	B-protein
+is	O
+presented	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+5	O
+),	O
+its	O
+binding	B-site
+site	I-site
+exhibits	O
+important	O
+differences	O
+that	O
+may	O
+impact	O
+the	O
+catalytic	O
+mechanism	O
+.	O
+The	O
+BhCel5B	B-protein
+binding	B-site
+site	I-site
+is	O
+V	B-protein_state
+-	I-protein_state
+shaped	I-protein_state
+and	O
+deeper	O
+than	O
+the	O
+BlCel5B	B-protein
+binding	B-site
+site	I-site
+(	O
+Figs	O
+5	O
+and	O
+6	O
+).	O
+This	O
+is	O
+due	O
+to	O
+the	O
+loop	B-structure_element
+between	O
+residues	O
+F177	B-residue_name_number
+and	O
+R185	B-residue_name_number
+from	O
+BhCel5B	B-protein
+(	O
+absent	B-protein_state
+in	O
+the	O
+BlCel5B	B-protein
+),	O
+which	O
+contains	O
+residue	O
+W181	B-residue_name_number
+that	O
+forms	O
+part	O
+of	O
+the	O
+binding	B-site
+cleft	I-site
+(	O
+Fig	O
+.	O
+6	O
+).	O
+Consistently	O
+,	O
+although	O
+BhCel5B	B-protein
+CBM46	B-structure_element
+is	O
+important	O
+for	O
+β	B-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+glucan	I-chemical
+hydrolysis	O
+(	O
+BhCel5B	B-protein
+is	O
+about	O
+60	O
+-	O
+fold	O
+less	O
+active	B-protein_state
+without	B-protein_state
+CBM46	B-structure_element
+),	O
+the	O
+truncated	B-protein_state
+enzyme	O
+is	O
+completely	O
+active	B-protein_state
+against	O
+xyloglucan	B-chemical
+,	O
+suggesting	O
+that	O
+the	O
+CBM46	B-structure_element
+,	O
+in	O
+this	O
+case	O
+,	O
+is	O
+necessary	O
+for	O
+the	O
+binding	O
+to	O
+specific	O
+substrates	O
+.	O
+A	O
+closer	O
+inspection	O
+of	O
+results	O
+of	O
+the	O
+phylogenetic	B-experimental_method
+analysis	I-experimental_method
+,	O
+more	O
+specifically	O
+of	O
+the	O
+clade	O
+composed	O
+by	O
+GH5_4	B-protein_type
+enzymes	O
+with	O
+trimodular	B-protein_state
+architecture	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4C	O
+),	O
+reveals	O
+subclades	O
+whose	O
+main	O
+characteristic	O
+is	O
+the	O
+varying	O
+length	O
+of	O
+the	O
+loop	B-structure_element
+located	O
+between	O
+residues	O
+161	B-residue_range
+and	I-residue_range
+163	I-residue_range
+(	O
+BlCel5B	B-protein
+residue	O
+numbering	O
+).	O
+Therefore	O
+,	O
+our	O
+results	O
+show	O
+that	O
+BlCel5B	B-protein
+represents	O
+a	O
+smaller	O
+group	O
+of	O
+enzymes	O
+that	O
+are	O
+completely	O
+dependent	O
+on	O
+its	O
+AMs	B-structure_element
+for	O
+hydrolysis	O
+of	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+polysaccharides	B-chemical
+,	O
+and	O
+that	O
+the	O
+underlying	O
+mechanism	O
+may	O
+rely	O
+on	O
+large	O
+-	O
+scale	O
+interdomain	O
+motions	O
+.	O
+The	O
+amino	O
+acid	O
+sequence	O
+of	O
+the	O
+BlCel5B	B-protein
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+is	O
+recognized	O
+by	O
+BLASTP	B-experimental_method
+as	O
+belonging	O
+to	O
+CBM_X2	B-structure_element
+,	O
+a	O
+poorly	O
+described	O
+group	O
+that	O
+has	O
+been	O
+compared	O
+with	O
+CBM	B-structure_element
+-	I-structure_element
+like	I-structure_element
+accessory	I-structure_element
+modules	I-structure_element
+without	O
+a	O
+defined	O
+function	O
+.	O
+Despite	O
+the	O
+similarity	O
+of	O
+BlCel5B	B-protein
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+to	O
+CBMs	B-structure_element
+,	O
+it	O
+lacks	O
+an	O
+identifiable	O
+aromatic	O
+residue	O
+-	O
+rich	O
+carbohydrate	B-site
+-	I-site
+binding	I-site
+site	I-site
+.	O
+Nonetheless	O
+,	O
+according	O
+to	O
+our	O
+results	O
+,	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+seems	O
+to	O
+play	O
+an	O
+important	O
+function	O
+as	O
+a	O
+structural	B-structure_element
+hinge	I-structure_element
+,	O
+dynamically	O
+holding	O
+the	O
+CBM46	B-structure_element
+and	O
+CD	B-structure_element
+in	O
+positions	O
+that	O
+are	O
+appropriate	O
+for	O
+enzymatic	O
+activity	O
+.	O
+Based	O
+on	O
+the	O
+results	O
+of	O
+our	O
+crystallographic	B-experimental_method
+,	I-experimental_method
+computer	I-experimental_method
+simulation	I-experimental_method
+,	O
+and	O
+SAXS	B-experimental_method
+structural	I-experimental_method
+analyses	I-experimental_method
+,	O
+as	O
+well	O
+as	O
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutagenesis	I-experimental_method
+and	O
+activity	B-experimental_method
+assays	I-experimental_method
+,	O
+we	O
+propose	O
+a	O
+molecular	O
+mechanism	O
+for	O
+BlCel5B	B-protein
+substrate	O
+binding	O
+,	O
+which	O
+might	O
+apply	O
+to	O
+other	O
+GH5_4	B-protein_type
+subfamily	O
+enzymes	O
+that	O
+share	O
+this	O
+tri	B-structure_element
+-	I-structure_element
+modular	I-structure_element
+architecture	O
+.	O
+BlCel5B	B-protein
+can	O
+be	O
+found	O
+in	O
+several	O
+different	O
+conformational	O
+states	O
+ranging	O
+from	O
+CBM46	B-structure_element
+/	O
+CD	B-structure_element
+closed	B-protein_state
+(	O
+or	O
+occluded	B-protein_state
+)	O
+to	O
+extended	B-protein_state
+conformations	O
+(	O
+Fig	O
+.	O
+7	O
+).	O
+In	O
+extended	B-protein_state
+configurations	O
+,	O
+the	O
+substrate	O
+may	O
+dock	O
+at	O
+the	O
+shallow	O
+substrate	B-site
+binding	I-site
+site	I-site
+of	O
+CD	B-structure_element
+in	O
+one	O
+of	O
+the	O
+semi	B-protein_state
+-	I-protein_state
+closed	I-protein_state
+conformations	O
+of	O
+the	O
+enzyme	O
+;	O
+however	O
+,	O
+its	O
+binding	O
+is	O
+properly	O
+stabilized	O
+for	O
+hydrolysis	O
+only	O
+with	O
+the	O
+aid	O
+of	O
+induced	O
+-	O
+fit	O
+repositioning	O
+mediated	O
+by	O
+CBM46	B-structure_element
+.	O
+After	O
+cleavage	O
+,	O
+the	O
+intrinsic	O
+dynamics	O
+of	O
+BlCel5B	B-protein
+would	O
+eventually	O
+allow	O
+the	O
+opening	O
+of	O
+the	O
+active	B-site
+site	I-site
+for	O
+product	O
+release	O
+.	O
+The	O
+proposed	O
+mechanism	O
+is	O
+consistent	O
+with	O
+our	O
+mutagenesis	B-experimental_method
+and	I-experimental_method
+enzymatic	I-experimental_method
+activity	I-experimental_method
+assays	I-experimental_method
+,	O
+which	O
+show	O
+that	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+and	O
+CBM46	B-structure_element
+are	O
+indispensable	O
+for	O
+BlCel5B	B-protein
+catalytic	O
+activity	O
+and	O
+,	O
+together	O
+with	O
+the	O
+CD	B-structure_element
+,	O
+form	O
+the	O
+unique	B-protein_state
+catalytic	B-structure_element
+domain	I-structure_element
+of	O
+the	O
+enzyme	O
+.	O
+These	O
+experiments	O
+reveal	O
+a	O
+novel	O
+function	O
+for	O
+CBMs	B-structure_element
+in	O
+which	O
+they	O
+are	O
+intimately	O
+involved	O
+in	O
+the	O
+assembly	O
+of	O
+the	O
+active	B-site
+site	I-site
+and	O
+catalytic	O
+process	O
+.	O
+Computer	B-experimental_method
+simulations	I-experimental_method
+suggest	O
+that	O
+large	O
+-	O
+scale	O
+motions	O
+of	O
+the	O
+CBM46	B-structure_element
+and	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+domains	I-structure_element
+mediate	O
+conformational	O
+selection	O
+and	O
+final	O
+induced	O
+-	O
+fit	O
+adjustments	O
+to	O
+trap	O
+the	O
+substrate	O
+at	O
+the	O
+active	B-site
+site	I-site
+and	O
+promote	O
+hydrolysis	O
+.	O
+SAXS	B-experimental_method
+data	O
+support	O
+the	O
+modeling	B-experimental_method
+results	O
+,	O
+providing	O
+compelling	O
+evidence	O
+for	O
+highly	B-protein_state
+mobile	I-protein_state
+domains	O
+in	O
+solution	O
+.	O
+Crystal	B-evidence
+models	I-evidence
+of	O
+BlCel5B	B-protein
+.	O
+Complete	O
+structure	B-evidence
+is	O
+shown	O
+as	O
+a	O
+cartoon	O
+illustration	O
+in	O
+(	O
+a	O
+)	O
+and	O
+a	O
+van	O
+der	O
+Waals	O
+surface	O
+in	O
+(	O
+b	O
+).	O
+The	O
+CD	B-structure_element
+module	O
+(	O
+red	O
+)	O
+has	O
+a	O
+typical	O
+TIM	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+fold	I-structure_element
+,	O
+and	O
+its	O
+substrate	B-site
+-	I-site
+binding	I-site
+site	I-site
+is	O
+adjacent	O
+to	O
+CBM46	B-structure_element
+(	O
+blue	O
+).	O
+Despite	O
+the	O
+proximity	O
+of	O
+the	O
+binding	B-site
+site	I-site
+in	O
+the	O
+crystallographic	O
+model	O
+,	O
+the	O
+CBM46	B-structure_element
+residues	O
+W479	B-residue_name_number
+and	O
+W481	B-residue_name_number
+are	O
+distant	O
+from	O
+the	O
+substrate	O
+cellotetraose	B-chemical
+(	O
+yellow	O
+).	O
+The	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+domain	I-structure_element
+(	O
+green	O
+)	O
+has	O
+a	O
+lateral	O
+position	O
+,	O
+serving	O
+as	O
+a	O
+connector	O
+between	O
+the	O
+CD	B-structure_element
+and	O
+CBM46	B-structure_element
+.	O
+(	O
+c	O
+)	O
+A	O
+superposition	B-experimental_method
+of	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+domain	I-structure_element
+and	O
+CBM46	B-structure_element
+illustrates	O
+their	O
+structural	O
+similarity	O
+,	O
+with	O
+most	O
+of	O
+the	O
+structural	O
+differences	O
+present	O
+in	O
+the	O
+loop	B-structure_element
+highlighted	O
+by	O
+a	O
+red	O
+circle	O
+.	O
+(	O
+d	O
+)	O
+Cellotetraose	B-chemical
+occupies	O
+subsites	B-site
+-	I-site
+1	I-site
+to	I-site
+-	I-site
+3	I-site
+and	O
+is	O
+primarily	O
+coordinated	B-bond_interaction
+by	O
+the	O
+residues	O
+represented	O
+in	O
+gray	O
+.	O
+BlCel5B	B-protein
+enzymatic	B-experimental_method
+activity	I-experimental_method
+characterization	I-experimental_method
+.	O
+(	O
+a	O
+)	O
+MALDI	B-experimental_method
+/	I-experimental_method
+TOF	I-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+spectra	B-evidence
+of	O
+the	O
+products	O
+released	O
+after	O
+incubation	O
+of	O
+BlCel5B	B-protein
+and	O
+its	O
+two	O
+deletion	B-experimental_method
+constructs	I-experimental_method
+(	O
+ΔCBM46	B-mutant
+and	O
+ΔIg	B-mutant
+-	I-mutant
+CBM46	I-mutant
+)	O
+with	O
+the	O
+substrate	O
+cellopentaose	B-chemical
+(	O
+C5	B-chemical
+).	O
+The	O
+first	O
+three	O
+spectra	B-evidence
+show	O
+the	O
+substrate	O
+,	O
+enzyme	O
+and	O
+buffer	O
+controls	O
+.	O
+The	O
+forth	O
+spectrum	B-evidence
+reveals	O
+that	O
+full	B-protein_state
+length	I-protein_state
+BlCel5B	B-protein
+is	O
+capable	O
+of	O
+enzymatic	O
+hydrolysis	O
+of	O
+C5	B-chemical
+into	O
+smaller	O
+oligosaccharides	B-chemical
+such	O
+as	O
+C4	B-chemical
+,	O
+C3	B-chemical
+and	O
+C2	B-chemical
+.	O
+The	O
+last	O
+two	O
+spectra	B-evidence
+show	O
+that	O
+the	O
+C	O
+-	O
+terminal	O
+deletions	O
+eliminate	B-protein_state
+the	I-protein_state
+enzyme	I-protein_state
+activity	I-protein_state
+.	O
+BlCel5B	B-protein
+activities	O
+on	O
+CMC	B-chemical
+as	O
+functions	O
+of	O
+pH	O
+and	O
+temperature	O
+are	O
+shown	O
+in	O
+(	O
+b	O
+)	O
+and	O
+(	O
+c	O
+),	O
+respectively	O
+.	O
+(	O
+d	O
+)	O
+Michaelis	B-evidence
+-	I-evidence
+Menten	I-evidence
+curve	I-evidence
+using	O
+CMC	B-chemical
+as	O
+a	O
+substrate	O
+.	O
+Open	B-protein_state
+-	O
+close	B-protein_state
+transitions	O
+of	O
+BlCel5B	B-protein
+.	O
+(	O
+a	O
+)	O
+BlCel5B	B-protein
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+substrate	O
+and	O
+(	O
+b	O
+)	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+cellooctaose	B-chemical
+,	O
+as	O
+observed	O
+in	O
+our	O
+aMD	B-experimental_method
+simulations	I-experimental_method
+.	O
+The	O
+distance	B-evidence
+between	O
+the	O
+α	O
+carbon	O
+of	O
+residues	O
+I120	B-residue_name_number
+(	O
+CD	B-structure_element
+)	O
+and	O
+E477	B-residue_name_number
+(	O
+CBM46	B-structure_element
+),	O
+illustrated	O
+as	O
+spheres	O
+in	O
+(	O
+a	O
+),	O
+is	O
+plotted	O
+in	O
+(	O
+c	O
+),	O
+revealing	O
+a	O
+transition	O
+by	O
+the	O
+decrease	O
+in	O
+the	O
+distance	B-evidence
+from	O
+40	O
+Å	O
+to	O
+7	O
+Å	O
+(	O
+substrate	B-protein_state
+-	I-protein_state
+free	I-protein_state
+)	O
+or	O
+20	O
+Å	O
+(	O
+in	O
+presence	B-protein_state
+of	I-protein_state
+cellooctaose	B-chemical
+).	O
+For	O
+the	O
+substrate	B-protein_state
+-	I-protein_state
+free	I-protein_state
+enzyme	O
+,	O
+the	O
+red	O
+line	O
+refers	O
+to	O
+a	O
+1	O
+μs	O
+-	O
+long	O
+aMD	B-experimental_method
+;	O
+for	O
+the	O
+BlCel5B	B-complex_assembly
+-	I-complex_assembly
+cellooctaose	I-complex_assembly
+complex	O
+,	O
+the	O
+first	O
+500	O
+ns	O
+refers	O
+to	O
+aMD	B-experimental_method
+(	O
+in	O
+blue	O
+)	O
+and	O
+the	O
+second	O
+500	O
+ns	O
+to	O
+conventional	O
+MD	B-experimental_method
+(	O
+in	O
+turquoise	O
+).	O
+(	O
+d	O
+)	O
+A	O
+snapshot	O
+of	O
+the	O
+BlCel5B	B-complex_assembly
+-	I-complex_assembly
+cellooctaose	I-complex_assembly
+complex	O
+,	O
+highlighting	O
+the	O
+tryptophan	B-residue_name
+residues	O
+that	O
+interact	O
+with	O
+the	O
+glucan	B-chemical
+chain	O
+in	O
+subsites	B-site
+−	I-site
+4	I-site
+to	I-site
++	I-site
+4	I-site
+.	O
+Residues	O
+W479	B-residue_name_number
+and	O
+W481	B-residue_name_number
+belong	O
+to	O
+CBM46	B-structure_element
+and	O
+only	O
+become	O
+available	O
+for	O
+substrate	O
+interactions	O
+in	O
+the	O
+closed	B-protein_state
+configuration	O
+of	O
+BlCel5B	B-protein
+.	O
+Large	O
+-	O
+scale	O
+movements	O
+of	O
+BlCel5B	B-protein
+modules	O
+and	O
+superposition	B-experimental_method
+of	O
+their	O
+representative	O
+conformations	O
+with	O
+the	O
+SAXS	B-experimental_method
+envelope	B-evidence
+.	O
+(	O
+a	O
+)	O
+BlCel5B	B-protein
+structure	B-evidence
+showing	O
+the	O
+distance	B-evidence
+between	O
+the	O
+backbone	O
+beads	O
+of	O
+residues	O
+I120	B-residue_name_number
+and	O
+E477	B-residue_name_number
+,	O
+which	O
+are	O
+centrally	O
+located	O
+in	O
+CD	B-structure_element
+and	O
+CBM46	B-structure_element
+,	O
+respectively	O
+,	O
+as	O
+a	O
+metric	O
+for	O
+the	O
+relative	O
+disposition	O
+between	O
+the	O
+two	O
+domains	O
+.	O
+(	O
+b	O
+)	O
+Time	O
+history	O
+of	O
+the	O
+I120	B-residue_name_number
+-	O
+E477	B-residue_name_number
+distance	B-evidence
+computed	O
+using	O
+CG	B-experimental_method
+-	I-experimental_method
+MD	I-experimental_method
+simulations	I-experimental_method
+.	O
+Different	O
+colors	O
+separated	O
+by	O
+vertical	O
+lines	O
+correspond	O
+to	O
+independent	O
+simulations	B-experimental_method
+of	O
+approximately	O
+120	O
+μs	O
+.	O
+(	O
+c	O
+)	O
+The	O
+distance	B-evidence
+distribution	I-evidence
+indicates	O
+three	O
+major	O
+peaks	O
+:	O
+closed	B-protein_state
+or	O
+occluded	B-protein_state
+CBM46	B-structure_element
+/	O
+CD	B-structure_element
+conformations	O
+(	O
+I	O
+);	O
+semi	B-protein_state
+-	I-protein_state
+open	I-protein_state
+(	O
+II	O
+),	O
+which	O
+is	O
+similar	O
+to	O
+the	O
+crystallographic	B-evidence
+structure	I-evidence
+;	O
+and	O
+extended	B-protein_state
+conformers	O
+(	O
+III	O
+).	O
+(	O
+d	O
+)	O
+Superimposition	B-experimental_method
+of	O
+the	O
+three	O
+representative	O
+molecular	O
+conformations	O
+of	O
+BlCel5B	B-protein
+with	O
+the	O
+SAXS	B-experimental_method
+model	B-evidence
+.	O
+(	O
+e	O
+)	O
+Average	O
+structures	B-evidence
+obtained	O
+from	O
+the	O
+simulation	B-experimental_method
+segments	O
+corresponding	O
+to	O
+population	O
+groups	O
+I	O
+-	O
+III	O
+,	O
+which	O
+are	O
+individually	O
+superposed	B-experimental_method
+on	O
+the	O
+SAXS	B-experimental_method
+envelope	B-evidence
+.	O
+Comparison	B-experimental_method
+of	O
+the	O
+binding	B-site
+site	I-site
+shape	O
+of	O
+GH5_4	B-protein_type
+enzymes	O
+available	O
+on	O
+the	O
+Protein	O
+Data	O
+Bank	O
+.	O
+(	O
+a	O
+)	O
+BlCel5B	B-protein
+in	O
+the	O
+crystallographic	B-experimental_method
+and	O
+closed	B-protein_state
+configuration	O
+;	O
+(	O
+b	O
+)	O
+Bacillus	B-species
+halodurans	I-species
+Cel5B	B-protein
+(	O
+BhCel5B	B-protein
+)	O
+(	O
+PDB	O
+id	O
+:	O
+4V2X	O
+)	O
+(	O
+c	O
+)	O
+Piromyces	B-species
+rhizinflata	I-species
+GH5	B-protein_type
+endoglucanase	B-protein_type
+(	O
+PDB	O
+id	O
+:	O
+3AYR	O
+);	O
+(	O
+d	O
+)	O
+Clostridium	B-species
+cellulolyticum	I-species
+GH5	B-protein_type
+endoglucanase	B-protein_type
+(	O
+PDB	O
+id	O
+:	O
+1EDG	O
+);	O
+(	O
+e	O
+)	O
+Clostridium	B-species
+cellulovorans	I-species
+GH5	B-protein_type
+endoglucanase	B-protein_type
+(	O
+PDB	O
+id	O
+:	O
+3NDY	O
+);	O
+(	O
+f	O
+)	O
+Bacteroides	B-species
+ovatus	I-species
+GH5	B-protein_type
+xyloglucanase	B-protein_type
+(	O
+PDB	O
+id	O
+:	O
+3ZMR	O
+);	O
+(	O
+g	O
+)	O
+Paenibacillus	B-species
+pabuli	I-species
+GH5	B-protein_type
+xyloglucanase	B-protein_type
+(	O
+PDB	O
+id	O
+:	O
+2JEP	O
+);	O
+(	O
+h	O
+)	O
+Prevotella	B-species
+bryantii	I-species
+GH5	B-protein_type
+endoglucanase	B-protein_type
+(	O
+PDB	O
+id	O
+:	O
+3VDH	O
+);	O
+(	O
+i	O
+)	O
+Ruminiclostridium	B-species
+thermocellum	I-species
+multifunctional	O
+GH5	B-protein_type
+cellulase	B-protein_type
+,	O
+xylanase	B-protein_type
+and	O
+mannase	B-protein_type
+(	O
+PDB	O
+id	O
+:	O
+4IM4	O
+);	O
+(	O
+j	O
+)	O
+Bacteroidetes	B-taxonomy_domain
+bacterium	I-taxonomy_domain
+AC2a	B-protein_type
+endocellulase	B-protein_type
+(	O
+PDB	O
+id	O
+:	O
+4YHE	O
+).	O
+Comparison	B-experimental_method
+of	O
+the	O
+binding	B-site
+cleft	I-site
+of	O
+the	O
+BlCel5B	B-protein
+and	O
+BhCel5B	B-protein
+.	O
+The	O
+main	O
+difference	O
+between	O
+BlCel5B	B-protein
+and	O
+BhCel5B	B-protein
+is	O
+that	O
+the	O
+latter	O
+exhibits	O
+a	O
+deeper	O
+cleft	B-site
+due	O
+to	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+residue	O
+W181	B-residue_name_number
+in	O
+the	O
+loop	B-structure_element
+between	O
+F177	B-residue_name_number
+and	O
+R185	B-residue_name_number
+.	O
+We	O
+conjecture	O
+that	O
+this	O
+difference	O
+in	O
+the	O
+binding	B-site
+site	I-site
+architecture	O
+relates	O
+to	O
+the	O
+importance	O
+that	O
+the	O
+CBM46	B-structure_element
+plays	O
+in	O
+the	O
+BlCel5B	B-protein
+enzymatic	O
+mechanism	O
+.	O
+Proposed	O
+molecular	O
+mechanism	O
+of	O
+BlCel5B	B-protein
+conformational	O
+selection	O
+.	O
+As	O
+suggested	O
+by	O
+the	O
+simulations	B-experimental_method
+and	O
+SAXS	B-experimental_method
+data	O
+,	O
+BlCel5B	B-protein
+spans	O
+multiple	O
+conformations	O
+ranging	O
+from	O
+closed	B-protein_state
+to	O
+extended	B-protein_state
+CBM46	B-structure_element
+/	O
+CD	B-structure_element
+states	O
+.	O
+In	O
+a	O
+given	O
+open	B-protein_state
+state	O
+,	O
+the	O
+substrate	O
+may	O
+reach	O
+the	O
+active	B-site
+site	I-site
+and	O
+become	O
+entrapped	O
+by	O
+the	O
+capping	O
+of	O
+CBM46	B-structure_element
+onto	O
+CD	B-structure_element
+and	O
+induced	O
+-	O
+fit	O
+conformational	O
+adjustments	O
+.	O
+After	O
+hydrolysis	O
+,	O
+the	O
+reaction	O
+product	O
+is	O
+released	O
+to	O
+yield	O
+apo	B-protein_state
+-	O
+BlCel5B	B-protein
+,	O
+which	O
+becomes	O
+ready	O
+for	O
+a	O
+new	O
+cycle	O
+.	O
+Activity	O
+of	O
+BlCel5B	B-protein
+constructs	O
+against	O
+tested	O
+substrates	O
+.	O
+Substrate	O
+(	O
+1	O
+%)	O
+Relative	O
+Activity	O
+(%)	O
+WT	B-protein_state
+*	O
+W479A	B-mutant
+W481A	B-mutant
+ΔCBM46	B-mutant
+ΔIg	B-mutant
+-	I-mutant
+CBM46	I-mutant
+β	B-chemical
+-	I-chemical
+glucan	I-chemical
+100	O
+79	O
+.	O
+1	O
+63	O
+.	O
+6	O
+nd	O
+nd	O
+CMC	B-chemical
+25	O
+.	O
+5	O
+12	O
+.	O
+2	O
+2	O
+.	O
+4	O
+nd	O
+nd	O
+Lichenan	B-chemical
+52	O
+.	O
+4	O
+41	O
+28	O
+.	O
+6	O
+nd	O
+nd	O
+Xyloglucan	B-chemical
+45	O
+.	O
+2	O
+41	O
+.	O
+2	O
+30	O
+.	O
+8	O
+nd	O
+nd	O
+Azo	B-chemical
+-	I-chemical
+Avicel	I-chemical
+nd	O
+**	O
+nd	O
+nd	O
+nd	O
+nd	O
+Arabinoxylan	B-chemical
+nd	O
+nd	O
+nd	O
+nd	O
+nd	O
+Galactomannan	B-chemical
+nd	O
+nd	O
+nd	O
+nd	O
+nd	O
+1	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+β	I-chemical
+-	I-chemical
+mannan	I-chemical
+nd	O
+nd	O
+nd	O
+nd	O
+nd	O
+*	O
+WT	B-protein_state
+=	O
+wild	B-protein_state
+type	I-protein_state
+.	O

annotation_IOB/PMC4980666.tsv ADDED Viewed

	@@ -0,0 +1,5903 @@

+N	B-chemical
+-	I-chemical
+acylhydrazone	I-chemical
+inhibitors	O
+of	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+PA	B-protein
+endonuclease	B-protein_type
+with	O
+versatile	O
+metal	O
+binding	O
+modes	O
+Influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+PA	B-protein
+endonuclease	B-protein_type
+has	O
+recently	O
+emerged	O
+as	O
+an	O
+attractive	O
+target	O
+for	O
+the	O
+development	O
+of	O
+novel	O
+antiviral	O
+therapeutics	O
+.	O
+This	O
+is	O
+an	O
+enzyme	O
+with	O
+divalent	O
+metal	O
+ion	O
+(	O
+s	O
+)	O
+(	O
+Mg2	B-chemical
++	I-chemical
+or	O
+Mn2	B-chemical
++)	I-chemical
+in	O
+its	O
+catalytic	B-site
+site	I-site
+:	O
+chelation	B-bond_interaction
+of	O
+these	O
+metal	O
+cofactors	O
+is	O
+an	O
+attractive	O
+strategy	O
+to	O
+inhibit	O
+enzymatic	O
+activity	O
+.	O
+Here	O
+we	O
+report	O
+the	O
+activity	O
+of	O
+a	O
+series	O
+of	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+in	O
+an	O
+enzymatic	B-experimental_method
+assay	I-experimental_method
+with	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+endonuclease	B-protein_type
+,	O
+as	O
+well	O
+as	O
+in	O
+cell	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+influenza	I-experimental_method
+vRNP	I-experimental_method
+reconstitution	I-experimental_method
+and	O
+virus	B-experimental_method
+yield	I-experimental_method
+assays	I-experimental_method
+.	O
+Several	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+were	O
+found	O
+to	O
+have	O
+promising	O
+anti	O
+-	O
+influenza	B-taxonomy_domain
+activity	O
+in	O
+the	O
+low	O
+micromolar	O
+concentration	O
+range	O
+and	O
+good	O
+selectivity	O
+.	O
+Computational	B-experimental_method
+docking	I-experimental_method
+studies	I-experimental_method
+are	O
+carried	O
+on	O
+to	O
+investigate	O
+the	O
+key	O
+features	O
+that	O
+determine	O
+inhibition	O
+of	O
+the	O
+endonuclease	B-protein_type
+enzyme	O
+by	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+.	O
+Moreover	O
+,	O
+we	O
+here	O
+describe	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+one	O
+of	O
+the	O
+most	O
+active	O
+inhibitors	O
+,	O
+revealing	O
+its	O
+interactions	O
+within	O
+the	O
+protein	O
+’	O
+s	O
+active	B-site
+site	I-site
+.	O
+Influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+is	O
+an	O
+enveloped	B-taxonomy_domain
+virus	I-taxonomy_domain
+with	O
+a	O
+segmented	O
+negative	B-chemical
+-	I-chemical
+oriented	I-chemical
+single	I-chemical
+-	I-chemical
+stranded	I-chemical
+RNA	I-chemical
+genome	O
+,	O
+belonging	O
+to	O
+the	O
+Orthomyxoviridae	B-taxonomy_domain
+.	O
+Seasonal	O
+influenza	B-taxonomy_domain
+A	I-taxonomy_domain
+and	O
+B	B-taxonomy_domain
+viruses	B-taxonomy_domain
+affect	O
+each	O
+year	O
+approximately	O
+5	O
+–	O
+10	O
+%	O
+of	O
+the	O
+adult	O
+and	O
+20	O
+–	O
+30	O
+%	O
+of	O
+the	O
+paediatric	O
+population	O
+,	O
+and	O
+there	O
+is	O
+a	O
+permanent	O
+risk	O
+of	O
+sudden	O
+influenza	B-taxonomy_domain
+pandemics	O
+,	O
+such	O
+as	O
+the	O
+notorious	O
+‘	O
+Spanish	O
+flu	O
+’	O
+in	O
+1918	O
+and	O
+the	O
+swine	O
+-	O
+origin	O
+H1N1	B-species
+pandemic	O
+in	O
+2009	O
+.	O
+Two	O
+classes	O
+of	O
+anti	O
+-	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+drugs	O
+are	O
+available	O
+,	O
+acting	O
+on	O
+the	O
+viral	B-taxonomy_domain
+M2	B-protein_type
+ion	I-protein_type
+-	I-protein_type
+channel	I-protein_type
+(	O
+amantadine	B-chemical
+and	O
+rimantadine	B-chemical
+)	O
+or	O
+on	O
+the	O
+viral	B-taxonomy_domain
+neuraminidase	B-protein_type
+(	O
+zanamivir	B-chemical
+and	O
+oseltamivir	B-chemical
+).	O
+The	O
+M2	B-protein_type
+inhibitors	O
+have	O
+limited	O
+clinical	O
+utility	O
+due	O
+to	O
+their	O
+central	O
+nervous	O
+system	O
+side	O
+effects	O
+and	O
+widespread	O
+resistance	O
+,	O
+as	O
+in	O
+the	O
+case	O
+of	O
+the	O
+2009	O
+pandemic	O
+H1N1	B-species
+virus	B-taxonomy_domain
+;	O
+resistance	O
+is	O
+also	O
+a	O
+growing	O
+concern	O
+for	O
+oseltamivir	B-chemical
+.	O
+The	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+polymerase	B-protein_type
+complex	O
+is	O
+composed	O
+of	O
+three	O
+subunits	O
+:	O
+PB1	B-protein
+,	O
+PB2	B-protein
+and	O
+PA	B-protein
+.	O
+The	O
+PA	B-protein
+subunit	B-structure_element
+performs	O
+the	O
+‘	O
+cap	O
+-	O
+snatching	O
+’	O
+endonuclease	B-protein_type
+reaction	O
+,	O
+the	O
+PB2	B-protein
+subunit	B-structure_element
+is	O
+responsible	O
+for	O
+initial	O
+binding	O
+of	O
+the	O
+capped	B-chemical
+RNAs	I-chemical
+,	O
+while	O
+the	O
+actual	O
+RNA	B-chemical
+synthesis	O
+is	O
+performed	O
+by	O
+the	O
+PB1	B-protein
+protein	O
+.	O
+Given	O
+its	O
+crucial	O
+role	O
+in	O
+the	O
+viral	B-taxonomy_domain
+life	O
+cycle	O
+,	O
+the	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+polymerase	B-protein_type
+is	O
+widely	O
+recognized	O
+as	O
+a	O
+superior	O
+target	O
+for	O
+antiviral	O
+drug	O
+development	O
+and	O
+,	O
+in	O
+particular	O
+,	O
+inhibition	O
+of	O
+the	O
+PA	B-protein
+endonuclease	B-protein_type
+has	O
+deserved	O
+much	O
+attention	O
+in	O
+recent	O
+years	O
+.	O
+The	O
+endonuclease	B-protein_type
+catalytic	B-site
+site	I-site
+resides	O
+in	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+of	O
+PA	B-protein
+(	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+;	O
+residues	O
+1	B-residue_range
+~	I-residue_range
+195	I-residue_range
+).	O
+It	O
+comprises	O
+a	O
+histidine	B-residue_name
+(	O
+His41	B-residue_name_number
+)	O
+and	O
+a	O
+cluster	O
+of	O
+three	O
+strictly	B-protein_state
+conserved	I-protein_state
+acidic	B-protein_state
+residues	O
+(	O
+Glu80	B-residue_name_number
+,	O
+Asp108	B-residue_name_number
+,	O
+Glu119	B-residue_name_number
+),	O
+which	O
+coordinate	B-bond_interaction
+(	O
+together	O
+with	O
+Ile120	B-residue_name_number
+)	O
+one	O
+,	O
+two	O
+,	O
+or	O
+three	O
+manganese	B-chemical
+or	O
+magnesium	B-chemical
+ions	O
+.	O
+Since	O
+the	O
+intracellular	O
+concentration	O
+of	O
+Mg2	B-chemical
++	I-chemical
+is	O
+at	O
+least	O
+1000	O
+-	O
+fold	O
+higher	O
+than	O
+that	O
+of	O
+Mn2	B-chemical
++,	I-chemical
+magnesium	B-chemical
+may	O
+be	O
+more	O
+biologically	O
+relevant	O
+.	O
+A	O
+controversy	O
+about	O
+number	O
+and	O
+type	O
+of	O
+metal	O
+ions	O
+exists	O
+also	O
+for	O
+the	O
+active	B-site
+site	I-site
+of	O
+HIV	B-species
+-	I-species
+1	I-species
+integrase	B-protein_type
+.	O
+HIV	B-species
+-	I-species
+1	I-species
+integrase	B-protein_type
+inhibitors	O
+are	O
+a	O
+paradigm	O
+for	O
+the	O
+innovative	O
+drug	O
+concept	O
+that	O
+is	O
+based	O
+on	O
+coordination	O
+with	O
+the	O
+metal	B-chemical
+cofactor	O
+(	O
+s	O
+)	O
+of	O
+viral	B-taxonomy_domain
+enzymes	O
+:	O
+similarly	O
+,	O
+several	O
+PA	B-protein
+-	O
+binding	O
+agents	O
+with	O
+metal	O
+-	O
+chelating	O
+properties	O
+have	O
+been	O
+identified	O
+as	O
+influenza	B-taxonomy_domain
+endonuclease	B-protein_type
+inhibitors	O
+(	O
+Fig	O
+.	O
+1	O
+),	O
+including	O
+2	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+dioxobutanoic	I-chemical
+acid	I-chemical
+derivatives	O
+,	O
+flutimide	B-chemical
+and	O
+its	O
+derivatives	O
+,	O
+2	B-chemical
+-	I-chemical
+hydroxyphenyl	I-chemical
+amide	I-chemical
+derivatives	O
+,	O
+as	O
+well	O
+as	O
+tetramic	B-chemical
+acids	I-chemical
+,	O
+5	B-chemical
+-	I-chemical
+hydroxypyrimidin	I-chemical
+-	I-chemical
+4	I-chemical
+-	I-chemical
+one	I-chemical
+derivatives	O
+,	O
+marchantins	B-chemical
+and	O
+green	B-taxonomy_domain
+tea	I-taxonomy_domain
+catechins	B-chemical
+,	O
+like	O
+epigallocatechin	B-chemical
+-	I-chemical
+3	I-chemical
+-	I-chemical
+gallate	I-chemical
+(	O
+EGCG	B-chemical
+,	O
+Fig	O
+.	O
+1	O
+).	O
+In	O
+recent	O
+years	O
+,	O
+we	O
+focused	O
+our	O
+research	O
+on	O
+chemical	O
+scaffolds	O
+that	O
+are	O
+able	O
+to	O
+chelate	O
+metal	O
+ions	O
+of	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+,	O
+resulting	O
+in	O
+inhibition	O
+of	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+replication	O
+.	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+represent	O
+an	O
+appealing	O
+class	O
+of	O
+chelating	O
+ligands	O
+with	O
+a	O
+broad	O
+spectrum	B-evidence
+of	O
+biological	O
+activities	O
+,	O
+such	O
+as	O
+activity	O
+against	O
+HIV	B-taxonomy_domain
+,	O
+hepatitis	B-taxonomy_domain
+A	I-taxonomy_domain
+,	O
+vaccinia	B-taxonomy_domain
+and	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+.	O
+In	O
+the	O
+present	O
+work	O
+,	O
+we	O
+report	O
+the	O
+biological	O
+activity	O
+of	O
+a	O
+series	O
+of	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+(	O
+Fig	O
+.	O
+2	O
+),	O
+as	O
+determined	O
+in	O
+an	O
+enzymatic	B-experimental_method
+assay	I-experimental_method
+with	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+endonuclease	B-protein_type
+as	O
+well	O
+as	O
+in	O
+cell	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+influenza	I-experimental_method
+viral	I-experimental_method
+ribonucleoprotein	I-experimental_method
+(	I-experimental_method
+vRNP	I-experimental_method
+)	I-experimental_method
+reconstitution	I-experimental_method
+and	O
+virus	B-experimental_method
+yield	I-experimental_method
+assays	I-experimental_method
+.	O
+Several	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+were	O
+found	O
+to	O
+have	O
+promising	O
+anti	O
+-	O
+influenza	B-taxonomy_domain
+activity	O
+with	O
+50	B-evidence
+%	I-evidence
+effective	I-evidence
+concentration	I-evidence
+values	O
+(	O
+EC50	B-evidence
+)	O
+in	O
+the	O
+range	O
+of	O
+3	O
+–	O
+20	O
+μM	O
+and	O
+good	O
+selectivity	O
+(	O
+Table	O
+1	O
+and	O
+Fig	O
+.	O
+3	O
+).	O
+Computational	B-experimental_method
+docking	I-experimental_method
+studies	I-experimental_method
+of	O
+two	O
+candidate	O
+ligands	O
+in	O
+the	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+active	B-site
+site	I-site
+gave	O
+information	O
+about	O
+the	O
+features	O
+that	O
+could	O
+determine	O
+inhibition	O
+of	O
+endonuclease	B-protein_type
+activity	O
+.	O
+Moreover	O
+,	O
+we	O
+describe	O
+the	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+crystal	I-evidence
+structure	I-evidence
+of	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+one	O
+of	O
+the	O
+most	O
+active	O
+inhibitors	O
+.	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+1	B-chemical
+–	I-chemical
+27	I-chemical
+(	O
+Fig	O
+.	O
+2	O
+)	O
+were	O
+prepared	O
+in	O
+high	O
+yields	O
+by	O
+following	O
+literature	O
+methods	O
+(	O
+Fig	O
+.	O
+2A	O
+);	O
+they	O
+were	O
+characterized	O
+by	O
+spectroscopic	O
+tools	O
+,	O
+mass	B-experimental_method
+spectrometry	I-experimental_method
+and	O
+elemental	B-experimental_method
+analysis	I-experimental_method
+.	O
+Even	O
+if	O
+isomerism	O
+around	O
+the	O
+C	O
+=	O
+N	O
+bond	O
+is	O
+possible	O
+,	O
+1	B-chemical
+–	I-chemical
+27	I-chemical
+are	O
+present	O
+in	O
+the	O
+E	O
+form	O
+in	O
+solution	O
+,	O
+as	O
+evidenced	O
+by	O
+the	O
+chemical	O
+shift	O
+values	O
+of	O
+the	O
+HC	O
+=	O
+N	O
+and	O
+NH	O
+protons	O
+in	O
+the	O
+1H	B-experimental_method
+-	I-experimental_method
+NMR	I-experimental_method
+spectrum	B-evidence
+.	O
+Exceptions	O
+are	O
+represented	O
+by	O
+the	O
+alkyl	O
+-	O
+derivatives	O
+3	B-chemical
+and	O
+4	B-chemical
+(	O
+2	O
+:	O
+1	O
+and	O
+5	O
+:	O
+3	O
+E	O
+:	O
+Z	O
+ratio	O
+,	O
+respectively	O
+).	O
+If	O
+R	O
+’	O
+(	O
+Fig	O
+.	O
+2A	O
+)	O
+is	O
+a	O
+2	O
+-	O
+hydroxy	O
+substituted	O
+phenyl	O
+ring	O
+,	O
+the	O
+corresponding	O
+acylhydrazones	B-chemical
+can	O
+coordinate	B-bond_interaction
+one	O
+or	O
+,	O
+depending	O
+on	O
+denticity	O
+,	O
+two	O
+metal	O
+centers	O
+(	O
+modes	O
+A	O
+and	O
+B	O
+in	O
+Fig	O
+.	O
+4	O
+).	O
+Starting	O
+from	O
+N	B-chemical
+’-(	I-chemical
+2	I-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+dihydroxybenzylidene	I-chemical
+)-	I-chemical
+semicarbazide	I-chemical
+(	O
+1	B-chemical
+)	O
+and	O
+its	O
+methoxy	O
+-	O
+analogue	O
+(	O
+2	B-chemical
+),	O
+we	O
+modified	O
+the	O
+acylhydrazonic	O
+substituent	O
+R	O
+”	O
+(	O
+3	B-chemical
+–	I-chemical
+8	I-chemical
+,	O
+18	B-chemical
+,	O
+19	B-chemical
+,	O
+Fig	O
+.	O
+2A	O
+).	O
+In	O
+18	B-chemical
+and	O
+19	B-chemical
+,	O
+also	O
+the	O
+gallic	B-chemical
+moiety	O
+can	O
+be	O
+involved	O
+in	O
+the	O
+chelation	B-bond_interaction
+of	O
+the	O
+metal	O
+cofactors	O
+(	O
+mode	O
+C	O
+,	O
+Fig	O
+.	O
+4	O
+).	O
+In	O
+order	O
+to	O
+investigate	O
+the	O
+role	O
+of	O
+hydroxyl	O
+substituents	O
+9	B-chemical
+–	I-chemical
+11	I-chemical
+,	O
+13	B-chemical
+–	I-chemical
+17	I-chemical
+,	O
+20	B-chemical
+–	I-chemical
+23	I-chemical
+and	O
+27	B-chemical
+were	O
+also	O
+synthesized	O
+.	O
+Compound	O
+12	B-chemical
+was	O
+synthesized	O
+in	O
+order	O
+to	O
+confirm	O
+the	O
+crucial	O
+influence	O
+of	O
+the	O
+gallic	B-chemical
+moiety	O
+.	O
+Finally	O
+,	O
+26	B-chemical
+was	O
+here	O
+considered	O
+,	O
+because	O
+it	O
+is	O
+an	O
+inhibitor	O
+of	O
+HIV	B-taxonomy_domain
+RNase	B-protein
+H	I-protein
+,	O
+another	O
+enzyme	O
+with	O
+two	O
+magnesium	B-chemical
+ions	O
+in	O
+its	O
+active	B-site
+site	I-site
+.	O
+Since	O
+the	O
+inhibitory	O
+activity	O
+of	O
+the	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+could	O
+be	O
+related	O
+to	O
+chelation	B-bond_interaction
+of	O
+the	O
+divalent	O
+metal	B-chemical
+cofactor	O
+(	O
+s	O
+)	O
+in	O
+the	O
+influenza	B-taxonomy_domain
+PA	B-protein
+-	O
+Nter	B-structure_element
+active	B-site
+site	I-site
+,	O
+we	O
+investigated	O
+the	O
+coordination	O
+properties	O
+of	O
+one	O
+model	O
+ligand	O
+(	O
+i	O
+.	O
+e	O
+.	O
+19	B-chemical
+,	O
+H2L	B-chemical
+)	O
+towards	O
+Mg2	B-chemical
++.	I-chemical
+Different	O
+reaction	O
+conditions	O
+were	O
+used	O
+(	O
+1	O
+:	O
+1	O
+and	O
+1	O
+:	O
+2	O
+metal	O
+to	O
+ligand	O
+ratio	O
+,	O
+up	O
+to	O
+4	O
+equivalents	O
+of	O
+triethylamine	B-chemical
+),	O
+but	O
+in	O
+any	O
+case	O
+the	O
+same	O
+chemical	O
+species	O
+Mg	B-chemical
+(	I-chemical
+HL	I-chemical
+)	I-chemical
+2	I-chemical
+∙	I-chemical
+4H2O	I-chemical
+was	O
+recovered	O
+and	O
+conveniently	O
+characterized	O
+.	O
+The	O
+use	O
+of	O
+a	O
+coordinating	O
+solvent	O
+as	O
+d6	B-chemical
+-	I-chemical
+DMSO	I-chemical
+causes	O
+partial	O
+decoordination	O
+of	O
+the	O
+ligand	O
+,	O
+but	O
+the	O
+1H	B-experimental_method
+-	I-experimental_method
+NMR	I-experimental_method
+spectrum	B-evidence
+in	O
+MeOD	O
+,	O
+instead	O
+,	O
+shows	O
+only	O
+the	O
+signals	O
+attributable	O
+to	O
+the	O
+complex	O
+.	O
+In	O
+the	O
+13C	B-experimental_method
+-	I-experimental_method
+NMR	I-experimental_method
+spectrum	B-evidence
+,	O
+the	O
+signal	O
+of	O
+the	O
+C	O
+=	O
+O	O
+quaternary	O
+carbon	O
+is	O
+practically	O
+unaffected	O
+by	O
+complexation	O
+,	O
+suggesting	O
+that	O
+the	O
+C	O
+=	O
+O	O
+group	O
+is	O
+weakly	O
+involved	O
+in	O
+the	O
+coordination	O
+to	O
+the	O
+metal	O
+ion	O
+.	O
+This	O
+is	O
+confirmed	O
+,	O
+in	O
+the	O
+IR	B-experimental_method
+spectrum	B-evidence
+,	O
+by	O
+the	O
+shift	O
+of	O
+about	O
+20	O
+cm	O
+−	O
+1	O
+of	O
+the	O
+C	O
+=	O
+O	O
+absorption	O
+,	O
+while	O
+a	O
+shift	O
+of	O
+30	O
+–	O
+50	O
+cm	O
+−	O
+1	O
+is	O
+expected	O
+when	O
+the	O
+carbonylic	O
+oxygen	O
+is	O
+tightly	O
+bound	O
+to	O
+the	O
+metal	O
+ion	O
+.	O
+ESI	B-experimental_method
+-	I-experimental_method
+mass	I-experimental_method
+spectra	B-evidence
+and	O
+elemental	B-experimental_method
+analysis	I-experimental_method
+confirmed	O
+the	O
+formula	O
+Mg	B-chemical
+(	I-chemical
+HL	I-chemical
+)	I-chemical
+2	I-chemical
+∙	I-chemical
+4H2O	I-chemical
+.	O
+The	O
+interaction	O
+between	O
+the	O
+N	B-chemical
+-	I-chemical
+acylhydrazone	I-chemical
+ligands	O
+and	O
+the	O
+magnesium	B-chemical
+cation	O
+was	O
+investigated	O
+also	O
+by	O
+means	O
+of	O
+UV	B-experimental_method
+-	I-experimental_method
+visible	I-experimental_method
+spectroscopy	I-experimental_method
+(	O
+UV	B-experimental_method
+-	I-experimental_method
+visible	I-experimental_method
+titrations	I-experimental_method
+of	O
+23	B-chemical
+and	O
+19	B-chemical
+with	O
+increasing	B-experimental_method
+amount	I-experimental_method
+of	O
+Mg	B-chemical
+(	I-chemical
+CH3COO	I-chemical
+)	I-chemical
+2	I-chemical
+are	O
+shown	O
+in	O
+Figure	O
+S1	O
+).	O
+The	O
+spectrum	B-evidence
+of	O
+19	B-chemical
+includes	O
+a	O
+band	O
+at	O
+313	O
+nm	O
+assignable	O
+to	O
+n	O
+-	O
+π	O
+*	O
+transitions	O
+of	O
+the	O
+C	O
+=	O
+N	O
+and	O
+C	O
+=	O
+O	O
+groups	O
+.	O
+By	O
+adding	O
+increasing	O
+equivalents	O
+of	O
+Mg	B-chemical
+(	I-chemical
+CH3COO	I-chemical
+)	I-chemical
+2	I-chemical
+,	O
+the	O
+absorption	O
+around	O
+400	O
+nm	O
+increases	O
+,	O
+and	O
+a	O
+new	O
+band	O
+appears	O
+with	O
+a	O
+maximum	O
+at	O
+397	O
+nm	O
+.	O
+When	O
+the	O
+same	O
+experiment	O
+was	O
+performed	O
+with	O
+23	B-chemical
+,	O
+a	O
+different	O
+behavior	O
+was	O
+observed	O
+.	O
+Increasing	O
+concentration	O
+of	O
+Mg2	B-chemical
++,	I-chemical
+in	O
+fact	O
+,	O
+caused	O
+a	O
+diminution	O
+in	O
+the	O
+maximum	O
+absorption	O
+,	O
+an	O
+isosbestic	O
+point	O
+is	O
+visible	O
+at	O
+about	O
+345	O
+nm	O
+,	O
+but	O
+a	O
+new	O
+band	O
+at	O
+400	O
+nm	O
+does	O
+not	O
+appear	O
+.	O
+Ligands	O
+19	B-chemical
+and	O
+23	B-chemical
+coordinate	B-bond_interaction
+the	O
+Mg2	B-chemical
++	I-chemical
+ions	O
+in	O
+different	O
+ways	O
+:	O
+19	B-chemical
+chelates	O
+the	O
+metal	O
+ion	O
+by	O
+using	O
+the	O
+deprotonated	O
+salicyl	O
+oxygen	O
+and	O
+the	O
+iminic	O
+nitrogen	O
+,	O
+while	O
+for	O
+23	B-chemical
+,	O
+the	O
+gallic	O
+moiety	O
+is	O
+supposed	O
+to	O
+be	O
+involved	O
+(	O
+Fig	O
+.	O
+4A	O
+,	O
+B	O
+versus	O
+C	O
+),	O
+leading	O
+to	O
+different	O
+,	O
+less	O
+extensive	O
+,	O
+modifications	O
+of	O
+the	O
+UV	B-experimental_method
+spectrum	B-evidence
+.	O
+Inhibition	O
+of	O
+the	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+enzyme	O
+All	O
+the	O
+compounds	O
+were	O
+tested	O
+for	O
+their	O
+ability	O
+to	O
+inhibit	O
+the	O
+influenza	B-taxonomy_domain
+endonuclease	B-protein_type
+in	O
+an	O
+enzymatic	B-experimental_method
+plasmid	I-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+assay	I-experimental_method
+with	O
+recombinant	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+,	O
+as	O
+well	O
+as	O
+in	O
+cell	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+influenza	I-experimental_method
+methods	I-experimental_method
+(	O
+i	O
+.	O
+e	O
+.	O
+virus	B-experimental_method
+yield	I-experimental_method
+and	I-experimental_method
+vRNP	I-experimental_method
+reconstitution	I-experimental_method
+assays	I-experimental_method
+).	O
+The	O
+results	O
+are	O
+shown	O
+in	O
+Table	O
+1	O
+and	O
+summarized	O
+in	O
+Fig	O
+.	O
+3	O
+to	O
+visualize	O
+the	O
+structure	O
+-	O
+activity	O
+relationships	O
+;	O
+Figure	O
+S2	O
+shows	O
+the	O
+dose	B-evidence
+-	I-evidence
+response	I-evidence
+curves	I-evidence
+for	O
+three	O
+representative	O
+compounds	O
+(	O
+i	O
+.	O
+e	O
+.	O
+10	B-chemical
+,	O
+13	B-chemical
+and	O
+23	B-chemical
+)	O
+in	O
+either	O
+the	O
+PA	B-experimental_method
+-	I-experimental_method
+enzyme	I-experimental_method
+or	I-experimental_method
+vRNP	I-experimental_method
+reconstitution	I-experimental_method
+assay	I-experimental_method
+.	O
+The	O
+moderate	O
+activity	O
+(	O
+IC50	B-evidence
+=	O
+24	O
+μM	O
+)	O
+of	O
+N	B-chemical
+’-	I-chemical
+2	I-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+dihydroxybenzylidene	I-chemical
+semicarbazide	I-chemical
+(	O
+1	B-chemical
+)	O
+was	O
+completely	O
+lost	O
+when	O
+the	O
+NH2	O
+moiety	O
+was	O
+replaced	O
+by	O
+a	O
+hydrophobic	O
+heptyl	O
+chain	O
+(	O
+3	B-chemical
+),	O
+but	O
+it	O
+is	O
+less	O
+affected	O
+when	O
+a	O
+phenyl	O
+or	O
+a	O
+2	O
+-	O
+hydroxyphenyl	O
+is	O
+present	O
+(	O
+5	B-chemical
+and	O
+7	B-chemical
+,	O
+IC50	B-evidence
+=	O
+84	O
+and	O
+54	O
+μM	O
+,	O
+respectively	O
+).	O
+When	O
+the	O
+hydroxyl	O
+in	O
+position	O
+3	O
+on	O
+R1	O
+(	O
+2	B-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+dihydroxybenzylidene	I-chemical
+)	O
+was	O
+replaced	O
+by	O
+a	O
+methoxy	O
+group	O
+(	O
+2	B-chemical
+-	I-chemical
+hydroxy	I-chemical
+-	I-chemical
+3	I-chemical
+-	I-chemical
+methoxybenzylidene	I-chemical
+),	O
+the	O
+activity	O
+disappeared	O
+(	O
+compounds	O
+2	B-chemical
+,	O
+4	B-chemical
+,	O
+6	B-chemical
+and	O
+8	B-chemical
+).	O
+The	O
+activity	O
+is	O
+unaffected	O
+(	O
+IC50	B-evidence
+values	O
+ranging	O
+from	O
+45	O
+to	O
+75	O
+μM	O
+)	O
+when	O
+going	O
+from	O
+two	O
+hydroxyls	O
+in	O
+R1	O
+(	O
+7	B-chemical
+)	O
+to	O
+compounds	O
+with	O
+three	O
+hydroxyls	O
+(	O
+i	O
+.	O
+e	O
+.	O
+9	B-chemical
+,	O
+10	B-chemical
+and	O
+11	B-chemical
+).	O
+Similarly	O
+,	O
+11	B-chemical
+(	O
+R1	O
+=	O
+3	O
+,	O
+4	O
+,	O
+5	O
+-	O
+trihydroxyphenyl	O
+,	O
+R2	O
+=	O
+2	O
+-	O
+hydroxyphenyl	O
+)	O
+had	O
+comparable	O
+activity	O
+as	O
+27	B-chemical
+(	O
+R1	O
+=	O
+3	O
+,	O
+4	O
+,	O
+5	O
+-	O
+trihydroxyphenyl	O
+,	O
+R2	O
+=	O
+NH2	O
+).	O
+Within	O
+the	O
+series	O
+carrying	O
+a	O
+2	O
+-	O
+hydroxyphenyl	O
+R2	O
+group	O
+,	O
+the	O
+activity	O
+of	O
+11	B-chemical
+is	O
+particularly	O
+intriguing	O
+.	O
+11	B-chemical
+does	O
+not	O
+have	O
+the	O
+possibility	O
+to	O
+chelate	O
+in	O
+a	O
+tridentate	O
+ONO	O
+fashion	O
+(	O
+mode	O
+A	O
+in	O
+Fig	O
+.	O
+4	O
+),	O
+but	O
+it	O
+can	O
+coordinate	B-bond_interaction
+two	O
+cations	O
+by	O
+means	O
+of	O
+its	O
+three	O
+OH	O
+groups	O
+in	O
+R1	O
+(	O
+mode	O
+C	O
+,	O
+Fig	O
+.	O
+4	O
+).	O
+Note	O
+that	O
+a	O
+similar	O
+chelating	O
+mode	O
+was	O
+observed	O
+in	O
+a	O
+crystal	B-evidence
+structure	I-evidence
+,	O
+solved	O
+by	O
+Cusack	O
+and	O
+coworkers	O
+,	O
+of	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+endonuclease	B-protein_type
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+the	O
+inhibitor	O
+EGCG	B-chemical
+.	O
+The	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+inhibitory	O
+activity	O
+strongly	O
+depends	O
+on	O
+the	O
+number	O
+and	O
+position	O
+of	O
+hydroxyl	O
+substituents	O
+in	O
+R1	O
+and	O
+R2	O
+:	O
+this	O
+is	O
+clearly	O
+highlighted	O
+by	O
+the	O
+data	O
+obtained	O
+with	O
+compounds	O
+13	B-chemical
+–	I-chemical
+23	I-chemical
+,	O
+in	O
+which	O
+R2	O
+is	O
+a	O
+3	O
+,	O
+4	O
+,	O
+5	O
+-	O
+trihydroxyphenyl	O
+(	O
+gallic	O
+)	O
+group	O
+,	O
+the	O
+most	O
+active	O
+scaffold	O
+in	O
+our	O
+series	O
+.	O
+The	O
+analogue	O
+carrying	O
+an	O
+unsubstituted	O
+aromatic	O
+ring	O
+as	O
+R1	O
+(	O
+compound	O
+13	B-chemical
+)	O
+had	O
+moderate	O
+activity	O
+(	O
+IC50	B-evidence
+=	O
+69	O
+μM	O
+).	O
+When	O
+one	O
+OH	O
+was	O
+added	O
+at	O
+position	O
+2	O
+of	O
+the	O
+R1	O
+ring	O
+(	O
+14	B-chemical
+),	O
+the	O
+activity	O
+was	O
+lost	O
+.	O
+Adding	O
+a	O
+second	O
+OH	O
+substituent	O
+at	O
+position	O
+5	O
+resulted	O
+in	O
+strong	O
+activity	O
+(	O
+compound	O
+15	B-chemical
+,	O
+IC50	B-evidence
+=	O
+9	O
+μM	O
+);	O
+medium	O
+activity	O
+for	O
+a	O
+3	O
+-	O
+OH	O
+(	O
+18	B-chemical
+;	O
+IC50	B-evidence
+=	O
+83	O
+μM	O
+),	O
+and	O
+marginal	O
+activity	O
+when	O
+the	O
+second	O
+OH	O
+is	O
+at	O
+position	O
+4	O
+(	O
+17	B-chemical
+,	O
+IC50	B-evidence
+≥	O
+370	O
+μM	O
+).	O
+The	O
+addition	O
+of	O
+a	O
+3	O
+-	O
+methoxy	O
+group	O
+(	O
+19	B-chemical
+)	O
+abolished	O
+all	O
+inhibitory	O
+activity	O
+.	O
+This	O
+cannot	O
+be	O
+related	O
+to	O
+variations	O
+in	O
+the	O
+chelating	O
+features	O
+displayed	O
+by	O
+the	O
+R1	O
+moiety	O
+,	O
+since	O
+compounds	O
+14	B-chemical
+–	I-chemical
+19	I-chemical
+all	O
+have	O
+,	O
+in	O
+theory	O
+,	O
+the	O
+capacity	O
+to	O
+chelate	O
+one	O
+metal	O
+ion	O
+through	O
+the	O
+ortho	O
+-	O
+OH	O
+and	O
+iminic	O
+nitrogen	O
+(	O
+mode	O
+A	O
+in	O
+Fig	O
+.	O
+4	O
+).	O
+Moreover	O
+,	O
+compound	O
+18	B-chemical
+can	O
+,	O
+in	O
+principle	O
+,	O
+chelate	O
+the	O
+two	O
+M2	B-chemical
++	I-chemical
+ions	O
+in	O
+the	O
+active	B-site
+site	I-site
+according	O
+to	O
+mode	O
+B	O
+(	O
+Fig	O
+.	O
+4	O
+),	O
+yet	O
+it	O
+(	O
+IC50	B-evidence
+=	O
+83	O
+μM	O
+)	O
+has	O
+nine	O
+-	O
+fold	O
+lower	O
+activity	O
+than	O
+15	B-chemical
+,	O
+that	O
+does	O
+not	O
+possess	O
+this	O
+two	O
+-	O
+metal	O
+chelating	O
+feature	O
+.	O
+Therefore	O
+,	O
+we	O
+hypothesized	O
+that	O
+the	O
+inhibitory	O
+activity	O
+of	O
+the	O
+series	O
+containing	O
+the	O
+gallic	O
+moiety	O
+is	O
+determined	O
+by	O
+:	O
+(	O
+i	O
+)	O
+the	O
+capacity	O
+of	O
+the	O
+moiety	O
+R2	O
+to	O
+chelate	O
+two	O
+metal	O
+ions	O
+in	O
+the	O
+active	B-site
+site	I-site
+of	O
+the	O
+enzyme	O
+,	O
+according	O
+to	O
+mode	O
+C	O
+(	O
+Fig	O
+.	O
+4	O
+);	O
+and	O
+(	O
+ii	O
+)	O
+the	O
+presence	O
+and	O
+position	O
+of	O
+one	O
+or	O
+more	O
+hydroxyl	O
+substituents	O
+in	O
+R1	O
+,	O
+which	O
+may	O
+possibly	O
+result	O
+in	O
+ligand	O
+-	O
+protein	O
+interactions	O
+(	O
+e	O
+.	O
+g	O
+.	O
+through	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+).	O
+This	O
+assumption	O
+was	O
+supported	O
+by	O
+molecular	B-experimental_method
+docking	I-experimental_method
+calculations	I-experimental_method
+and	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+analysis	I-experimental_method
+of	O
+inhibitor	O
+23	B-chemical
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+PA	B-protein
+-	O
+Nter	B-structure_element
+(	O
+vide	O
+infra	O
+).	O
+Substitution	O
+of	O
+the	O
+5	O
+-	O
+hydroxyl	O
+in	O
+15	B-chemical
+by	O
+a	O
+methoxy	O
+group	O
+(	O
+16	B-chemical
+)	O
+causes	O
+a	O
+dramatic	O
+drop	O
+in	O
+activity	O
+(	O
+IC50	B-evidence
+=	O
+9	O
+and	O
+454	O
+μM	O
+for	O
+15	B-chemical
+and	O
+16	B-chemical
+,	O
+respectively	O
+).	O
+In	O
+particular	O
+,	O
+all	O
+the	O
+compounds	O
+with	O
+a	O
+trihydroxylated	O
+phenyl	O
+group	O
+as	O
+R1	O
+(	O
+i	O
+.	O
+e	O
+.	O
+20	B-chemical
+,	O
+21	B-chemical
+,	O
+22	B-chemical
+and	O
+23	B-chemical
+)	O
+were	O
+able	O
+to	O
+inhibit	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+quite	O
+potently	O
+.	O
+The	O
+lowest	O
+IC50	B-evidence
+values	O
+were	O
+obtained	O
+for	O
+21	B-chemical
+and	O
+23	B-chemical
+(	O
+IC50	B-evidence
+=	O
+13	O
+and	O
+7	O
+μM	O
+,	O
+respectively	O
+),	O
+which	O
+both	O
+have	O
+one	O
+of	O
+their	O
+three	O
+hydroxyl	O
+groups	O
+at	O
+position	O
+5	O
+.	O
+The	O
+most	O
+active	O
+compound	O
+in	O
+this	O
+series	O
+was	O
+23	B-chemical
+,	O
+which	O
+lacks	O
+the	O
+hydroxyl	O
+group	O
+at	O
+position	O
+2	O
+of	O
+R1	O
+,	O
+further	O
+confirming	O
+that	O
+this	O
+function	O
+is	O
+undesirable	O
+or	O
+even	O
+detrimental	O
+for	O
+inhibitory	O
+activity	O
+against	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+,	O
+as	O
+already	O
+noticed	O
+above	O
+for	O
+14	B-chemical
+.	O
+Consistent	O
+with	O
+a	O
+crucial	O
+role	O
+of	O
+the	O
+R2	O
+gallic	O
+moiety	O
+in	O
+metal	O
+chelation	B-bond_interaction
+,	O
+the	O
+strong	O
+activity	O
+of	O
+15	B-chemical
+was	O
+completely	O
+lost	O
+in	O
+its	O
+3	O
+,	O
+4	O
+,	O
+5	O
+-	O
+trimethoxy	O
+analogue	O
+12	B-chemical
+.	O
+On	O
+the	O
+other	O
+hand	O
+,	O
+the	O
+R2	O
+gallic	O
+containing	O
+compounds	O
+displayed	O
+moderate	O
+activity	O
+(	O
+IC50	B-evidence
+values	O
+around	O
+40	O
+μM	O
+)	O
+when	O
+R1	O
+was	O
+absent	O
+(	O
+i	O
+.	O
+e	O
+.	O
+the	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+,	I-chemical
+5	I-chemical
+-	I-chemical
+trihydroxybenzohydrazide	I-chemical
+28	B-chemical
+,	O
+Fig	O
+.	O
+2	O
+),	O
+or	O
+composed	O
+of	O
+an	O
+extended	O
+ring	O
+system	O
+(	O
+26	B-chemical
+)	O
+or	O
+a	O
+pyrrole	O
+ring	O
+(	O
+25	B-chemical
+).	O
+Still	O
+lower	O
+activity	O
+was	O
+seen	O
+with	O
+the	O
+pyridine	O
+analogue	O
+24	B-chemical
+.	O
+Evidently	O
+,	O
+the	O
+3	O
+,	O
+4	O
+,	O
+5	O
+-	O
+trihydroxybenzyl	O
+moiety	O
+at	O
+R2	O
+is	O
+fundamental	O
+but	O
+not	O
+sufficient	O
+to	O
+ensure	O
+potent	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+endonuclease	B-protein_type
+inhibition	O
+,	O
+since	O
+the	O
+interactions	O
+of	O
+R1	O
+with	O
+the	O
+amino	O
+acid	O
+side	O
+chains	O
+of	O
+the	O
+protein	O
+appear	O
+crucial	O
+in	O
+modulating	O
+activity	O
+.	O
+Inhibition	O
+of	O
+vRNP	B-complex_assembly
+activity	O
+or	O
+virus	B-taxonomy_domain
+replication	O
+in	O
+cells	O
+To	O
+determine	O
+the	O
+anti	O
+-	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+activity	O
+of	O
+compounds	O
+1	B-chemical
+–	I-chemical
+28	I-chemical
+in	O
+cell	O
+culture	O
+,	O
+we	O
+performed	O
+an	O
+influenza	B-experimental_method
+vRNP	I-experimental_method
+reconstitution	I-experimental_method
+assay	I-experimental_method
+in	O
+human	B-species
+embryonic	O
+kidney	O
+293	O
+T	O
+(	O
+HEK293T	O
+)	O
+cells	O
+,	O
+then	O
+subjected	O
+the	O
+active	O
+compounds	O
+(	O
+i	O
+.	O
+e	O
+.	O
+EC50	B-evidence
+<	O
+100	O
+μM	O
+)	O
+to	O
+a	O
+virus	B-experimental_method
+yield	I-experimental_method
+assay	I-experimental_method
+in	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+-	O
+infected	O
+Madin	O
+-	O
+Darby	O
+canine	O
+kidney	O
+(	O
+MDCK	O
+)	O
+cells	O
+(	O
+Table	O
+1	O
+and	O
+Fig	O
+.	O
+3	O
+).	O
+For	O
+some	O
+N	B-chemical
+-	I-chemical
+acylhydrazone	I-chemical
+compounds	O
+,	O
+we	O
+observed	O
+quite	O
+potent	O
+and	O
+selective	O
+activity	O
+in	O
+the	O
+vRNP	B-experimental_method
+reconstitution	I-experimental_method
+assay	I-experimental_method
+.	O
+This	O
+indicates	O
+that	O
+they	O
+are	O
+able	O
+to	O
+inhibit	O
+viral	B-taxonomy_domain
+RNA	B-chemical
+synthesis	O
+and	O
+suggests	O
+that	O
+they	O
+could	O
+be	O
+classified	O
+as	O
+original	O
+PA	B-protein
+inhibitors	O
+.	O
+Values	O
+for	O
+EC50	B-evidence
+(	O
+vRNP	B-complex_assembly
+)	O
+or	O
+EC90	B-evidence
+(	O
+virus	B-taxonomy_domain
+yield	O
+)	O
+in	O
+the	O
+range	O
+of	O
+0	O
+.	O
+4	O
+–	O
+18	O
+μM	O
+were	O
+obtained	O
+for	O
+compounds	O
+15	B-chemical
+and	O
+20	B-chemical
+–	I-chemical
+23	I-chemical
+,	O
+which	O
+all	O
+carry	O
+a	O
+3	O
+,	O
+4	O
+,	O
+5	O
+-	O
+trihydroxyphenyl	O
+as	O
+R2	O
+,	O
+and	O
+possess	O
+either	O
+two	O
+(	O
+15	B-chemical
+)	O
+or	O
+three	O
+(	O
+20	B-chemical
+–	O
+23	B-chemical
+)	O
+hydroxyl	O
+substituents	O
+in	O
+the	O
+R1	O
+moiety	O
+.	O
+As	O
+in	O
+the	O
+enzymatic	B-experimental_method
+PA	I-experimental_method
+-	I-experimental_method
+Nter	I-experimental_method
+assays	I-experimental_method
+,	O
+the	O
+compounds	O
+having	O
+R2	O
+as	O
+a	O
+gallic	O
+moiety	O
+(	O
+Fig	O
+.	O
+3	O
+:	O
+21	B-chemical
+,	O
+22	B-chemical
+and	O
+23	B-chemical
+)	O
+showed	O
+slightly	O
+higher	O
+activity	O
+than	O
+the	O
+compounds	O
+carrying	O
+a	O
+2	O
+-	O
+hydroxyl	O
+R2	O
+group	O
+(	O
+9	B-chemical
+,	O
+10	B-chemical
+and	O
+11	B-chemical
+);	O
+10	B-chemical
+and	O
+22	B-chemical
+have	O
+substantially	O
+the	O
+same	O
+EC50	B-evidence
+in	O
+the	O
+vRNP	B-experimental_method
+reconstitution	I-experimental_method
+assay	I-experimental_method
+in	O
+HEK293T	O
+cells	O
+.	O
+The	O
+hydrazide	B-chemical
+28	B-chemical
+displayed	O
+weak	O
+(	O
+virus	B-taxonomy_domain
+yield	O
+)	O
+to	O
+moderate	O
+(	O
+vRNP	B-experimental_method
+reconstitution	I-experimental_method
+)	O
+activity	O
+,	O
+albeit	O
+less	O
+than	O
+the	O
+most	O
+active	O
+molecules	O
+in	O
+the	O
+3	O
+,	O
+4	O
+,	O
+5	O
+-	O
+trihydroxyphenyl	O
+series	O
+(	O
+i	O
+.	O
+e	O
+.	O
+18	B-chemical
+and	O
+21	B-chemical
+–	I-chemical
+23	I-chemical
+).	O
+Even	O
+if	O
+there	O
+are	O
+no	O
+data	O
+indicating	O
+that	O
+the	O
+compounds	O
+reported	O
+in	O
+the	O
+paper	O
+are	O
+subject	O
+to	O
+hydrolysis	O
+,	O
+the	O
+activity	O
+of	O
+28	B-chemical
+could	O
+raise	O
+the	O
+concern	O
+that	O
+for	O
+some	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+the	O
+antiviral	O
+activity	O
+in	O
+cell	O
+culture	O
+may	O
+be	O
+related	O
+to	O
+their	O
+intracellular	O
+hydrolysis	O
+.	O
+However	O
+,	O
+this	O
+is	O
+unlikely	O
+,	O
+since	O
+the	O
+antiviral	O
+potency	O
+showed	O
+large	O
+differences	O
+(	O
+i	O
+.	O
+e	O
+.	O
+EC50	B-evidence
+values	O
+between	O
+0	O
+.	O
+42	O
+and	O
+29	O
+μM	O
+)	O
+for	O
+compounds	O
+with	O
+the	O
+same	O
+R2	O
+but	O
+different	O
+R1	O
+groups	O
+,	O
+meaning	O
+that	O
+R1	O
+does	O
+play	O
+a	O
+role	O
+in	O
+modulating	O
+the	O
+antiviral	O
+effect	O
+.	O
+Most	O
+compounds	O
+carrying	O
+as	O
+R1	O
+a	O
+2	B-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+dihydroxybenzylidene	I-chemical
+(	O
+i	O
+.	O
+e	O
+.	O
+3	B-chemical
+,	O
+5	B-chemical
+and	O
+7	B-chemical
+)	O
+or	O
+2	B-chemical
+-	I-chemical
+hydroxy	I-chemical
+-	I-chemical
+3	I-chemical
+-	I-chemical
+methoxybenzylidene	I-chemical
+moiety	O
+(	O
+i	O
+.	O
+e	O
+.	O
+4	B-chemical
+,	O
+6	B-chemical
+and	O
+8	B-chemical
+)	O
+showed	O
+relatively	O
+high	O
+cytotoxicity	O
+in	O
+the	O
+vRNP	B-experimental_method
+assay	I-experimental_method
+,	O
+with	O
+CC50	B-evidence
+values	O
+below	O
+50	O
+μM	O
+and	O
+a	O
+selectivity	B-evidence
+index	I-evidence
+(	O
+ratio	O
+of	O
+CC50	B-evidence
+to	O
+EC50	B-evidence
+)	O
+below	O
+8	O
+.	O
+Two	O
+notable	O
+exceptions	O
+are	O
+18	B-chemical
+and	O
+19	B-chemical
+(	O
+containing	O
+a	O
+2	B-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+dihydroxybenzylidene	I-chemical
+or	O
+2	B-chemical
+-	I-chemical
+hydroxy	I-chemical
+-	I-chemical
+3	I-chemical
+-	I-chemical
+methoxybenzylidene	I-chemical
+R1	O
+,	O
+respectively	O
+)	O
+which	O
+were	O
+not	O
+cytotoxic	O
+at	O
+200	O
+μM	O
+and	O
+displayed	O
+favorable	O
+antiviral	O
+selectivity	O
+.	O
+Some	O
+N	B-chemical
+-	I-chemical
+acylhydrazone	I-chemical
+compounds	O
+were	O
+devoid	O
+of	O
+activity	O
+in	O
+the	O
+enzymatic	B-experimental_method
+assay	I-experimental_method
+,	O
+yet	O
+showed	O
+good	O
+to	O
+moderate	O
+efficacy	O
+in	O
+cell	O
+culture	O
+(	O
+e	O
+.	O
+g	O
+.	O
+14	B-chemical
+and	O
+19	B-chemical
+,	O
+having	O
+EC50	B-evidence
+values	O
+of	O
+2	O
+.	O
+2	O
+and	O
+7	O
+.	O
+1	O
+μM	O
+,	O
+respectively	O
+).	O
+For	O
+most	O
+of	O
+the	O
+active	O
+compounds	O
+(	O
+i	O
+.	O
+e	O
+.	O
+9	B-chemical
+,	O
+11	B-chemical
+,	O
+13	B-chemical
+,	O
+15	B-chemical
+–	I-chemical
+21	I-chemical
+,	O
+23	B-chemical
+,	O
+24	B-chemical
+and	O
+26	B-chemical
+)	O
+a	O
+fair	O
+correlation	O
+was	O
+seen	O
+for	O
+the	O
+two	O
+cell	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+assays	I-experimental_method
+,	O
+since	O
+the	O
+EC50	B-evidence
+values	O
+obtained	O
+in	O
+the	O
+vRNP	B-experimental_method
+assay	I-experimental_method
+were	O
+maximum	O
+5	O
+-	O
+fold	O
+different	O
+from	O
+the	O
+EC90	B-evidence
+values	O
+in	O
+the	O
+virus	B-experimental_method
+yield	I-experimental_method
+assay	I-experimental_method
+.	O
+On	O
+the	O
+other	O
+hand	O
+,	O
+this	O
+difference	O
+was	O
+8	O
+-	O
+fold	O
+or	O
+more	O
+for	O
+7	B-chemical
+,	O
+10	B-chemical
+,	O
+14	B-chemical
+,	O
+22	B-chemical
+,	O
+25	B-chemical
+and	O
+28	B-chemical
+.	O
+Some	O
+N	B-chemical
+-	I-chemical
+acylhydrazone	I-chemical
+compounds	O
+showed	O
+good	O
+to	O
+moderate	O
+efficacy	O
+in	O
+the	O
+vRNP	B-experimental_method
+assay	I-experimental_method
+(	O
+e	O
+.	O
+g	O
+.	O
+14	B-chemical
+and	O
+19	B-chemical
+,	O
+having	O
+EC50	B-evidence
+values	O
+of	O
+2	O
+.	O
+3	O
+and	O
+5	O
+.	O
+7	O
+μM	O
+,	O
+respectively	O
+),	O
+yet	O
+were	O
+devoid	O
+of	O
+activity	O
+in	O
+the	O
+enzymatic	B-experimental_method
+assay	I-experimental_method
+.	O
+This	O
+observation	O
+suggests	O
+that	O
+they	O
+may	O
+inhibit	O
+the	O
+viral	B-taxonomy_domain
+polymerase	B-protein_type
+in	O
+an	O
+endonuclease	B-protein_type
+-	O
+independent	O
+manner	O
+.	O
+To	O
+achieve	O
+a	O
+clear	O
+insight	O
+into	O
+the	O
+antiviral	O
+profile	O
+of	O
+the	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+,	O
+specific	O
+mechanistic	O
+experiments	O
+are	O
+currently	O
+ongoing	O
+in	O
+our	O
+laboratory	O
+,	O
+in	O
+which	O
+we	O
+are	O
+analyzing	O
+in	O
+full	O
+depth	O
+their	O
+effects	O
+on	O
+virus	B-taxonomy_domain
+entry	O
+,	O
+polymerase	B-protein_type
+-	O
+dependent	O
+RNA	B-chemical
+synthesis	O
+or	O
+the	O
+late	O
+stage	O
+(	O
+maturation	O
+and	O
+release	O
+)	O
+of	O
+the	O
+virus	B-taxonomy_domain
+replication	O
+cycle	O
+.	O
+Docking	B-experimental_method
+studies	I-experimental_method
+In	O
+order	O
+to	O
+explore	O
+the	O
+possible	O
+binding	O
+mode	O
+of	O
+the	O
+synthesized	O
+compounds	O
+,	O
+docking	B-experimental_method
+simulations	I-experimental_method
+by	O
+GOLD	B-experimental_method
+program	I-experimental_method
+were	O
+performed	O
+by	O
+using	O
+the	O
+structural	O
+coordinates	O
+(	O
+PDB	O
+code	O
+4AWM	O
+)	O
+for	O
+the	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+endonuclease	B-protein_type
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+EGCG	B-chemical
+.	O
+Considering	O
+that	O
+the	O
+position	O
+of	O
+the	O
+side	O
+-	O
+chains	O
+of	O
+some	O
+residues	O
+changes	O
+depending	O
+on	O
+which	O
+pocket	O
+the	O
+ligand	O
+is	O
+occupying	O
+,	O
+we	O
+superimposed	B-experimental_method
+some	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+structures	I-evidence
+of	O
+complexes	O
+between	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+endonuclease	B-protein_type
+and	O
+known	O
+active	O
+ligands	O
+.	O
+It	O
+was	O
+observed	O
+that	O
+the	O
+side	O
+-	O
+chain	O
+of	O
+amino	O
+acid	O
+Tyr24	B-residue_name_number
+shows	O
+greater	O
+movement	O
+than	O
+the	O
+other	O
+residues	O
+and	O
+for	O
+this	O
+reason	O
+we	O
+considered	O
+it	O
+as	O
+a	O
+flexible	B-protein_state
+residue	O
+during	O
+the	O
+docking	B-experimental_method
+procedure	I-experimental_method
+.	O
+First	O
+,	O
+test	B-experimental_method
+docking	I-experimental_method
+calculations	I-experimental_method
+,	O
+using	O
+EGCG	B-chemical
+,	O
+L	B-chemical
+-	I-chemical
+742	I-chemical
+,	I-chemical
+001	I-chemical
+and	O
+2	B-chemical
+-(	I-chemical
+4	I-chemical
+-(	I-chemical
+1H	I-chemical
+-	I-chemical
+tetrazol	I-chemical
+-	I-chemical
+5	I-chemical
+-	I-chemical
+yl	I-chemical
+)	I-chemical
+phenyl	I-chemical
+)-	I-chemical
+5	I-chemical
+-	I-chemical
+hydroxypyrimidin	I-chemical
+-	I-chemical
+4	I-chemical
+(	I-chemical
+3H	I-chemical
+)-	I-chemical
+one	I-chemical
+(	O
+Fig	O
+.	O
+1	O
+),	O
+were	O
+carried	O
+out	O
+to	O
+compare	O
+experimental	O
+and	O
+predicted	O
+binding	O
+modes	O
+and	O
+validate	O
+docking	B-experimental_method
+procedure	I-experimental_method
+.	O
+Their	O
+best	O
+docking	O
+poses	O
+agreed	O
+well	O
+with	O
+the	O
+experimental	O
+binding	O
+modes	O
+(	O
+rmsd	B-evidence
+values	O
+of	O
+0	O
+.	O
+8	O
+,	O
+1	O
+.	O
+2	O
+and	O
+0	O
+.	O
+7	O
+,	O
+respectively	O
+).	O
+Next	O
+,	O
+docking	B-experimental_method
+of	O
+several	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+was	O
+performed	O
+and	O
+this	O
+generated	O
+a	O
+number	O
+of	O
+possible	O
+binding	O
+conformations	O
+,	O
+highlighting	O
+that	O
+the	O
+active	B-site
+site	I-site
+cavity	I-site
+of	O
+the	O
+PA	B-protein
+endonuclease	B-protein_type
+is	O
+quite	O
+spacious	O
+,	O
+as	O
+already	O
+demonstrated	O
+by	O
+crystallographic	B-experimental_method
+studies	I-experimental_method
+,	O
+and	O
+confirming	O
+the	O
+ability	O
+of	O
+this	O
+scaffold	O
+to	O
+chelate	O
+the	O
+two	O
+M2	B-chemical
++	I-chemical
+ions	O
+in	O
+different	O
+ways	O
+(	O
+Mode	O
+A	O
+-	O
+C	O
+in	O
+Fig	O
+.	O
+4	O
+).	O
+Figure	O
+5	O
+displays	O
+the	O
+first	O
+(	O
+panel	O
+A	O
+)	O
+and	O
+second	O
+(	O
+panel	O
+B	O
+)	O
+GOLD	B-experimental_method
+cluster	I-experimental_method
+docked	I-experimental_method
+solutions	O
+for	O
+compound	O
+23	B-chemical
+.	O
+These	O
+two	O
+complex	O
+structures	B-evidence
+represent	O
+the	O
+largest	O
+clusters	O
+with	O
+similar	O
+fitness	O
+values	O
+(	O
+59	O
+.	O
+20	O
+and	O
+58	O
+.	O
+65	O
+,	O
+respectively	O
+).	O
+In	O
+both	O
+cases	O
+,	O
+23	B-chemical
+appears	O
+able	O
+to	O
+coordinate	B-bond_interaction
+the	O
+two	O
+M2	B-chemical
++	I-chemical
+ions	O
+in	O
+the	O
+active	B-site
+site	I-site
+through	O
+the	O
+three	O
+contiguous	O
+OH	O
+groups	O
+(	O
+Fig	O
+.	O
+5	O
+).	O
+In	O
+addition	O
+,	O
+23	B-chemical
+was	O
+predicted	O
+to	O
+form	O
+two	O
+hydrogen	B-bond_interaction
+bonding	I-bond_interaction
+interactions	I-bond_interaction
+,	O
+i	O
+.	O
+e	O
+.	O
+with	O
+the	O
+catalytic	B-protein_state
+Lys134	B-residue_name_number
+on	O
+the	O
+one	O
+side	O
+and	O
+Glu26	B-residue_name_number
+on	O
+the	O
+other	O
+side	O
+.	O
+Furthermore	O
+,	O
+in	O
+these	O
+two	O
+different	O
+binding	O
+modes	O
+,	O
+23	B-chemical
+forms	O
+π	B-bond_interaction
+–	I-bond_interaction
+π	I-bond_interaction
+interactions	I-bond_interaction
+with	O
+the	O
+aromatic	O
+ring	O
+of	O
+Tyr24	B-residue_name_number
+,	O
+in	O
+a	O
+fashion	O
+similar	O
+to	O
+that	O
+described	O
+for	O
+other	O
+endonuclease	B-protein_type
+inhibitors	O
+,	O
+i	O
+.	O
+e	O
+.	O
+EGCG	B-chemical
+and	O
+L	B-chemical
+-	I-chemical
+742	I-chemical
+,	I-chemical
+001	I-chemical
+.	O
+The	O
+best	O
+docked	O
+conformation	O
+for	O
+compound	O
+15	B-chemical
+(	O
+Fig	O
+.	O
+6	O
+,	O
+fitness	B-evidence
+value	I-evidence
+68	O
+.	O
+56	O
+),	O
+which	O
+has	O
+an	O
+activity	O
+slightly	O
+lower	O
+than	O
+23	O
+,	O
+reveals	O
+a	O
+different	O
+role	O
+for	O
+the	O
+gallic	O
+moiety	O
+.	O
+The	O
+ligand	O
+seems	O
+to	O
+form	O
+two	O
+hydrogen	B-bond_interaction
+bonding	I-bond_interaction
+interactions	I-bond_interaction
+with	O
+Tyr130	B-residue_name_number
+as	O
+well	O
+as	O
+a	O
+cation	B-bond_interaction
+–	I-bond_interaction
+π	I-bond_interaction
+interaction	I-bond_interaction
+with	O
+Lys134	B-residue_name_number
+.	O
+Tyr130	B-residue_name_number
+lies	O
+in	O
+a	O
+pocket	B-site
+that	O
+also	O
+contains	O
+Arg124	B-residue_name_number
+,	O
+a	O
+residue	O
+that	O
+was	O
+proposed	O
+to	O
+have	O
+a	O
+crucial	O
+role	O
+in	O
+binding	O
+of	O
+the	O
+RNA	B-chemical
+substrate	O
+.	O
+Compound	O
+15	B-chemical
+appears	O
+further	O
+stabilized	O
+by	O
+hydrogen	B-bond_interaction
+bonding	I-bond_interaction
+interactions	I-bond_interaction
+between	O
+two	O
+hydroxyl	O
+groups	O
+and	O
+Arg82	B-residue_name_number
+and	O
+Asp108	B-residue_name_number
+.	O
+In	O
+this	O
+case	O
+,	O
+chelation	B-bond_interaction
+of	O
+the	O
+two	O
+M2	B-chemical
++	I-chemical
+ions	O
+is	O
+carried	O
+out	O
+by	O
+involving	O
+the	O
+imine	O
+group	O
+(	O
+mode	O
+A	O
+in	O
+Fig	O
+.	O
+4	O
+).	O
+It	O
+is	O
+important	O
+to	O
+highlight	O
+that	O
+compounds	O
+23	B-chemical
+and	O
+15	B-chemical
+,	O
+although	O
+in	O
+different	O
+ways	O
+,	O
+both	O
+are	O
+able	O
+to	O
+chelate	O
+the	O
+metal	O
+cofactors	O
+and	O
+to	O
+establish	O
+interactions	O
+with	O
+highly	B-protein_state
+conserved	I-protein_state
+aminoacids	O
+(	O
+Tyr24	B-residue_name_number
+,	O
+Glu26	B-residue_name_number
+,	O
+Arg124	B-residue_name_number
+,	O
+Tyr130	B-residue_name_number
+and	O
+Lys134	B-residue_name_number
+)	O
+that	O
+are	O
+very	O
+important	O
+for	O
+both	O
+endonuclease	B-protein_type
+activity	O
+and	O
+transcription	O
+in	O
+vitro	O
+.	O
+The	O
+crucial	O
+role	O
+of	O
+such	O
+interactions	O
+is	O
+underlined	O
+by	O
+the	O
+differences	O
+in	O
+activity	O
+between	O
+15	B-chemical
+(	O
+IC50	B-evidence
+=	O
+9	O
+.	O
+0	O
+μM	O
+)	O
+and	O
+19	B-chemical
+(>	O
+500	O
+μM	O
+):	O
+their	O
+coordinating	O
+features	O
+are	O
+similar	O
+,	O
+since	O
+both	O
+coordinate	B-bond_interaction
+to	O
+the	O
+divalent	O
+metal	O
+ion	O
+through	O
+the	O
+phenolic	O
+oxygen	O
+,	O
+the	O
+iminic	O
+nitrogen	O
+and	O
+the	O
+carbonylic	O
+oxygen	O
+(	O
+mode	O
+A	O
+in	O
+Fig	O
+.	O
+4	O
+),	O
+but	O
+the	O
+biological	O
+activity	O
+could	O
+be	O
+related	O
+to	O
+their	O
+different	O
+ability	O
+to	O
+engage	O
+interactions	O
+with	O
+the	O
+protein	O
+environment	O
+.	O
+Crystallographic	B-experimental_method
+Studies	I-experimental_method
+Attempts	O
+were	O
+made	O
+to	O
+co	B-experimental_method
+-	I-experimental_method
+crystallize	I-experimental_method
+PA	B-protein
+-	O
+Nter	B-structure_element
+with	O
+15	B-chemical
+,	O
+20	B-chemical
+,	O
+21	B-chemical
+and	O
+23	B-chemical
+in	O
+one	O
+to	O
+four	O
+molar	O
+excess	O
+.	O
+While	O
+crystals	B-evidence
+appeared	O
+and	O
+diffracted	O
+well	O
+,	O
+upon	O
+data	O
+processing	O
+,	O
+no	O
+or	O
+very	O
+little	O
+electron	B-evidence
+density	I-evidence
+for	O
+the	O
+inhibitors	O
+was	O
+observed	O
+.	O
+Attempts	O
+to	O
+soak	O
+apo	B-protein_state
+crystals	B-evidence
+in	O
+crystallization	O
+solution	O
+containing	O
+5	O
+mM	O
+inhibitor	O
+overnight	O
+also	O
+did	O
+not	O
+result	O
+in	O
+substantial	O
+electron	B-evidence
+density	I-evidence
+for	O
+the	O
+inhibitor	O
+.	O
+As	O
+a	O
+last	O
+resort	O
+,	O
+dry	O
+powder	O
+of	O
+the	O
+inhibitor	O
+was	O
+sprinkled	O
+over	O
+the	O
+crystallization	O
+drop	O
+containing	O
+apo	B-protein_state
+crystals	B-evidence
+and	O
+left	O
+over	O
+night	O
+.	O
+This	O
+experiment	O
+was	O
+successful	O
+for	O
+compound	O
+23	B-chemical
+,	O
+the	O
+crystals	B-evidence
+diffracted	O
+to	O
+2	O
+.	O
+15	O
+Å	O
+and	O
+diffraction	O
+data	O
+were	O
+collected	O
+(	O
+PDB	O
+ID	O
+5EGA	O
+).	O
+The	O
+refined	O
+structure	B-evidence
+shows	O
+unambiguous	O
+electron	B-evidence
+density	I-evidence
+for	O
+the	O
+inhibitor	O
+(	O
+Table	O
+S1	O
+and	O
+Fig	O
+.	O
+7	O
+).	O
+The	O
+complex	B-evidence
+structure	I-evidence
+confirms	O
+one	O
+of	O
+the	O
+two	O
+binding	O
+modes	O
+predicted	O
+by	O
+the	O
+docking	B-experimental_method
+simulations	I-experimental_method
+(	O
+Fig	O
+.	O
+5	O
+,	O
+panel	O
+B	O
+).	O
+The	O
+galloyl	O
+moiety	O
+chelates	O
+the	O
+manganese	B-chemical
+ions	O
+,	O
+while	O
+the	O
+trihydroxyphenyl	O
+group	O
+stacks	O
+against	O
+the	O
+Tyr24	B-residue_name_number
+side	O
+chain	O
+.	O
+It	O
+is	O
+interesting	O
+to	O
+note	O
+that	O
+two	O
+of	O
+these	O
+hydroxyl	O
+groups	O
+are	O
+in	O
+position	O
+to	O
+form	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+with	O
+the	O
+side	O
+chain	O
+of	O
+Glu26	B-residue_name_number
+and	O
+Lys34	B-residue_name_number
+(	O
+Fig	O
+.	O
+7	O
+).	O
+These	O
+interactions	O
+suggest	O
+that	O
+other	O
+functional	O
+groups	O
+,	O
+e	O
+.	O
+g	O
+.	O
+halogens	O
+,	O
+could	O
+be	O
+used	O
+in	O
+place	O
+of	O
+the	O
+hydroxyl	O
+groups	O
+for	O
+better	O
+interactions	O
+with	O
+Glu26	B-residue_name_number
+and	O
+Lys34	B-residue_name_number
+side	O
+chains	O
+,	O
+and	O
+the	O
+inhibitory	O
+potency	O
+of	O
+these	O
+compounds	O
+could	O
+be	O
+further	O
+improved	O
+.	O
+Chemical	O
+structures	O
+of	O
+some	O
+prototype	O
+inhibitors	O
+of	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+endonuclease	B-protein_type
+.	O
+Inhibitor	O
+activity	O
+in	O
+enzymatic	B-experimental_method
+assays	I-experimental_method
+(	O
+IC50	B-evidence
+,	O
+μM	O
+)	O
+as	O
+reported	O
+in	O
+:	O
+aref	O
+.,	O
+bref	O
+.,	O
+cref	O
+.,	O
+dref	O
+..	O
+General	O
+synthesis	O
+for	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+1	B-chemical
+–	I-chemical
+27	I-chemical
+and	O
+hydrazides	B-chemical
+28	B-chemical
+and	O
+29	B-chemical
+(	O
+A	O
+).	O
+Chemical	O
+structures	O
+of	O
+compounds	O
+1	B-chemical
+–	I-chemical
+27	I-chemical
+(	O
+B	O
+).	O
+Overview	O
+of	O
+the	O
+structure	O
+-	O
+activity	O
+relationship	O
+for	O
+compounds	O
+1	B-chemical
+–	I-chemical
+27	I-chemical
+.	O
+Scheme	O
+of	O
+possible	O
+binding	O
+modes	O
+of	O
+the	O
+studied	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+.	O
+First	O
+(	O
+A	O
+)	O
+and	O
+second	O
+(	O
+B	O
+)	O
+GOLD	B-experimental_method
+cluster	I-experimental_method
+docked	I-experimental_method
+solutions	O
+of	O
+compound	O
+23	B-chemical
+(	O
+orange	O
+and	O
+cyan	O
+,	O
+respectively	O
+)	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+PA	B-protein
+endonuclease	B-protein_type
+.	O
+Key	O
+residues	O
+of	O
+the	O
+pocket	B-site
+are	O
+presented	O
+using	O
+PyMOL	O
+[	O
+http	O
+://	O
+www	O
+.	O
+pymol	O
+.	O
+org	O
+]	O
+and	O
+LIGPLUS	B-experimental_method
+[	O
+Laskowski	O
+,	O
+R	O
+.	O
+A	O
+.;	O
+Swindells	O
+,	O
+M	O
+.	O
+B	O
+.	O
+Journal	O
+of	O
+chemical	O
+information	O
+and	O
+modeling	O
+2011	O
+,	O
+51	O
+,	O
+2778	O
+].	O
+Hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+are	O
+illustrated	O
+by	O
+dotted	O
+lines	O
+,	O
+while	O
+the	O
+divalent	O
+metal	O
+ions	O
+are	O
+shown	O
+as	O
+purple	O
+spheres	O
+.	O
+Schematic	O
+drawings	O
+of	O
+the	O
+interactions	O
+of	O
+the	O
+first	O
+(	O
+C	O
+)	O
+and	O
+second	O
+(	O
+D	O
+)	O
+GOLD	B-experimental_method
+cluster	I-experimental_method
+docked	I-experimental_method
+solutions	O
+generated	O
+using	O
+LIGPLUS	B-experimental_method
+.	O
+Dashed	O
+lines	O
+are	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+and	O
+‘	O
+eyelashes	O
+’	O
+show	O
+residues	O
+involved	O
+in	O
+hydrophobic	B-bond_interaction
+interactions	I-bond_interaction
+.	O
+(	O
+A	O
+)	O
+Binding	O
+mode	O
+of	O
+compound	O
+15	B-chemical
+(	O
+orange	O
+)	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+PA	B-protein
+endonuclease	B-protein_type
+.	O
+Hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+are	O
+illustrated	O
+by	O
+dotted	O
+lines	O
+while	O
+the	O
+divalent	O
+metal	O
+ions	O
+are	O
+shown	O
+as	O
+purple	O
+spheres	O
+.	O
+(	O
+B	O
+)	O
+Schematic	O
+drawing	O
+of	O
+the	O
+interactions	O
+of	O
+compound	O
+15	B-chemical
+generated	O
+using	O
+LIGPLUS	B-experimental_method
+.	O
+Crystal	B-evidence
+structure	I-evidence
+of	O
+PANΔLoop	B-mutant
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+compound	O
+23	B-chemical
+.	O
+Active	B-site
+site	I-site
+residues	O
+are	O
+shown	O
+in	O
+sticks	O
+with	O
+green	O
+carbons	O
+,	O
+manganese	B-chemical
+atoms	O
+are	O
+shown	O
+as	O
+purple	O
+spheres	O
+and	O
+water	B-chemical
+molecules	O
+as	O
+red	O
+spheres	O
+.	O
+Compound	O
+23	B-chemical
+is	O
+shown	O
+in	O
+sticks	O
+with	O
+yellow	O
+carbons	O
+.	O
+2Fo	B-evidence
+-	I-evidence
+Fc	I-evidence
+electron	I-evidence
+density	I-evidence
+map	I-evidence
+contoured	O
+at	O
+1σ	O
+is	O
+shown	O
+as	O
+blue	O
+mesh	O
+.	O
+Hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+and	O
+metal	B-bond_interaction
+coordination	I-bond_interaction
+are	O
+shown	O
+with	O
+dotted	O
+lines	O
+.	O
+The	O
+H	B-bond_interaction
+-	I-bond_interaction
+bond	I-bond_interaction
+distances	O
+from	O
+the	O
+side	O
+chain	O
+carboxyl	O
+group	O
+of	O
+Glu26	B-residue_name_number
+to	O
+p	O
+-	O
+OH	O
+and	O
+m	O
+-	O
+OH	O
+of	O
+the	O
+trihydroxyphenyl	O
+group	O
+of	O
+the	O
+inhibitor	O
+are	O
+2	O
+.	O
+7	O
+Å	O
+and	O
+3	O
+.	O
+0	O
+Å	O
+,	O
+respectively	O
+.	O
+The	O
+H	B-bond_interaction
+-	I-bond_interaction
+bond	I-bond_interaction
+distance	O
+from	O
+the	O
+side	O
+chain	O
+of	O
+Lys34	B-residue_name_number
+to	O
+p	O
+-	O
+OH	O
+of	O
+the	O
+trihydroxyphenyl	O
+group	O
+is	O
+3	O
+.	O
+6	O
+Å	O
+.	O
+The	O
+H	B-bond_interaction
+-	I-bond_interaction
+bond	I-bond_interaction
+distance	O
+to	O
+the	O
+water	B-chemical
+molecule	O
+from	O
+m	O
+-	O
+OH	O
+of	O
+the	O
+galloyl	O
+moiety	O
+is	O
+3	O
+.	O
+0	O
+Å	O
+,	O
+which	O
+in	O
+turn	O
+is	O
+H	B-bond_interaction
+-	I-bond_interaction
+bonded	I-bond_interaction
+to	O
+the	O
+side	O
+chain	O
+of	O
+Tyr130	B-residue_name_number
+with	O
+a	O
+distance	O
+of	O
+2	O
+.	O
+7	O
+Å	O
+.	O
+Crystal	B-evidence
+structure	I-evidence
+has	O
+been	O
+deposited	O
+in	O
+the	O
+RCSB	O
+Protein	O
+Data	O
+Bank	O
+with	O
+PDB	O
+ID	O
+:	O
+5EGA	O
+.	O
+Inhibitory	O
+activity	O
+of	O
+the	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+1	B-chemical
+–	I-chemical
+27	I-chemical
+and	O
+hydrazide	B-chemical
+28	B-chemical
+in	O
+the	O
+enzymatic	B-experimental_method
+assay	I-experimental_method
+with	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+PA	B-protein
+-	O
+Nter	B-structure_element
+endonuclease	B-protein_type
+,	O
+or	O
+in	O
+cellular	B-experimental_method
+influenza	I-experimental_method
+virus	I-experimental_method
+assays	I-experimental_method
+.	O
+Compound	B-experimental_method
+Enzyme	I-experimental_method
+assay	I-experimental_method
+with	O
+PA	B-protein
+-	O
+Ntera	O
+Virus	B-experimental_method
+yield	I-experimental_method
+assay	I-experimental_method
+in	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+-	O
+infected	O
+MDCK	O
+cellsb	O
+vRNP	B-experimental_method
+reconstitution	I-experimental_method
+assay	I-experimental_method
+in	O
+HEK293T	O
+cellsc	O
+Antiviral	O
+activity	O
+Cytotoxicity	O
+SId	O
+Activity	O
+Cytotoxicity	O
+IC50	B-evidence
+EC99	B-evidence
+EC90	B-evidence
+CC50	B-evidence
+EC50	B-evidence
+CC50	B-evidence
+(	O
+1	O
+)	O
+24	O
+NDf	O
+ND	O
+ND	O
+107	O
+>	O
+200	O
+(	O
+2	O
+)	O
+>	O
+500	O
+ND	O
+ND	O
+ND	O
+>	O
+100	O
+>	O
+200	O
+(	O
+3	O
+)	O
+>	O
+500	O
+ND	O
+ND	O
+>	O
+200	O
+5	O
+.	O
+9	O
+48	O
+(	O
+4	O
+)	O
+>	O
+500	O
+ND	O
+ND	O
+>	O
+200	O
+6	O
+.	O
+3	O
+33	O
+(	O
+5	O
+)	O
+67	O
+>	O
+25	O
+>	O
+25	O
+≥	O
+146	O
+2	O
+.	O
+6	O
+10	O
+(	O
+6	O
+)	O
+>	O
+500	O
+>	O
+50	O
+>	O
+50	O
+>	O
+200	O
+15	O
+14	O
+(	O
+7	O
+)	O
+54	O
+172	O
+100	O
+>	O
+200	O
+>	O
+2	O
+.	O
+0	O
+3	O
+.	O
+2	O
+8	O
+.	O
+9	O
+(	O
+8	O
+)	O
+>	O
+500	O
+>	O
+12	O
+.	O
+5	O
+>	O
+12	O
+.	O
+5	O
+>	O
+200	O
+1	O
+.	O
+9	O
+15	O
+(	O
+9	O
+)	O
+34	O
+16	O
+5	O
+.	O
+3	O
+>	O
+200	O
+>	O
+38	O
+5	O
+.	O
+5	O
+>	O
+200	O
+(	O
+10	O
+)	O
+68	O
+14	O
+8	O
+.	O
+5	O
+111	O
+>	O
+13	O
+0	O
+.	O
+40	O
+132	O
+(	O
+11	O
+)	O
+45	O
+30	O
+12	O
+>	O
+200	O
+>	O
+17	O
+5	O
+.	O
+6	O
+>	O
+200	O
+(	O
+12	O
+)	O
+>	O
+500	O
+>	O
+12	O
+.	O
+5	O
+>	O
+12	O
+.	O
+5	O
+>	O
+200	O
+20	O
+39	O
+(	O
+13	O
+)	O
+69	O
+71	O
+34	O
+>	O
+200	O
+>	O
+5	O
+.	O
+9	O
+6	O
+.	O
+3	O
+>	O
+200	O
+(	O
+14	O
+)	O
+>	O
+500	O
+63	O
+37	O
+>	O
+200	O
+>	O
+5	O
+.	O
+4	O
+2	O
+.	O
+3	O
+>	O
+200	O
+(	O
+15	O
+)	O
+8	O
+.	O
+9	O
+18	O
+7	O
+.	O
+5	O
+≥	O
+172	O
+≥	O
+23	O
+14	O
+>	O
+200	O
+(	O
+16	O
+)	O
+454	O
+67	O
+28	O
+>	O
+200	O
+>	O
+7	O
+.	O
+1	O
+5	O
+.	O
+2	O
+>	O
+200	O
+(	O
+17	O
+)	O
+482	O
+21	O
+8	O
+.	O
+1	O
+>	O
+200	O
+>	O
+25	O
+7	O
+.	O
+1	O
+>	O
+200	O
+(	O
+18	O
+)	O
+83	O
+6	O
+.	O
+2	O
+2	O
+.	O
+2	O
+>	O
+200	O
+>	O
+91	O
+3	O
+.	O
+3	O
+>	O
+200	O
+(	O
+19	O
+)	O
+>	O
+500	O
+53	O
+26	O
+>	O
+200	O
+>	O
+7	O
+.	O
+7	O
+5	O
+.	O
+7	O
+>	O
+200	O
+(	O
+20	O
+)	O
+18	O
+35	O
+11	O
+>	O
+200	O
+>	O
+18	O
+2	O
+.	O
+2	O
+>	O
+200	O
+(	O
+21	O
+)	O
+13	O
+8	O
+.	O
+3	O
+3	O
+.	O
+6	O
+>	O
+200	O
+>	O
+56	O
+2	O
+.	O
+5	O
+>	O
+200	O
+(	O
+22	O
+)	O
+75	O
+7	O
+.	O
+4	O
+3	O
+.	O
+4	O
+>	O
+200	O
+>	O
+59	O
+0	O
+.	O
+42	O
+>	O
+200	O
+(	O
+23	O
+)	O
+8	O
+.	O
+7	O
+11	O
+3	O
+.	O
+5	O
+>	O
+200	O
+>	O
+57	O
+3	O
+.	O
+1	O
+>	O
+200	O
+(	O
+24	O
+)	O
+131	O
+58	O
+26	O
+>	O
+200	O
+>	O
+7	O
+.	O
+7	O
+25	O
+>	O
+200	O
+(	O
+25	O
+)	O
+40	O
+132	O
+70	O
+>	O
+200	O
+>	O
+2	O
+.	O
+9	O
+4	O
+.	O
+1	O
+>	O
+200	O
+(	O
+26	O
+)	O
+30	O
+36	O
+13	O
+>	O
+200	O
+>	O
+15	O
+5	O
+.	O
+5	O
+>	O
+200	O
+(	O
+27	O
+)	O
+36	O
+ND	O
+ND	O
+ND	O
+21	O
+>	O
+200	O
+(	O
+28	O
+)	O
+40	O
+158	O
+85	O
+>	O
+200	O
+>	O
+2	O
+.	O
+4	O
+7	O
+.	O
+2	O
+>	O
+200	O
+DPBAe	O
+5	O
+.	O
+3	O
+ND	O
+ND	O
+ND	O
+ND	O
+ND	O
+Ribavirin	O
+ND	O
+13	O
+8	O
+.	O
+5	O
+>	O
+200	O
+>	O
+24	O
+9	O
+.	O
+4	O
+>	O
+200	O
+aRecombinant	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+was	O
+incubated	B-experimental_method
+with	O
+the	O
+ssDNA	B-chemical
+plasmid	O
+substrate	O
+,	O
+a	O
+Mn2	B-chemical
++-	I-chemical
+containing	O
+buffer	O
+and	O
+test	O
+compounds	O
+.	O
+The	O
+IC50	B-evidence
+represents	O
+the	O
+compound	O
+concentration	O
+(	O
+in	O
+μM	O
+)	O
+required	O
+to	O
+obtain	O
+50	O
+%	O
+inhibition	O
+of	O
+cleavage	O
+,	O
+calculated	O
+by	O
+nonlinear	B-experimental_method
+least	I-experimental_method
+-	I-experimental_method
+squares	I-experimental_method
+regression	I-experimental_method
+analysis	I-experimental_method
+(	O
+using	O
+GraphPad	O
+Prism	O
+software	O
+)	O
+of	O
+the	O
+results	O
+from	O
+2	O
+–	O
+4	O
+independent	O
+experiments	O
+.	O
+bMDCK	O
+cells	O
+were	O
+infected	O
+with	O
+influenza	B-taxonomy_domain
+A	I-taxonomy_domain
+virus	B-taxonomy_domain
+(	O
+strain	O
+A	O
+/	O
+PR	O
+/	O
+8	O
+/	O
+34	O
+)	O
+and	O
+incubated	O
+with	O
+the	O
+compounds	O
+during	O
+24	O
+h	O
+.	O
+The	O
+virus	B-taxonomy_domain
+yield	O
+in	O
+the	O
+supernatant	O
+was	O
+assessed	O
+by	O
+real	B-experimental_method
+-	I-experimental_method
+time	I-experimental_method
+qPCR	I-experimental_method
+.	O
+The	O
+EC99	B-evidence
+and	O
+EC90	B-evidence
+values	O
+represent	O
+the	O
+compound	O
+concentrations	O
+(	O
+in	O
+μM	O
+)	O
+producing	O
+a	O
+2	O
+-	O
+log10	O
+or	O
+1	O
+-	O
+log10	O
+reduction	O
+in	O
+virus	B-taxonomy_domain
+titer	O
+,	O
+respectively	O
+,	O
+determined	O
+in	O
+2	O
+–	O
+3	O
+independent	O
+experiments	O
+.	O
+The	O
+cytotoxicity	O
+,	O
+assessed	O
+in	O
+uninfected	O
+MDCK	O
+cells	O
+,	O
+was	O
+expressed	O
+as	O
+the	O
+CC50	B-evidence
+value	O
+(	O
+50	O
+%	O
+cytotoxic	O
+concentration	O
+,	O
+determined	O
+with	O
+the	O
+MTS	B-experimental_method
+cell	I-experimental_method
+viability	I-experimental_method
+assay	I-experimental_method
+,	O
+in	O
+μM	O
+).	O
+cHEK293T	O
+cells	O
+were	O
+co	B-experimental_method
+-	I-experimental_method
+transfected	I-experimental_method
+with	O
+the	O
+four	O
+vRNP	B-complex_assembly
+-	O
+reconstituting	O
+plasmids	O
+and	O
+the	O
+luciferase	O
+reporter	O
+plasmid	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+the	O
+test	O
+compounds	O
+.	O
+The	O
+EC50	B-evidence
+represents	O
+the	O
+compound	O
+concentration	O
+(	O
+in	O
+μM	O
+)	O
+producing	O
+50	O
+%	O
+reduction	O
+in	O
+vRNP	B-complex_assembly
+-	O
+driven	O
+firefly	O
+reporter	O
+signal	O
+,	O
+estimated	O
+at	O
+24	O
+h	O
+after	O
+transfection	O
+.	O
+The	O
+EC50	B-evidence
+value	O
+was	O
+derived	O
+from	O
+data	O
+from	O
+2	O
+–	O
+4	O
+independent	O
+experiments	O
+,	O
+by	O
+nonlinear	B-experimental_method
+least	I-experimental_method
+-	I-experimental_method
+squares	I-experimental_method
+regression	I-experimental_method
+analysis	I-experimental_method
+(	O
+using	O
+GraphPad	O
+Prism	O
+software	O
+).	O
+The	O
+CC50	B-evidence
+(	O
+in	O
+μM	O
+),	O
+i	O
+.	O
+e	O
+.	O
+the	O
+50	O
+%	O
+cytotoxic	O
+concentration	O
+,	O
+was	O
+determined	O
+in	O
+untransfected	O
+HEK293T	O
+cells	O
+by	O
+MTS	B-experimental_method
+cell	I-experimental_method
+viability	I-experimental_method
+assay	I-experimental_method
+.	O
+dSI	B-evidence
+,	O
+selectivity	B-evidence
+index	I-evidence
+,	O
+defined	O
+as	O
+the	O
+ratio	O
+between	O
+the	O
+CC50	B-evidence
+and	O
+EC90	B-evidence
+.	O
+eDPBA	B-chemical
+,	O
+2	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+dioxo	I-chemical
+-	I-chemical
+4	I-chemical
+-	I-chemical
+phenylbutanoic	I-chemical
+acid	I-chemical
+.	O

annotation_IOB/PMC4981400.tsv ADDED Viewed

	@@ -0,0 +1,3348 @@

+Crystal	B-evidence
+Structure	I-evidence
+of	O
+the	O
+SPOC	B-structure_element
+Domain	O
+of	O
+the	O
+Arabidopsis	B-taxonomy_domain
+Flowering	B-protein_type
+Regulator	I-protein_type
+FPA	B-protein
+The	O
+Arabidopsis	B-taxonomy_domain
+protein	O
+FPA	B-protein
+controls	O
+flowering	O
+time	O
+by	O
+regulating	O
+the	O
+alternative	O
+3	O
+′-	O
+end	O
+processing	O
+of	O
+the	O
+FLOWERING	B-gene
+LOCUS	I-gene
+(	O
+FLC	B-gene
+)	O
+antisense	B-chemical
+RNA	I-chemical
+.	O
+FPA	B-protein
+belongs	O
+to	O
+the	O
+split	B-protein_type
+ends	I-protein_type
+(	O
+SPEN	B-protein_type
+)	O
+family	O
+of	O
+proteins	O
+,	O
+which	O
+contain	O
+N	O
+-	O
+terminal	O
+RNA	B-structure_element
+recognition	I-structure_element
+motifs	I-structure_element
+(	O
+RRMs	B-structure_element
+)	O
+and	O
+a	O
+SPEN	B-structure_element
+paralog	I-structure_element
+and	I-structure_element
+ortholog	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+(	O
+SPOC	B-structure_element
+)	O
+domain	O
+.	O
+The	O
+SPOC	B-structure_element
+domain	O
+is	O
+highly	B-protein_state
+conserved	I-protein_state
+among	O
+FPA	B-protein
+homologs	O
+in	O
+plants	B-taxonomy_domain
+,	O
+but	O
+the	O
+conservation	O
+with	O
+the	O
+domain	O
+in	O
+other	O
+SPEN	B-protein_type
+proteins	O
+is	O
+much	O
+lower	O
+.	O
+We	O
+have	O
+determined	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+Arabidopsis	B-species
+thaliana	I-species
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+at	O
+2	O
+.	O
+7	O
+Å	O
+resolution	O
+.	O
+The	O
+overall	O
+structure	B-evidence
+is	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+SPOC	B-structure_element
+domain	O
+in	O
+human	B-species
+SMRT	B-protein
+/	I-protein
+HDAC1	I-protein
+Associated	I-protein
+Repressor	I-protein
+Protein	I-protein
+(	O
+SHARP	B-protein
+),	O
+although	O
+there	O
+are	O
+also	O
+substantial	O
+conformational	O
+differences	O
+between	O
+them	O
+.	O
+Structural	B-experimental_method
+and	I-experimental_method
+sequence	I-experimental_method
+analyses	I-experimental_method
+identify	O
+a	O
+surface	B-site
+patch	I-site
+that	O
+is	O
+conserved	B-protein_state
+among	O
+plant	B-taxonomy_domain
+FPA	B-protein
+homologs	O
+.	O
+Mutations	B-experimental_method
+of	O
+two	O
+residues	O
+in	O
+this	O
+surface	B-site
+patch	I-site
+did	O
+not	O
+disrupt	O
+FPA	B-protein
+functions	O
+,	O
+suggesting	O
+that	O
+either	O
+the	O
+SPOC	B-structure_element
+domain	O
+is	O
+not	O
+required	O
+for	O
+the	O
+role	O
+of	O
+FPA	B-protein
+in	O
+regulating	O
+RNA	B-chemical
+3	O
+′-	O
+end	O
+formation	O
+or	O
+the	O
+functions	O
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+cannot	O
+be	O
+disrupted	O
+by	O
+the	O
+combination	O
+of	O
+mutations	O
+,	O
+in	O
+contrast	O
+to	O
+observations	O
+with	O
+the	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+.	O
+Eukaryotic	B-taxonomy_domain
+messenger	B-chemical
+RNAs	I-chemical
+(	O
+mRNAs	B-chemical
+)	O
+are	O
+made	O
+as	O
+precursors	O
+through	O
+transcription	O
+by	O
+RNA	B-complex_assembly
+polymerase	I-complex_assembly
+II	I-complex_assembly
+(	O
+Pol	B-complex_assembly
+II	I-complex_assembly
+),	O
+and	O
+these	O
+primary	O
+transcripts	O
+undergo	O
+extensive	O
+processing	O
+,	O
+including	O
+3	O
+′-	O
+end	O
+cleavage	O
+and	O
+polyadenylation	O
+.	O
+In	O
+addition	O
+,	O
+alternative	O
+3	O
+′-	O
+end	O
+cleavage	O
+and	O
+polyadenylation	O
+is	O
+an	O
+essential	O
+and	O
+ubiquitous	O
+process	O
+in	O
+eukaryotes	B-taxonomy_domain
+.	O
+Recently	O
+,	O
+the	O
+split	B-protein_type
+ends	I-protein_type
+(	O
+SPEN	B-protein_type
+)	O
+family	O
+of	O
+proteins	O
+was	O
+identified	O
+as	O
+RNA	B-protein_type
+binding	I-protein_type
+proteins	I-protein_type
+that	O
+regulate	O
+alternative	O
+3	O
+′-	O
+end	O
+cleavage	O
+and	O
+polyadenylation	O
+.	O
+They	O
+are	O
+characterized	O
+by	O
+possessing	O
+N	O
+-	O
+terminal	O
+RNA	B-structure_element
+recognition	I-structure_element
+motifs	I-structure_element
+(	O
+RRMs	B-structure_element
+)	O
+and	O
+a	O
+conserved	B-protein_state
+SPEN	B-structure_element
+paralog	I-structure_element
+and	I-structure_element
+ortholog	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+(	O
+SPOC	B-structure_element
+)	O
+domain	O
+(	O
+Fig	O
+1A	O
+).	O
+The	O
+SPOC	B-structure_element
+domain	O
+is	O
+believed	O
+to	O
+mediate	O
+protein	O
+-	O
+protein	O
+interactions	O
+and	O
+has	O
+diverse	O
+functions	O
+among	O
+SPEN	B-protein_type
+family	O
+proteins	O
+,	O
+but	O
+the	O
+molecular	O
+mechanism	O
+of	O
+these	O
+functions	O
+is	O
+not	O
+well	O
+understood	O
+.	O
+Sequence	B-evidence
+conservation	I-evidence
+of	O
+SPOC	B-structure_element
+domains	O
+.	O
+Domain	O
+organization	O
+of	O
+A	B-species
+.	I-species
+thaliana	I-species
+FPA	B-protein
+.	O
+(	O
+B	O
+).	O
+Sequence	B-experimental_method
+alignment	I-experimental_method
+of	O
+the	O
+SPOC	B-structure_element
+domains	O
+of	O
+Arabidopsis	B-species
+thaliana	I-species
+FPA	B-protein
+,	O
+human	B-species
+RBM15	B-protein
+,	O
+Drosophila	B-taxonomy_domain
+SPEN	B-protein_type
+,	O
+mouse	B-taxonomy_domain
+MINT	B-protein
+,	O
+and	O
+human	B-species
+SHARP	B-protein
+.	O
+Residues	O
+in	O
+surface	B-site
+patch	I-site
+1	I-site
+are	O
+indicated	O
+with	O
+the	O
+orange	O
+dots	O
+,	O
+and	O
+those	O
+in	O
+surface	B-site
+patch	I-site
+2	I-site
+with	O
+the	O
+green	O
+dots	O
+.	O
+The	O
+secondary	O
+structure	O
+elements	O
+in	O
+the	O
+structure	B-evidence
+of	O
+FPA	B-protein
+SPOC	B-structure_element
+are	O
+labeled	O
+.	O
+Residues	O
+that	O
+are	O
+strictly	B-protein_state
+conserved	I-protein_state
+among	O
+the	O
+five	O
+proteins	O
+are	O
+shown	O
+in	O
+white	O
+with	O
+a	O
+red	O
+background	O
+,	O
+and	O
+those	O
+that	O
+are	O
+mostly	B-protein_state
+conserved	I-protein_state
+in	O
+red	O
+.	O
+FPA	B-protein
+,	O
+a	O
+SPEN	B-protein_type
+family	O
+protein	O
+in	O
+Arabidopsis	B-species
+thaliana	I-species
+and	O
+other	O
+plants	B-taxonomy_domain
+,	O
+was	O
+found	O
+to	O
+regulate	O
+the	O
+3	O
+′-	O
+end	O
+alternative	O
+cleavage	O
+and	O
+polyadenylation	O
+of	O
+the	O
+antisense	B-chemical
+RNAs	I-chemical
+of	O
+FLOWERING	B-gene
+LOCUS	I-gene
+(	O
+FLC	B-gene
+),	O
+a	O
+flowering	O
+repressor	O
+gene	O
+.	O
+FPA	B-protein
+promotes	O
+the	O
+3	O
+′-	O
+end	O
+processing	O
+of	O
+class	O
+I	O
+FLC	B-gene
+antisense	B-chemical
+RNAs	I-chemical
+,	O
+which	O
+includes	O
+the	O
+proximal	O
+polyadenylation	B-site
+site	I-site
+.	O
+This	O
+is	O
+associated	O
+with	O
+histone	B-protein_type
+demethylase	I-protein_type
+activity	O
+and	O
+down	O
+-	O
+regulation	O
+of	O
+FLC	B-gene
+transcription	O
+.	O
+Although	O
+a	O
+SPOC	B-structure_element
+domain	O
+is	O
+found	O
+in	O
+all	O
+the	O
+SPEN	B-protein_type
+family	O
+proteins	O
+,	O
+its	O
+sequence	O
+conservation	O
+is	O
+rather	O
+low	O
+.	O
+For	O
+example	O
+,	O
+the	O
+sequence	O
+identity	O
+between	O
+the	O
+SPOC	B-structure_element
+domains	O
+of	O
+A	B-species
+.	I-species
+thaliana	I-species
+FPA	B-protein
+and	O
+human	B-species
+SMRT	B-protein
+/	I-protein
+HDAC1	I-protein
+Associated	I-protein
+Repressor	I-protein
+Protein	I-protein
+(	O
+SHARP	B-protein
+)	O
+is	O
+only	O
+19	O
+%	O
+(	O
+Fig	O
+1B	O
+).	O
+Currently	O
+,	O
+the	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+is	O
+the	O
+only	O
+one	O
+with	O
+structural	O
+information	O
+.	O
+As	O
+a	O
+first	O
+step	O
+toward	O
+understanding	O
+the	O
+molecular	O
+basis	O
+for	O
+the	O
+regulation	O
+of	O
+alternative	O
+3	O
+′-	O
+end	O
+processing	O
+and	O
+flowering	O
+by	O
+FPA	B-protein
+,	O
+we	O
+have	O
+determined	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+SPOC	B-structure_element
+domain	O
+of	O
+A	B-species
+.	I-species
+thaliana	I-species
+FPA	B-protein
+at	O
+2	O
+.	O
+7	O
+Å	O
+resolution	O
+.	O
+The	O
+overall	O
+structure	B-evidence
+is	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+,	O
+although	O
+there	O
+are	O
+also	O
+substantial	O
+conformational	O
+differences	O
+between	O
+them	O
+.	O
+The	O
+structure	B-evidence
+reveals	O
+a	O
+surface	B-site
+patch	I-site
+that	O
+is	O
+conserved	B-protein_state
+among	O
+FPA	B-protein
+homologs	O
+.	O
+Structure	B-evidence
+of	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+SPOC	B-structure_element
+domain	O
+of	O
+A	B-species
+.	I-species
+thaliana	I-species
+FPA	B-protein
+has	O
+been	O
+determined	O
+at	O
+2	O
+.	O
+7	O
+Å	O
+resolution	O
+using	O
+the	O
+selenomethionyl	B-experimental_method
+single	I-experimental_method
+-	I-experimental_method
+wavelength	I-experimental_method
+anomalous	I-experimental_method
+dispersion	I-experimental_method
+method	I-experimental_method
+.	O
+The	O
+expression	O
+construct	O
+contained	O
+residues	O
+433	B-residue_range
+–	I-residue_range
+565	I-residue_range
+of	O
+FPA	B-protein
+,	O
+but	O
+only	O
+residues	O
+439	B-residue_range
+–	I-residue_range
+460	I-residue_range
+and	O
+465	B-residue_range
+–	I-residue_range
+565	I-residue_range
+are	O
+ordered	O
+in	O
+the	O
+crystal	B-evidence
+.	O
+The	O
+atomic	B-evidence
+model	I-evidence
+has	O
+good	O
+agreement	O
+with	O
+the	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+diffraction	I-evidence
+data	I-evidence
+and	O
+the	O
+expected	O
+bond	O
+lengths	O
+,	O
+bond	O
+angles	O
+and	O
+other	O
+geometric	O
+parameters	O
+(	O
+Table	O
+1	O
+).	O
+All	O
+the	O
+residues	O
+are	O
+located	O
+in	O
+the	O
+favored	O
+regions	O
+of	O
+the	O
+Ramachandran	B-evidence
+plot	I-evidence
+(	O
+data	O
+not	O
+shown	O
+).	O
+The	O
+structure	B-evidence
+has	O
+been	O
+deposited	O
+in	O
+the	O
+Protein	O
+Data	O
+Bank	O
+,	O
+with	O
+accession	O
+code	O
+5KXF	O
+.	O
+Resolution	O
+range	O
+(	O
+Å	O
+)	O
+1	O
+50	O
+–	O
+2	O
+.	O
+7	O
+(	O
+2	O
+.	O
+8	O
+–	O
+2	O
+.	O
+7	O
+)	O
+Number	O
+of	O
+observations	O
+78	O
+,	O
+008	O
+Rmerge	O
+(%)	O
+10	O
+.	O
+5	O
+(	O
+45	O
+.	O
+3	O
+)	O
+I	O
+/	O
+σI	O
+24	O
+.	O
+1	O
+(	O
+6	O
+.	O
+3	O
+)	O
+Redundancy	O
+Completeness	O
+(%)	O
+100	O
+(	O
+100	O
+)	O
+R	B-evidence
+factor	I-evidence
+(%)	O
+19	O
+.	O
+2	O
+(	O
+25	O
+.	O
+0	O
+)	O
+Free	B-evidence
+R	I-evidence
+factor	I-evidence
+(%)	O
+25	O
+.	O
+4	O
+(	O
+35	O
+.	O
+4	O
+)	O
+Rms	O
+deviation	O
+in	O
+bond	O
+lengths	O
+(	O
+Å	O
+)	O
+0	O
+.	O
+017	O
+Rms	O
+deviation	O
+in	O
+bond	O
+angles	O
+(°)	O
+1	O
+.	O
+9	O
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+contains	O
+a	O
+seven	B-structure_element
+-	I-structure_element
+stranded	I-structure_element
+,	I-structure_element
+mostly	I-structure_element
+anti	I-structure_element
+-	I-structure_element
+parallel	I-structure_element
+β	I-structure_element
+-	I-structure_element
+barrel	I-structure_element
+(	O
+β1	B-structure_element
+-	I-structure_element
+β7	I-structure_element
+)	O
+and	O
+three	O
+helices	B-structure_element
+(	O
+αA	B-structure_element
+-	I-structure_element
+αC	I-structure_element
+)	O
+(	O
+Fig	O
+2A	O
+).	O
+Only	O
+two	O
+of	O
+the	O
+neighboring	O
+strands	B-structure_element
+,	O
+β1	B-structure_element
+and	O
+β3	B-structure_element
+,	O
+are	O
+parallel	O
+to	O
+each	O
+other	O
+.	O
+Helix	B-structure_element
+αB	B-structure_element
+covers	O
+one	O
+end	O
+of	O
+the	O
+barrel	B-structure_element
+,	O
+while	O
+helices	B-structure_element
+αA	B-structure_element
+and	O
+αC	B-structure_element
+are	O
+located	O
+next	O
+to	O
+each	O
+other	O
+at	O
+one	O
+side	O
+of	O
+the	O
+barrel	B-structure_element
+(	O
+Fig	O
+2B	O
+).	O
+The	O
+other	O
+end	O
+of	O
+the	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+is	O
+covered	O
+by	O
+the	O
+loop	B-structure_element
+connecting	O
+strands	B-structure_element
+β2	B-structure_element
+and	O
+β3	B-structure_element
+,	O
+which	O
+contains	O
+the	O
+disordered	B-protein_state
+461	B-residue_range
+–	I-residue_range
+464	I-residue_range
+segment	O
+.	O
+The	O
+center	O
+of	O
+the	O
+barrel	B-structure_element
+is	O
+filled	O
+with	O
+hydrophobic	O
+side	O
+chains	O
+and	O
+is	O
+not	O
+accessible	O
+to	O
+the	O
+solvent	O
+.	O
+Crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+SPOC	B-structure_element
+domain	O
+of	O
+A	B-species
+.	I-species
+thaliana	I-species
+FPA	B-protein
+.	O
+Schematic	O
+drawing	O
+of	O
+the	O
+structure	B-evidence
+of	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+,	O
+colored	O
+from	O
+blue	O
+at	O
+the	O
+N	O
+terminus	O
+to	O
+red	O
+at	O
+the	O
+C	O
+terminus	O
+.	O
+The	O
+view	O
+is	O
+from	O
+the	O
+side	O
+of	O
+the	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+.	O
+The	O
+disordered	B-protein_state
+segment	O
+(	O
+residues	O
+460	B-residue_range
+–	I-residue_range
+465	I-residue_range
+)	O
+is	O
+indicated	O
+with	O
+the	O
+dotted	O
+line	O
+.	O
+Structure	B-evidence
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+,	O
+viewed	O
+from	O
+the	O
+end	O
+of	O
+the	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+,	O
+after	O
+90	O
+°	O
+rotation	O
+around	O
+the	O
+horizontal	O
+axis	O
+from	O
+panel	O
+A	O
+.	O
+All	O
+structure	O
+figures	O
+were	O
+produced	O
+with	O
+PyMOL	O
+(	O
+www	O
+.	O
+pymol	O
+.	O
+org	O
+).	O
+Comparisons	B-experimental_method
+to	I-experimental_method
+structural	I-experimental_method
+homologs	I-experimental_method
+of	O
+the	O
+SPOC	B-structure_element
+domain	O
+Only	O
+five	O
+structural	O
+homologs	O
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+were	O
+found	O
+in	O
+the	O
+Protein	O
+Data	O
+Bank	O
+with	O
+the	O
+DaliLite	B-experimental_method
+server	I-experimental_method
+,	O
+suggesting	O
+that	O
+the	O
+SPOC	B-structure_element
+domain	O
+structure	B-evidence
+is	O
+relatively	O
+unique	O
+.	O
+The	O
+top	O
+hit	O
+is	O
+the	O
+SPOC	B-structure_element
+domain	O
+of	O
+human	B-species
+SHARP	B-protein
+(	O
+Fig	O
+3A	O
+),	O
+with	O
+a	O
+Z	B-evidence
+score	I-evidence
+of	O
+12	O
+.	O
+3	O
+.	O
+The	O
+other	O
+four	O
+structural	O
+homologs	O
+include	O
+the	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+domain	O
+of	O
+the	O
+proteins	O
+Ku70	B-protein
+and	O
+Ku80	B-protein
+(	O
+Z	B-evidence
+score	I-evidence
+11	O
+.	O
+4	O
+)	O
+(	O
+Fig	O
+3B	O
+),	O
+a	O
+domain	O
+in	O
+the	O
+chromodomain	B-protein_type
+protein	I-protein_type
+Chp1	B-protein
+(	O
+Z	B-evidence
+score	I-evidence
+10	O
+.	O
+8	O
+)	O
+(	O
+Fig	O
+3C	O
+),	O
+and	O
+the	O
+activator	B-structure_element
+interacting	I-structure_element
+domain	I-structure_element
+(	O
+ACID	B-structure_element
+)	O
+of	O
+the	O
+Med25	B-protein
+subunit	O
+of	O
+the	O
+Mediator	O
+complex	O
+(	O
+Z	B-evidence
+score	I-evidence
+8	O
+.	O
+5	O
+)	O
+(	O
+Fig	O
+3D	O
+).	O
+The	O
+next	O
+structural	O
+homolog	O
+has	O
+a	O
+Z	B-evidence
+score	I-evidence
+of	O
+3	O
+.	O
+0	O
+.	O
+Structural	O
+homologs	O
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+.	O
+Overlay	B-experimental_method
+of	O
+the	O
+structures	B-evidence
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+(	O
+cyan	O
+)	O
+and	O
+the	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+(	O
+gray	O
+).	O
+The	O
+bound	O
+position	O
+of	O
+a	O
+doubly	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+peptide	B-chemical
+from	O
+SMRT	B-protein
+is	O
+shown	O
+in	O
+magenta	O
+.	O
+Overlay	B-experimental_method
+of	O
+the	O
+structures	B-evidence
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+(	O
+cyan	O
+)	O
+and	O
+the	O
+Ku70	B-protein
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+domain	O
+(	O
+gray	O
+).	O
+Ku80	B-protein
+contains	O
+a	O
+homologous	O
+domain	O
+(	O
+green	O
+),	O
+which	O
+forms	O
+a	O
+hetero	B-oligomeric_state
+-	I-oligomeric_state
+dimer	I-oligomeric_state
+with	O
+that	O
+in	O
+Ku70	B-protein
+.	O
+The	O
+two	O
+domains	O
+,	O
+and	O
+inserted	O
+segments	O
+on	O
+them	O
+,	O
+mediate	O
+the	O
+binding	O
+of	O
+dsDNA	B-chemical
+(	O
+orange	O
+).	O
+The	O
+red	O
+rectangle	O
+highlights	O
+the	O
+region	O
+of	O
+contact	O
+between	O
+the	O
+two	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+domains	O
+.	O
+Overlay	B-experimental_method
+of	O
+the	O
+structures	B-evidence
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+(	O
+cyan	O
+)	O
+and	O
+the	O
+homologous	O
+domain	O
+in	O
+Chp1	B-protein
+(	O
+gray	O
+).	O
+The	O
+binding	O
+partner	O
+of	O
+Chp1	B-protein
+,	O
+Tas3	B-protein
+,	O
+is	O
+shown	O
+in	O
+green	O
+.	O
+The	O
+red	O
+rectangle	O
+indicates	O
+the	O
+region	O
+equivalent	O
+to	O
+the	O
+binding	B-site
+site	I-site
+of	O
+the	O
+SMART	B-protein
+phosphopeptide	B-ptm
+in	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+,	O
+where	O
+a	O
+loop	B-structure_element
+of	O
+Tas3	B-protein
+is	O
+also	O
+located	O
+.	O
+(	O
+D	O
+).	O
+Overlay	B-experimental_method
+of	O
+the	O
+structures	B-evidence
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+(	O
+cyan	O
+)	O
+and	O
+the	O
+Med25	B-protein
+ACID	B-structure_element
+(	O
+gray	O
+).	O
+SHARP	B-protein
+is	O
+a	O
+transcriptional	B-protein_type
+co	I-protein_type
+-	I-protein_type
+repressor	I-protein_type
+in	O
+the	O
+nuclear	B-protein_type
+receptor	I-protein_type
+and	O
+Notch	B-protein
+/	O
+RBP	B-protein
+-	I-protein
+Jκ	I-protein
+signaling	O
+pathways	O
+.	O
+The	O
+SPOC	B-structure_element
+domain	O
+of	O
+SHARP	B-protein
+interacts	O
+directly	O
+with	O
+silencing	B-protein
+mediator	I-protein
+for	I-protein
+retinoid	I-protein
+and	I-protein
+thyroid	I-protein
+receptor	I-protein
+(	O
+SMRT	B-protein
+),	O
+nuclear	B-protein_type
+receptor	I-protein_type
+co	I-protein_type
+-	I-protein_type
+repressor	I-protein_type
+(	O
+N	B-protein_type
+-	I-protein_type
+CoR	I-protein_type
+),	O
+HDAC	B-protein
+,	O
+and	O
+other	O
+components	O
+to	O
+represses	O
+transcription	O
+.	O
+While	O
+the	O
+overall	O
+structure	B-evidence
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+is	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+,	O
+there	O
+are	O
+noticeable	O
+differences	O
+in	O
+the	O
+positioning	O
+of	O
+the	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+and	O
+the	O
+helices	B-structure_element
+,	O
+and	O
+most	O
+of	O
+the	O
+loops	B-structure_element
+have	O
+substantially	O
+different	O
+conformations	O
+as	O
+well	O
+(	O
+Fig	O
+3A	O
+).	O
+In	O
+addition	O
+,	O
+the	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+has	O
+three	O
+extra	O
+helices	B-structure_element
+.	O
+One	O
+of	O
+them	O
+covers	O
+the	O
+other	O
+end	O
+of	O
+the	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+,	O
+and	O
+the	O
+other	O
+two	O
+shield	O
+an	O
+additional	O
+surface	O
+of	O
+the	O
+side	O
+of	O
+the	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+from	O
+solvent	O
+.	O
+A	O
+doubly	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+peptide	B-chemical
+from	O
+SMRT	B-protein
+is	O
+bound	B-protein_state
+to	I-protein_state
+the	O
+side	O
+of	O
+the	O
+barrel	B-structure_element
+,	O
+near	O
+strands	B-structure_element
+β1	B-structure_element
+and	O
+β3	B-structure_element
+(	O
+Fig	O
+3A	O
+).	O
+Such	O
+a	O
+binding	O
+mode	O
+probably	O
+would	O
+not	O
+be	O
+possible	O
+in	O
+FPA	B-protein
+,	O
+as	O
+the	O
+peptide	B-chemical
+would	O
+clash	O
+with	O
+the	O
+β1	B-structure_element
+-	I-structure_element
+β2	I-structure_element
+loop	I-structure_element
+.	O
+The	O
+Ku70	B-complex_assembly
+-	I-complex_assembly
+Ku80	I-complex_assembly
+hetero	B-oligomeric_state
+-	I-oligomeric_state
+dimer	I-oligomeric_state
+is	O
+involved	O
+in	O
+DNA	O
+double	O
+-	O
+strand	O
+break	O
+repair	O
+and	O
+the	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+domain	O
+contributes	O
+to	O
+DNA	B-chemical
+binding	O
+.	O
+In	O
+fact	O
+,	O
+the	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+domains	O
+of	O
+Ku70	B-protein
+and	O
+Ku80	B-protein
+form	O
+a	O
+hetero	B-oligomeric_state
+-	I-oligomeric_state
+dimer	I-oligomeric_state
+,	O
+primarily	O
+through	O
+interactions	O
+between	O
+the	O
+loops	B-structure_element
+connecting	O
+the	O
+third	B-structure_element
+and	I-structure_element
+fourth	I-structure_element
+strands	I-structure_element
+of	O
+the	O
+barrel	B-structure_element
+(	O
+Fig	O
+3B	O
+).	O
+The	O
+open	O
+ends	O
+of	O
+the	O
+two	O
+β	B-structure_element
+-	I-structure_element
+barrels	I-structure_element
+face	O
+the	O
+DNA	B-site
+binding	I-site
+sites	I-site
+,	O
+and	O
+contact	O
+the	O
+phosphodiester	O
+backbone	O
+of	O
+the	O
+dsDNA	B-chemical
+.	O
+In	O
+addition	O
+,	O
+a	O
+long	B-structure_element
+insert	I-structure_element
+connecting	O
+strands	B-structure_element
+β2	B-structure_element
+and	O
+β3	B-structure_element
+in	O
+the	O
+two	O
+domains	O
+form	O
+an	O
+arch	B-structure_element
+-	I-structure_element
+like	I-structure_element
+structure	I-structure_element
+,	O
+encircling	O
+the	O
+dsDNA	B-chemical
+.	O
+Chp1	B-protein
+is	O
+a	O
+subunit	O
+of	O
+the	O
+RNA	B-complex_assembly
+-	I-complex_assembly
+induced	I-complex_assembly
+initiation	I-complex_assembly
+of	I-complex_assembly
+transcriptional	I-complex_assembly
+gene	I-complex_assembly
+silencing	I-complex_assembly
+(	O
+RITS	B-complex_assembly
+)	O
+complex	O
+.	O
+The	O
+partner	O
+of	O
+Chp1	B-protein
+,	O
+Tas3	B-protein
+,	O
+is	O
+bound	O
+between	O
+the	O
+barrel	B-structure_element
+domain	I-structure_element
+and	O
+the	O
+second	B-structure_element
+domain	I-structure_element
+of	O
+Chp1	B-protein
+,	O
+and	O
+the	O
+linker	B-structure_element
+between	O
+the	O
+two	O
+domains	O
+is	O
+also	O
+crucial	O
+for	O
+this	O
+interaction	O
+(	O
+Fig	O
+3C	O
+).	O
+It	O
+is	O
+probably	O
+unlikely	O
+that	O
+the	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+itself	O
+is	O
+sufficient	O
+to	O
+bind	O
+Tas3	B-protein
+.	O
+Interestingly	O
+,	O
+a	O
+loop	B-structure_element
+in	O
+Tas3	B-protein
+contacts	O
+strand	B-structure_element
+β3	B-structure_element
+of	O
+the	O
+barrel	B-structure_element
+domain	I-structure_element
+,	O
+at	O
+a	O
+location	O
+somewhat	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+N	O
+-	O
+terminal	O
+segment	O
+of	O
+the	O
+SMRT	B-protein
+peptide	B-chemical
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+(	O
+Fig	O
+3A	O
+).	O
+Mediator	B-protein_type
+is	O
+a	O
+coactivator	O
+complex	O
+that	O
+promotes	O
+transcription	O
+by	O
+Pol	B-complex_assembly
+II	I-complex_assembly
+.	O
+The	O
+Med25	B-protein
+subunit	O
+ACID	B-structure_element
+is	O
+the	O
+target	O
+of	O
+the	O
+potent	O
+activator	O
+VP16	B-protein
+of	O
+the	O
+herpes	B-species
+simplex	I-species
+virus	I-species
+.	O
+The	O
+structure	B-evidence
+of	O
+ACID	B-structure_element
+contains	O
+a	O
+helix	B-structure_element
+at	O
+the	O
+C	O
+-	O
+terminus	O
+as	O
+well	O
+as	O
+an	O
+extended	O
+β1	B-structure_element
+-	I-structure_element
+β2	I-structure_element
+loop	I-structure_element
+.	O
+Nonetheless	O
+,	O
+the	O
+binding	B-site
+site	I-site
+for	O
+VP16	B-protein
+has	O
+been	O
+mapped	O
+to	O
+roughly	O
+the	O
+same	O
+surface	B-site
+patch	I-site
+,	O
+near	O
+strands	B-structure_element
+β1	B-structure_element
+and	O
+β3	B-structure_element
+,	O
+that	O
+is	O
+used	O
+by	O
+the	O
+SHARP	B-protein
+and	O
+Tas3	B-protein
+SPOC	B-structure_element
+domains	O
+for	O
+binding	O
+their	O
+partners	O
+.	O
+A	O
+conserved	B-protein_state
+surface	B-site
+patch	I-site
+in	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+An	O
+analysis	O
+of	O
+the	O
+SPOC	B-structure_element
+domain	O
+indicates	O
+a	O
+large	O
+surface	B-site
+patch	I-site
+near	O
+strands	B-structure_element
+β1	B-structure_element
+,	O
+β3	B-structure_element
+,	O
+β5	B-structure_element
+and	O
+β6	B-structure_element
+that	O
+is	O
+conserved	B-protein_state
+among	O
+plant	B-taxonomy_domain
+FPA	B-protein
+homologs	O
+(	O
+Fig	O
+4A	O
+).	O
+This	O
+surface	B-site
+patch	I-site
+can	O
+be	O
+broken	O
+into	O
+two	O
+sub	B-site
+-	I-site
+patches	I-site
+,	O
+with	O
+residues	O
+Lys447	B-residue_name_number
+(	O
+in	O
+strand	B-structure_element
+β1	B-structure_element
+),	O
+Arg477	B-residue_name_number
+(	O
+β3	B-structure_element
+),	O
+Tyr515	B-residue_name_number
+(	O
+αB	B-structure_element
+)	O
+and	O
+Arg521	B-residue_name_number
+(	O
+β5	B-structure_element
+)	O
+in	O
+one	O
+sub	B-site
+-	I-site
+patch	I-site
+,	O
+and	O
+residues	O
+His486	B-residue_name_number
+(	O
+αA	B-structure_element
+),	O
+Thr478	B-residue_name_number
+(	O
+β3	B-structure_element
+),	O
+Val524	B-residue_name_number
+(	O
+β5	B-structure_element
+)	O
+and	O
+Phe534	B-residue_name_number
+(	O
+β6	B-structure_element
+)	O
+in	O
+the	O
+other	O
+sub	B-site
+-	I-site
+patch	I-site
+(	O
+Fig	O
+4B	O
+).	O
+The	O
+first	B-site
+surface	I-site
+patch	I-site
+is	O
+electropositive	B-protein_state
+in	O
+nature	O
+(	O
+Fig	O
+4C	O
+),	O
+and	O
+residues	O
+Arg477	B-residue_name_number
+and	O
+Tyr515	B-residue_name_number
+are	O
+also	O
+conserved	B-protein_state
+in	O
+the	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+(	O
+Fig	O
+1B	O
+).	O
+In	O
+fact	O
+,	O
+one	O
+of	O
+the	O
+phosphorylated	B-protein_state
+residues	O
+of	O
+the	O
+SMRT	B-protein
+peptide	B-chemical
+interacts	O
+with	O
+this	O
+surface	B-site
+patch	I-site
+(	O
+Fig	O
+3A	O
+),	O
+suggesting	O
+that	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+might	O
+also	O
+interact	O
+with	O
+a	O
+phosphorylated	B-protein_state
+segment	O
+here	O
+.	O
+In	O
+comparison	O
+,	O
+the	O
+second	B-site
+surface	I-site
+patch	I-site
+is	O
+more	O
+hydrophobic	B-protein_state
+in	O
+nature	O
+(	O
+Fig	O
+4C	O
+).	O
+A	O
+conserved	B-protein_state
+surface	B-site
+patch	I-site
+of	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+.	O
+Two	O
+views	O
+of	O
+the	O
+molecular	O
+surface	O
+of	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+colored	O
+based	O
+on	O
+sequence	O
+conservation	O
+among	O
+plant	B-taxonomy_domain
+FPA	B-protein
+homologs	O
+.	O
+Residues	O
+in	O
+the	O
+conserved	B-protein_state
+surface	B-site
+patch	I-site
+of	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+.	O
+The	O
+side	O
+chains	O
+of	O
+the	O
+residues	O
+are	O
+shown	O
+in	O
+stick	O
+models	O
+,	O
+colored	O
+orange	O
+in	O
+the	O
+first	B-site
+sub	I-site
+-	I-site
+patch	I-site
+and	O
+green	O
+in	O
+the	O
+second	O
+.	O
+(	O
+C	O
+).	O
+Molecular	O
+surface	O
+of	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+colored	O
+based	O
+on	O
+electrostatic	O
+potential	O
+.	O
+Testing	O
+the	O
+requirement	O
+of	O
+specific	O
+conserved	O
+amino	O
+acids	O
+for	O
+FPA	B-protein
+functions	O
+We	O
+next	O
+examined	O
+the	O
+potential	O
+impact	O
+of	O
+the	O
+conserved	B-protein_state
+surface	B-site
+patch	I-site
+on	O
+FPA	B-protein
+function	O
+in	O
+vivo	O
+.	O
+We	O
+mutated	B-experimental_method
+two	O
+residues	O
+,	O
+Arg477	B-residue_name_number
+and	O
+Tyr515	B-residue_name_number
+,	O
+of	O
+the	O
+surface	B-site
+patch	I-site
+,	O
+which	O
+are	O
+also	O
+conserved	B-protein_state
+in	O
+the	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+(	O
+Fig	O
+1B	O
+)	O
+and	O
+were	O
+found	O
+to	O
+be	O
+functionally	O
+important	O
+.	O
+The	O
+mutations	B-experimental_method
+were	O
+introduced	B-experimental_method
+into	O
+a	O
+transgene	O
+designed	O
+to	O
+express	O
+FPA	B-protein
+from	O
+its	O
+native	O
+control	O
+elements	O
+(	O
+promoter	O
+,	O
+introns	O
+and	O
+3	O
+′	O
+UTR	O
+).	O
+The	O
+resulting	O
+transgenes	O
+were	O
+then	O
+stably	B-experimental_method
+transformed	I-experimental_method
+into	O
+an	O
+fpa	B-gene
+-	I-gene
+8	I-gene
+mutant	B-protein_state
+background	O
+so	O
+that	O
+the	O
+impact	O
+of	O
+the	O
+mutations	B-experimental_method
+on	O
+FPA	B-protein
+function	O
+could	O
+be	O
+assessed	O
+.	O
+Control	O
+transformation	O
+of	O
+the	O
+same	O
+expression	B-experimental_method
+constructs	I-experimental_method
+into	O
+fpa	B-gene
+-	I-gene
+8	I-gene
+designed	O
+to	O
+express	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+FPA	B-protein
+protein	O
+restored	O
+FPA	B-protein
+protein	O
+expression	B-evidence
+levels	I-evidence
+to	O
+near	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+levels	O
+(	O
+panel	O
+A	O
+in	O
+S1	O
+Fig	O
+)	O
+and	O
+rescued	O
+the	O
+function	O
+of	O
+FPA	B-protein
+in	O
+controlling	O
+RNA	B-chemical
+3	O
+′-	O
+end	O
+formation	O
+,	O
+for	O
+example	O
+in	O
+FPA	B-protein
+pre	B-chemical
+-	I-chemical
+mRNA	I-chemical
+(	O
+panel	O
+B	O
+in	O
+S1	O
+Fig	O
+).	O
+We	O
+examined	O
+independent	O
+transgenic	O
+lines	O
+expressing	O
+each	O
+R477A	B-mutant
+and	O
+Y515A	B-mutant
+mutation	B-experimental_method
+.	O
+In	O
+each	O
+case	O
+,	O
+we	O
+confirmed	O
+that	O
+detectable	O
+levels	O
+of	O
+FPA	B-protein
+protein	O
+expression	O
+were	O
+restored	O
+close	O
+to	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+levels	O
+in	O
+protein	B-experimental_method
+blot	I-experimental_method
+analyses	O
+using	O
+antibodies	O
+that	O
+specifically	O
+recognize	O
+FPA	B-protein
+(	O
+S2	O
+Fig	O
+).	O
+We	O
+then	O
+examined	O
+the	O
+impact	O
+of	O
+the	O
+surface	B-site
+patch	I-site
+mutations	B-experimental_method
+on	O
+FPA	B-protein
+’	O
+s	O
+function	O
+in	O
+controlling	O
+RNA	O
+3	O
+′-	O
+end	O
+formation	O
+by	O
+determining	O
+whether	O
+the	O
+mutant	B-protein_state
+proteins	O
+functioned	O
+in	O
+FPA	B-protein
+autoregulation	O
+and	O
+the	O
+repression	O
+of	O
+FLC	B-gene
+expression	O
+.	O
+FPA	B-protein
+autoregulates	O
+its	O
+expression	O
+by	O
+promoting	O
+cleavage	O
+and	O
+polyadenylation	O
+within	O
+intron	O
+1	O
+of	O
+its	O
+own	O
+pre	B-chemical
+-	I-chemical
+mRNA	I-chemical
+,	O
+resulting	O
+in	O
+a	O
+truncated	O
+transcript	O
+that	O
+does	O
+not	O
+encode	O
+functional	O
+protein	O
+.	O
+We	O
+used	O
+RNA	B-experimental_method
+gel	I-experimental_method
+blot	I-experimental_method
+analyses	I-experimental_method
+to	O
+reveal	O
+that	O
+in	O
+each	O
+of	O
+three	O
+independent	O
+transgenic	O
+lines	O
+for	O
+each	O
+single	O
+mutant	B-protein_state
+,	O
+rescue	O
+of	O
+proximally	O
+polyadenylated	O
+FPA	B-protein
+pre	B-chemical
+-	I-chemical
+mRNA	I-chemical
+can	O
+be	O
+detected	O
+(	O
+Fig	O
+5A	O
+and	O
+5B	O
+).	O
+We	O
+therefore	O
+conclude	O
+that	O
+neither	O
+of	O
+these	O
+mutations	O
+disrupted	O
+the	O
+ability	O
+of	O
+FPA	B-protein
+to	O
+promote	O
+RNA	O
+3	O
+′-	O
+end	O
+formation	O
+in	O
+its	O
+own	O
+transcript	O
+.	O
+Impact	O
+of	O
+individual	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+mutations	B-experimental_method
+on	O
+alternative	O
+polyadenylation	O
+of	O
+FPA	B-protein
+pre	B-chemical
+-	I-chemical
+mRNA	I-chemical
+.	O
+RNA	B-experimental_method
+gel	I-experimental_method
+blot	I-experimental_method
+analysis	O
+of	O
+WT	B-protein_state
+A	B-species
+.	I-species
+thaliana	I-species
+accession	O
+Columbia	O
+(	O
+Col	O
+-	O
+0	O
+)	O
+plants	B-taxonomy_domain
+fpa	B-gene
+-	I-gene
+8	I-gene
+and	O
+fpa	B-gene
+-	I-gene
+8	I-gene
+mutants	B-protein_state
+expressing	O
+either	O
+FPA	B-protein
+::	O
+FPA	B-mutant
+R477A	I-mutant
+(	O
+A	O
+),	O
+or	O
+FPA	B-protein
+::	O
+FPA	B-mutant
+Y515A	I-mutant
+(	O
+B	O
+)	O
+using	O
+poly	O
+(	O
+A	O
+)+	O
+purified	O
+mRNAs	B-chemical
+.	O
+A	O
+probe	O
+corresponding	O
+to	O
+the	O
+5	O
+’	O
+UTR	O
+region	O
+of	O
+FPA	B-protein
+mRNA	B-chemical
+was	O
+used	O
+to	O
+detect	O
+FPA	B-protein
+specific	O
+mRNAs	B-chemical
+.	O
+Proximally	O
+and	O
+distally	O
+polyadenylated	O
+FPA	B-protein
+transcripts	O
+are	O
+marked	O
+with	O
+arrows	O
+.	O
+The	O
+ratio	O
+of	O
+distal	O
+:	O
+proximal	O
+polyadenylated	O
+forms	O
+is	O
+given	O
+under	O
+each	O
+lane	O
+.	O
+(	O
+C	O
+,	O
+D	O
+)	O
+Impact	O
+of	O
+individual	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+mutations	B-experimental_method
+on	O
+FLC	B-gene
+transcript	O
+levels	O
+.	O
+qRT	B-experimental_method
+-	I-experimental_method
+PCR	I-experimental_method
+analysis	O
+was	O
+performed	O
+with	O
+total	O
+RNA	B-chemical
+purified	O
+from	O
+Col	O
+-	O
+0	O
+,	O
+fpa	B-gene
+-	I-gene
+8	I-gene
+,	O
+35S	O
+::	O
+FPA	B-protein
+:	O
+YFP	B-experimental_method
+and	O
+FPA	B-protein
+::	O
+FPA	B-mutant
+R477A	I-mutant
+(	O
+C	O
+),	O
+FPA	B-protein
+::	O
+FPA	B-mutant
+Y515A	I-mutant
+(	O
+D	O
+)	O
+plants	B-taxonomy_domain
+.	O
+Histograms	B-evidence
+show	O
+mean	O
+values	O
+±	O
+SE	O
+for	O
+three	O
+independent	O
+PCR	B-experimental_method
+amplifications	O
+of	O
+three	O
+biological	O
+replicates	O
+.	O
+We	O
+next	O
+examined	O
+whether	O
+the	O
+corresponding	O
+mutations	O
+disrupted	O
+the	O
+ability	O
+of	O
+FPA	B-protein
+to	O
+control	O
+FLC	B-gene
+expression	O
+.	O
+We	O
+used	O
+RT	B-experimental_method
+-	I-experimental_method
+qPCR	I-experimental_method
+to	O
+measure	O
+the	O
+expression	O
+of	O
+FLC	B-gene
+mRNA	B-chemical
+and	O
+found	O
+that	O
+in	O
+each	O
+independent	O
+transgenic	O
+line	O
+encoding	O
+each	O
+mutated	B-protein_state
+FPA	B-protein
+protein	O
+,	O
+the	O
+elevated	O
+levels	O
+of	O
+FLC	B-gene
+detected	O
+in	O
+fpa	B-gene
+-	I-gene
+8	I-gene
+mutants	B-protein_state
+were	O
+restored	O
+to	O
+near	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+levels	O
+by	O
+expression	O
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+conserved	B-protein_state
+patch	B-site
+mutant	B-protein_state
+proteins	O
+(	O
+Fig	O
+5C	O
+and	O
+5D	O
+).	O
+Since	O
+each	O
+surface	B-site
+patch	I-site
+mutation	B-experimental_method
+appeared	O
+to	O
+be	O
+insufficient	O
+to	O
+disrupt	O
+FPA	B-protein
+functions	O
+on	O
+its	O
+own	O
+,	O
+we	O
+combined	O
+both	O
+mutations	O
+into	O
+the	O
+same	O
+transgene	O
+.	O
+We	O
+could	O
+again	O
+confirm	O
+that	O
+near	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+levels	O
+of	O
+FPA	B-protein
+protein	O
+were	O
+expressed	O
+from	O
+three	O
+independent	O
+transgenic	O
+lines	O
+expressing	O
+the	O
+FPA	B-mutant
+R477A	I-mutant
+;	I-mutant
+Y515A	I-mutant
+doubly	B-protein_state
+mutated	I-protein_state
+protein	O
+in	O
+an	O
+fpa	B-gene
+-	I-gene
+8	I-gene
+mutant	B-protein_state
+background	O
+(	O
+S3	O
+Fig	O
+).	O
+We	O
+found	O
+that	O
+FPA	B-mutant
+R477A	I-mutant
+;	I-mutant
+Y515A	I-mutant
+protein	O
+functioned	O
+like	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+FPA	B-protein
+to	O
+restore	O
+FPA	B-protein
+pre	B-chemical
+-	I-chemical
+mRNA	I-chemical
+proximal	O
+polyadenylation	O
+(	O
+Fig	O
+6A	O
+)	O
+and	O
+FLC	B-gene
+expression	O
+to	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+levels	O
+(	O
+Fig	O
+6B	O
+).	O
+Impact	O
+of	O
+double	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+mutations	B-experimental_method
+on	O
+alternative	O
+polyadenylation	O
+of	O
+FPA	B-protein
+pre	B-chemical
+-	I-chemical
+mRNA	I-chemical
+and	O
+FLC	B-gene
+expression	O
+.	O
+(	O
+A	O
+)	O
+RNA	B-experimental_method
+gel	I-experimental_method
+blot	I-experimental_method
+analysis	O
+of	O
+WT	B-protein_state
+A	B-species
+.	I-species
+thaliana	I-species
+accession	O
+Columbia	O
+(	O
+Col	O
+-	O
+0	O
+)	O
+plants	B-taxonomy_domain
+fpa	B-gene
+-	I-gene
+8	I-gene
+and	O
+fpa	B-gene
+-	I-gene
+8	I-gene
+mutants	B-protein_state
+expressing	O
+FPA	B-protein
+::	O
+FPA	B-mutant
+R477A	I-mutant
+;	I-mutant
+Y515A	I-mutant
+using	O
+poly	O
+(	O
+A	O
+)+	O
+purified	O
+mRNAs	B-chemical
+.	O
+Black	O
+arrows	O
+indicate	O
+the	O
+proximally	O
+and	O
+distally	O
+polyadenylated	O
+FPA	B-protein
+mRNAs	B-chemical
+.	O
+qRT	B-experimental_method
+-	I-experimental_method
+PCR	I-experimental_method
+analysis	O
+was	O
+performed	O
+with	O
+total	O
+RNA	B-chemical
+purified	O
+from	O
+Col	O
+-	O
+0	O
+,	O
+fpa	B-gene
+-	I-gene
+8	I-gene
+,	O
+and	O
+FPA	B-protein
+::	O
+FPA	B-mutant
+R477A	I-mutant
+;	I-mutant
+Y515A	I-mutant
+plants	B-taxonomy_domain
+.	O
+Together	O
+our	O
+findings	O
+suggest	O
+that	O
+either	O
+the	O
+SPOC	B-structure_element
+domain	O
+is	O
+not	O
+required	O
+for	O
+the	O
+role	O
+of	O
+FPA	B-protein
+in	O
+regulating	O
+RNA	B-chemical
+3	O
+′-	O
+end	O
+formation	O
+,	O
+or	O
+that	O
+this	O
+combination	O
+of	O
+mutations	B-experimental_method
+is	O
+not	O
+sufficient	O
+to	O
+critically	O
+disrupt	O
+the	O
+function	O
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+.	O
+Since	O
+the	O
+corresponding	O
+mutations	B-experimental_method
+in	O
+the	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+do	O
+disrupt	O
+its	O
+recognition	O
+of	O
+unphosphorylated	B-protein_state
+SMRT	B-protein
+peptides	B-chemical
+,	O
+these	O
+observations	O
+may	O
+reinforce	O
+the	O
+idea	O
+that	O
+the	O
+features	O
+and	O
+functions	O
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+differ	O
+from	O
+those	O
+of	O
+the	O
+only	O
+other	O
+well	O
+-	O
+characterized	O
+SPOC	B-structure_element
+domain	O
+.	O

annotation_IOB/PMC4993997.tsv ADDED Viewed

	@@ -0,0 +1,7856 @@

+Structure	O
+and	O
+function	O
+of	O
+human	B-species
+Naa60	B-protein
+(	O
+NatF	B-complex_assembly
+),	O
+a	O
+Golgi	O
+-	O
+localized	O
+bi	O
+-	O
+functional	O
+acetyltransferase	B-protein_type
+N	B-ptm
+-	I-ptm
+terminal	I-ptm
+acetylation	I-ptm
+(	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+),	O
+carried	O
+out	O
+by	O
+N	B-protein_type
+-	I-protein_type
+terminal	I-protein_type
+acetyltransferases	I-protein_type
+(	O
+NATs	B-protein_type
+),	O
+is	O
+a	O
+conserved	O
+and	O
+primary	O
+modification	O
+of	O
+nascent	O
+peptide	B-chemical
+chains	O
+.	O
+Naa60	B-protein
+(	O
+also	O
+named	O
+NatF	B-complex_assembly
+)	O
+is	O
+a	O
+recently	O
+identified	O
+NAT	B-protein_type
+found	O
+only	O
+in	O
+multicellular	B-taxonomy_domain
+eukaryotes	I-taxonomy_domain
+.	O
+This	O
+protein	O
+was	O
+shown	O
+to	O
+locate	O
+on	O
+the	O
+Golgi	O
+apparatus	O
+and	O
+mainly	O
+catalyze	O
+the	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+of	O
+transmembrane	O
+proteins	O
+,	O
+and	O
+it	O
+also	O
+harbors	O
+lysine	B-protein_type
+Nε	I-protein_type
+-	I-protein_type
+acetyltransferase	I-protein_type
+(	O
+KAT	B-protein_type
+)	O
+activity	O
+to	O
+catalyze	O
+the	O
+acetylation	B-ptm
+of	O
+lysine	B-residue_name
+ε	O
+-	O
+amine	O
+.	O
+Here	O
+,	O
+we	O
+report	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+human	B-species
+Naa60	B-protein
+(	O
+hNaa60	B-protein
+)	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+Acetyl	B-chemical
+-	I-chemical
+Coenzyme	I-chemical
+A	I-chemical
+(	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+)	O
+or	O
+Coenzyme	B-chemical
+A	I-chemical
+(	O
+CoA	B-chemical
+).	O
+The	O
+hNaa60	B-protein
+protein	O
+contains	O
+an	O
+amphipathic	B-structure_element
+helix	I-structure_element
+following	O
+its	O
+GNAT	B-structure_element
+domain	I-structure_element
+that	O
+may	O
+contribute	O
+to	O
+Golgi	O
+localization	O
+of	O
+hNaa60	B-protein
+,	O
+and	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+adopted	O
+different	O
+conformations	O
+in	O
+the	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+and	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+crystal	B-evidence
+structures	I-evidence
+.	O
+Remarkably	O
+,	O
+we	O
+found	O
+that	O
+the	O
+side	O
+-	O
+chain	O
+of	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+can	O
+influence	O
+the	O
+position	O
+of	O
+the	O
+coenzyme	B-chemical
+,	O
+indicating	O
+a	O
+new	O
+regulatory	O
+mechanism	O
+involving	O
+enzyme	O
+,	O
+co	O
+-	O
+factor	O
+and	O
+substrates	O
+interactions	O
+.	O
+Moreover	O
+,	O
+structural	B-experimental_method
+comparison	I-experimental_method
+and	I-experimental_method
+biochemical	I-experimental_method
+studies	I-experimental_method
+indicated	O
+that	O
+Tyr	B-residue_name_number
+97	I-residue_name_number
+and	O
+His	B-residue_name_number
+138	I-residue_name_number
+are	O
+key	O
+residues	O
+for	O
+catalytic	O
+reaction	O
+and	O
+that	O
+a	O
+non	B-protein_state
+-	I-protein_state
+conserved	I-protein_state
+β3	B-structure_element
+-	I-structure_element
+β4	I-structure_element
+long	I-structure_element
+loop	I-structure_element
+participates	O
+in	O
+the	O
+regulation	O
+of	O
+hNaa60	B-protein
+activity	O
+.	O
+Acetylation	B-ptm
+is	O
+one	O
+of	O
+the	O
+most	O
+ubiquitous	O
+modifications	O
+that	O
+plays	O
+a	O
+vital	O
+role	O
+in	O
+many	O
+biological	O
+processes	O
+,	O
+such	O
+as	O
+transcriptional	O
+regulation	O
+,	O
+protein	O
+-	O
+protein	O
+interaction	O
+,	O
+enzyme	O
+activity	O
+,	O
+protein	O
+stability	O
+,	O
+antibiotic	O
+resistance	O
+,	O
+biological	O
+rhythm	O
+and	O
+so	O
+on	O
+.	O
+Protein	O
+acetylation	B-ptm
+can	O
+be	O
+grouped	O
+into	O
+lysine	B-ptm
+Nε	I-ptm
+-	I-ptm
+acetylation	I-ptm
+and	O
+peptide	B-chemical
+N	B-ptm
+-	I-ptm
+terminal	I-ptm
+acetylation	I-ptm
+(	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+).	O
+Generally	O
+,	O
+Nε	B-ptm
+-	I-ptm
+acetylation	I-ptm
+refers	O
+to	O
+the	O
+transfer	O
+of	O
+an	O
+acetyl	B-chemical
+group	O
+from	O
+an	O
+acetyl	B-chemical
+coenzyme	I-chemical
+A	I-chemical
+(	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+)	O
+to	O
+the	O
+ε	O
+-	O
+amino	O
+group	O
+of	O
+lysine	B-residue_name
+.	O
+This	O
+kind	O
+of	O
+modification	O
+is	O
+catalyzed	O
+by	O
+lysine	B-protein_type
+acetyltransferases	I-protein_type
+(	O
+KATs	B-protein_type
+),	O
+some	O
+of	O
+which	O
+are	O
+named	O
+histone	B-protein_type
+acetyltransferases	I-protein_type
+(	O
+HATs	B-protein_type
+)	O
+because	O
+early	O
+studies	O
+focused	O
+mostly	O
+on	O
+the	O
+post	O
+-	O
+transcriptional	O
+acetylation	B-ptm
+of	O
+histones	B-protein_type
+.	O
+Despite	O
+the	O
+prominent	O
+accomplishments	O
+in	O
+the	O
+field	O
+regarding	O
+Nε	B-ptm
+-	I-ptm
+acetylation	I-ptm
+by	O
+KATs	B-protein_type
+for	O
+over	O
+50	O
+years	O
+,	O
+the	O
+significance	O
+of	O
+the	O
+more	O
+evolutionarily	O
+conserved	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+is	O
+still	O
+inconclusive	O
+.	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+is	O
+an	O
+abundant	O
+and	O
+evolutionarily	O
+conserved	O
+modification	O
+occurring	O
+in	O
+bacteria	B-taxonomy_domain
+,	O
+archaea	B-taxonomy_domain
+and	O
+eukaryotes	B-taxonomy_domain
+.	O
+It	O
+is	O
+estimated	O
+that	O
+about	O
+80	O
+–	O
+90	O
+%	O
+of	O
+soluble	O
+human	B-species
+proteins	O
+and	O
+50	O
+–	O
+70	O
+%	O
+of	O
+yeast	B-taxonomy_domain
+proteins	O
+are	O
+subjected	O
+to	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+,	O
+where	O
+an	O
+acetyl	B-chemical
+moiety	O
+is	O
+transferred	O
+from	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+to	O
+the	O
+α	O
+-	O
+amino	O
+group	O
+of	O
+the	O
+first	O
+residue	O
+.	O
+Recently	O
+Nt	O
+-	O
+acetylome	O
+expands	O
+the	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+to	O
+transmembrane	O
+proteins	O
+.	O
+Unlike	O
+Nε	B-ptm
+-	I-ptm
+acetylation	I-ptm
+that	O
+can	O
+be	O
+eliminated	O
+by	O
+deacetylases	B-protein_type
+,	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+is	O
+considered	O
+irreversible	B-protein_state
+since	O
+no	O
+corresponding	O
+deacetylase	B-protein_type
+is	O
+found	O
+to	O
+date	O
+.	O
+Although	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+has	O
+been	O
+regarded	O
+as	O
+a	O
+co	O
+-	O
+translational	O
+modification	O
+traditionally	O
+,	O
+there	O
+is	O
+evidence	O
+that	O
+post	O
+-	O
+translational	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+exists	O
+.	O
+During	O
+the	O
+past	O
+decades	O
+,	O
+a	O
+large	O
+number	O
+of	O
+Nt	O
+-	O
+acetylome	O
+researches	O
+have	O
+shed	O
+light	O
+on	O
+the	O
+functional	O
+roles	O
+of	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+,	O
+including	O
+protein	O
+degradation	O
+,	O
+subcellular	O
+localization	O
+,	O
+protein	O
+-	O
+protein	O
+interaction	O
+,	O
+protein	O
+-	O
+membrane	O
+interaction	O
+,	O
+plant	B-taxonomy_domain
+development	O
+,	O
+stress	O
+-	O
+response	O
+and	O
+protein	O
+stability	O
+.	O
+The	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+is	O
+carried	O
+out	O
+by	O
+N	B-protein_type
+-	I-protein_type
+terminal	I-protein_type
+acetyltransferases	I-protein_type
+(	O
+NATs	B-protein_type
+)	O
+that	O
+belong	O
+to	O
+the	O
+GNAT	B-protein_type
+superfamily	I-protein_type
+.	O
+To	O
+date	O
+,	O
+six	O
+NATs	B-protein_type
+(	O
+NatA	B-complex_assembly
+/	O
+B	B-complex_assembly
+/	O
+C	B-complex_assembly
+/	O
+D	B-complex_assembly
+/	O
+E	B-complex_assembly
+/	O
+F	B-complex_assembly
+)	O
+have	O
+been	O
+identified	O
+in	O
+eukaryotes	B-taxonomy_domain
+.	O
+About	O
+40	O
+percent	O
+of	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+of	O
+soluble	O
+proteins	O
+in	O
+cells	O
+is	O
+catalyzed	O
+by	O
+NatA	B-complex_assembly
+complex	O
+which	O
+is	O
+composed	O
+of	O
+the	O
+catalytic	O
+subunit	O
+Naa10p	B-protein
+and	O
+the	O
+auxiliary	O
+subunit	O
+Naa15p	B-protein
+.	O
+NatE	B-complex_assembly
+was	O
+found	O
+to	O
+physically	O
+interact	O
+with	O
+the	O
+NatA	B-complex_assembly
+complex	O
+without	O
+any	O
+observation	O
+of	O
+impact	O
+on	O
+NatA	B-complex_assembly
+-	O
+activity	O
+.	O
+Two	O
+other	O
+multimeric	O
+complexes	O
+of	O
+NATs	B-protein_type
+are	O
+NatB	B-complex_assembly
+and	O
+NatC	B-complex_assembly
+which	O
+contain	O
+the	O
+catalytic	O
+subunits	O
+Naa20	B-protein
+and	O
+Naa30	B-protein
+and	O
+the	O
+auxiliary	O
+subunits	O
+Naa25	B-protein
+and	O
+Naa35	B-protein
+/	O
+Naa38	B-protein
+,	O
+respectively	O
+.	O
+Furthermore	O
+,	O
+only	O
+the	O
+catalytic	O
+subunits	O
+Naa40	B-protein
+and	O
+Naa60	B-protein
+were	O
+found	O
+for	O
+NatD	B-complex_assembly
+and	O
+NatF	B-complex_assembly
+,	O
+respectively	O
+.	O
+Besides	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+,	O
+accumulating	O
+reports	O
+have	O
+proposed	O
+Nε	B-ptm
+-	I-ptm
+acetylation	I-ptm
+carried	O
+out	O
+by	O
+NATs	B-protein_type
+.	O
+There	O
+is	O
+an	O
+evolutionary	O
+increasing	O
+in	O
+the	O
+degree	O
+of	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+between	O
+yeast	B-taxonomy_domain
+and	O
+human	B-species
+which	O
+could	O
+partly	O
+be	O
+explained	O
+by	O
+the	O
+contribution	O
+of	O
+NatF	B-complex_assembly
+.	O
+As	O
+the	O
+first	O
+N	B-protein_type
+-	I-protein_type
+terminal	I-protein_type
+acetyltransferase	I-protein_type
+discovered	O
+on	O
+an	O
+organelle	O
+,	O
+NatF	B-complex_assembly
+,	O
+encoded	O
+by	O
+NAA60	B-protein
+and	O
+also	O
+named	O
+as	O
+Histone	B-protein
+acetyltransferase	I-protein
+type	I-protein
+B	I-protein
+protein	I-protein
+4	I-protein
+(	O
+HAT4	B-protein
+),	O
+Naa60	B-protein
+or	O
+N	B-protein
+-	I-protein
+acetyltransferase	I-protein
+15	I-protein
+(	O
+NAT15	B-protein
+),	O
+is	O
+the	O
+youngest	O
+member	O
+of	O
+the	O
+NAT	B-protein_type
+family	O
+.	O
+Unlike	O
+other	O
+NATs	B-protein_type
+that	O
+are	O
+highly	B-protein_state
+conserved	I-protein_state
+among	O
+lower	B-taxonomy_domain
+and	O
+higher	B-taxonomy_domain
+eukaryotes	I-taxonomy_domain
+,	O
+NatF	B-complex_assembly
+only	O
+exists	O
+in	O
+higher	B-taxonomy_domain
+eukaryotes	I-taxonomy_domain
+.	O
+Subsequent	O
+researches	O
+indicated	O
+that	O
+NatF	B-complex_assembly
+displays	O
+its	O
+catalytic	O
+ability	O
+with	O
+both	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+and	O
+lysine	B-ptm
+Nε	I-ptm
+-	I-ptm
+acetylation	I-ptm
+.	O
+As	O
+an	O
+N	B-protein_type
+-	I-protein_type
+terminal	I-protein_type
+acetyltransferase	I-protein_type
+,	O
+NatF	B-complex_assembly
+can	O
+specifically	O
+catalyze	O
+acetylation	B-ptm
+of	O
+the	O
+N	O
+-	O
+terminal	O
+α	O
+-	O
+amine	O
+of	O
+most	O
+transmembrane	O
+proteins	O
+and	O
+has	O
+substrate	O
+preference	O
+towards	O
+proteins	O
+with	O
+Met	B-structure_element
+-	I-structure_element
+Lys	I-structure_element
+-,	I-structure_element
+Met	B-structure_element
+-	I-structure_element
+Val	I-structure_element
+-,	I-structure_element
+Met	B-structure_element
+-	I-structure_element
+Ala	I-structure_element
+-	I-structure_element
+and	O
+Met	B-structure_element
+-	I-structure_element
+Met	I-structure_element
+-	I-structure_element
+N	O
+-	O
+termini	O
+,	O
+thus	O
+partially	O
+overlapping	O
+substrate	O
+selectivity	O
+with	O
+NatC	B-complex_assembly
+and	O
+NatE	B-complex_assembly
+.	O
+On	O
+the	O
+other	O
+hand	O
+,	O
+NatF	B-complex_assembly
+,	O
+with	O
+its	O
+lysine	B-protein_type
+acetyltransferase	I-protein_type
+activity	O
+,	O
+mediates	O
+the	O
+lysine	B-ptm
+acetylation	I-ptm
+of	O
+free	O
+histone	B-protein_type
+H4	B-protein_type
+,	O
+including	O
+H4K20	B-protein_type
+,	O
+H4K79	B-protein_type
+and	O
+H4K91	B-protein_type
+.	O
+Another	O
+important	O
+feature	O
+of	O
+NatF	B-complex_assembly
+is	O
+that	O
+this	O
+protein	O
+is	O
+anchored	O
+on	O
+the	O
+Golgi	O
+apparatus	O
+through	O
+its	O
+C	O
+-	O
+terminal	O
+membrane	B-structure_element
+-	I-structure_element
+integrating	I-structure_element
+region	I-structure_element
+and	O
+takes	O
+part	O
+in	O
+the	O
+maintaining	O
+of	O
+Golgi	O
+integrity	O
+.	O
+With	O
+its	O
+unique	O
+intracellular	O
+organellar	O
+localization	O
+and	O
+substrate	O
+selectivity	O
+,	O
+NatF	B-complex_assembly
+appears	O
+to	O
+provide	O
+more	O
+evolutionary	O
+information	O
+among	O
+the	O
+NAT	B-protein_type
+family	O
+members	O
+.	O
+It	O
+was	O
+recently	O
+found	O
+that	O
+NatF	B-complex_assembly
+facilitates	O
+nucleosomes	B-complex_assembly
+assembly	O
+and	O
+that	O
+NAA60	B-protein
+knockdown	O
+in	O
+MCF7	O
+-	O
+cell	O
+inhibits	O
+cell	O
+proliferation	O
+,	O
+sensitizes	O
+cells	O
+to	O
+DNA	O
+damage	O
+and	O
+induces	O
+cell	O
+apoptosis	O
+.	O
+In	O
+Drosophila	B-taxonomy_domain
+cells	O
+,	O
+NAA60	B-protein
+knockdown	O
+induces	O
+chromosomal	O
+segregation	O
+defects	O
+during	O
+anaphase	O
+including	O
+lagging	O
+chromosomes	O
+and	O
+chromosomal	O
+bridges	O
+.	O
+Much	O
+recent	O
+attention	O
+has	O
+also	O
+been	O
+focused	O
+on	O
+the	O
+requirement	O
+of	O
+NatF	B-complex_assembly
+for	O
+regulation	O
+of	O
+organellar	O
+structure	O
+.	O
+In	O
+HeLa	O
+cells	O
+,	O
+NAA60	B-protein
+knockdown	O
+causes	O
+Golgi	O
+apparatus	O
+fragmentation	O
+which	O
+can	O
+be	O
+rescued	O
+by	O
+overexpression	B-experimental_method
+Naa60	B-protein
+.	O
+The	O
+systematic	O
+investigation	O
+of	O
+publicly	O
+available	O
+microarray	O
+data	O
+showed	O
+that	O
+NATs	B-protein_type
+share	O
+distinct	O
+tissue	O
+-	O
+specific	O
+expression	O
+patterns	O
+in	O
+Drosophila	B-taxonomy_domain
+and	O
+NatF	B-complex_assembly
+shows	O
+a	O
+higher	O
+expression	O
+level	O
+in	O
+central	O
+nervous	O
+system	O
+of	O
+Drosophila	B-taxonomy_domain
+.	O
+In	O
+this	O
+study	O
+,	O
+we	O
+solved	B-experimental_method
+the	O
+structures	B-evidence
+of	O
+human	B-species
+Naa60	B-protein
+(	O
+NatF	B-complex_assembly
+)	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+coenzyme	B-chemical
+.	O
+The	O
+hNaa60	B-protein
+protein	O
+contains	O
+a	O
+unique	O
+amphipathic	B-structure_element
+α	I-structure_element
+-	I-structure_element
+helix	I-structure_element
+(	O
+α5	B-structure_element
+)	O
+following	O
+its	O
+GNAT	B-structure_element
+domain	I-structure_element
+that	O
+might	O
+account	O
+for	O
+the	O
+Golgi	O
+localization	O
+of	O
+this	O
+protein	O
+.	O
+Crystal	B-evidence
+structures	I-evidence
+showed	O
+that	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+rotated	O
+about	O
+50	O
+degrees	O
+upon	O
+removing	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+the	O
+protein	O
+and	O
+this	O
+movement	O
+substantially	O
+changed	O
+the	O
+geometry	O
+of	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+.	O
+Remarkably	O
+,	O
+we	O
+find	O
+that	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+may	O
+participate	O
+in	O
+the	O
+proper	O
+positioning	O
+of	O
+the	O
+coenzyme	B-chemical
+for	O
+the	O
+transfer	O
+reaction	O
+to	O
+occur	O
+.	O
+Further	O
+structure	B-experimental_method
+comparison	I-experimental_method
+and	O
+biochemical	B-experimental_method
+studies	I-experimental_method
+also	O
+identified	O
+other	O
+key	O
+structural	O
+elements	O
+essential	O
+for	O
+the	O
+enzyme	O
+activity	O
+of	O
+Naa60	B-protein
+.	O
+Overall	O
+structure	B-evidence
+of	O
+hNaa60	B-protein
+In	O
+the	O
+effort	O
+to	O
+prepare	O
+the	O
+protein	O
+for	O
+structural	O
+studies	O
+,	O
+we	O
+tried	O
+a	O
+large	O
+number	O
+of	O
+hNaa60	B-protein
+constructs	O
+but	O
+all	O
+failed	O
+due	O
+to	O
+heavy	O
+precipitation	O
+or	O
+aggregation	O
+.	O
+Sequence	B-experimental_method
+alignment	I-experimental_method
+of	O
+Naa60	B-protein
+from	O
+different	O
+species	O
+revealed	O
+a	O
+Glu	B-structure_element
+-	I-structure_element
+Glu	I-structure_element
+-	I-structure_element
+Arg	I-structure_element
+(	O
+EER	B-structure_element
+)	O
+versus	O
+Val	B-structure_element
+-	I-structure_element
+Val	I-structure_element
+-	I-structure_element
+Pro	I-structure_element
+(	O
+VVP	B-structure_element
+)	O
+sequence	O
+difference	O
+near	O
+the	O
+N	O
+-	O
+terminus	O
+of	O
+the	O
+protein	O
+in	O
+Xenopus	B-species
+Laevis	I-species
+versus	O
+Homo	B-species
+sapiens	I-species
+(	O
+Fig	O
+.	O
+1A	O
+).	O
+Considering	O
+that	O
+terminal	O
+residues	O
+may	O
+lack	O
+higher	O
+-	O
+order	O
+structure	O
+and	O
+hydrophobic	O
+residues	O
+in	O
+this	O
+region	O
+may	O
+expose	O
+to	O
+solvent	O
+and	O
+hence	O
+cause	O
+protein	O
+aggregation	O
+,	O
+we	O
+mutated	B-experimental_method
+residues	O
+4	B-residue_range
+–	I-residue_range
+6	I-residue_range
+from	O
+VVP	B-mutant
+to	I-mutant
+EER	I-mutant
+for	O
+the	O
+purpose	O
+of	O
+improving	O
+solubility	O
+of	O
+this	O
+protein	O
+.	O
+According	O
+to	O
+previous	O
+studies	O
+,	O
+this	O
+N	O
+-	O
+terminal	O
+region	O
+should	O
+not	O
+interfere	O
+with	O
+hNaa60	B-protein
+’	O
+s	O
+Golgi	O
+localization	O
+.	O
+We	O
+tried	O
+many	O
+hNaa60	B-protein
+constructs	O
+with	O
+the	O
+three	O
+-	O
+residues	O
+mutation	B-experimental_method
+but	O
+only	O
+the	O
+truncated	B-protein_state
+variant	O
+1	B-residue_range
+-	I-residue_range
+199	I-residue_range
+and	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+protein	O
+behaved	O
+well	O
+.	O
+We	O
+obtained	O
+the	O
+crystal	B-evidence
+of	O
+the	O
+truncated	B-protein_state
+variant	O
+1	B-residue_range
+-	I-residue_range
+199	I-residue_range
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+CoA	B-chemical
+first	O
+,	O
+and	O
+after	O
+extensive	O
+trials	O
+we	O
+got	O
+the	O
+crystal	B-evidence
+of	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+protein	O
+(	O
+spanning	O
+residues	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+(	O
+Fig	O
+.	O
+1B	O
+,	O
+C	O
+).	O
+Hereafter	O
+,	O
+all	O
+deletions	O
+or	O
+point	O
+mutants	B-protein_state
+of	O
+hNaa60	B-protein
+we	O
+describe	O
+here	O
+are	O
+with	O
+the	O
+EER	B-structure_element
+mutation	B-experimental_method
+.	O
+The	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+and	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+were	O
+determined	O
+by	O
+molecular	B-experimental_method
+replacement	I-experimental_method
+and	O
+refined	O
+to	O
+1	O
+.	O
+38	O
+Å	O
+and	O
+1	O
+.	O
+60	O
+Å	O
+resolution	O
+,	O
+respectively	O
+(	O
+Table	O
+1	O
+).	O
+The	O
+electron	B-evidence
+density	I-evidence
+maps	I-evidence
+were	O
+of	O
+sufficient	O
+quality	O
+to	O
+trace	O
+residues	O
+1	B-residue_range
+-	I-residue_range
+211	I-residue_range
+of	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+and	O
+residues	O
+5	B-residue_range
+-	I-residue_range
+199	I-residue_range
+of	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+).	I-mutant
+The	O
+structure	B-evidence
+of	O
+hNaa60	B-protein
+protein	O
+contains	O
+a	O
+central	B-structure_element
+domain	I-structure_element
+exhibiting	O
+a	O
+classic	O
+GCN5	B-protein_type
+-	I-protein_type
+related	I-protein_type
+N	I-protein_type
+-	I-protein_type
+acetyltransferase	I-protein_type
+(	O
+GNAT	B-protein_type
+)	O
+folding	O
+,	O
+along	O
+with	O
+the	O
+extended	B-protein_state
+N	B-structure_element
+-	I-structure_element
+and	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+regions	I-structure_element
+(	O
+Fig	O
+.	O
+1B	O
+,	O
+C	O
+).	O
+The	O
+central	B-structure_element
+domain	I-structure_element
+includes	O
+nine	O
+β	B-structure_element
+strands	I-structure_element
+(	O
+β1	B-structure_element
+-	I-structure_element
+β9	I-structure_element
+)	O
+and	O
+four	O
+α	B-structure_element
+-	I-structure_element
+helixes	I-structure_element
+(	O
+α1	B-structure_element
+-	I-structure_element
+α4	I-structure_element
+)	O
+and	O
+is	O
+highly	B-protein_state
+similar	I-protein_state
+to	O
+the	O
+known	O
+hNaa50p	B-protein
+and	O
+other	O
+reported	O
+NATs	B-protein_type
+(	O
+Fig	O
+.	O
+1D	O
+).	O
+However	O
+,	O
+in	O
+hNaa60	B-protein
+,	O
+there	O
+is	O
+an	O
+extra	B-structure_element
+20	I-structure_element
+-	I-structure_element
+residue	I-structure_element
+loop	I-structure_element
+between	O
+β3	B-structure_element
+and	O
+β4	B-structure_element
+that	O
+forms	O
+a	O
+small	B-structure_element
+subdomain	I-structure_element
+with	O
+well	O
+-	O
+defined	O
+3D	O
+structure	O
+(	O
+Fig	O
+.	O
+1B	O
+–	O
+D	O
+).	O
+Furthermore	O
+,	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+strands	I-structure_element
+form	O
+an	O
+approximately	B-structure_element
+antiparallel	I-structure_element
+β	I-structure_element
+-	I-structure_element
+hairpin	I-structure_element
+structure	I-structure_element
+remarkably	O
+different	O
+from	O
+that	O
+in	O
+hNaa50p	B-protein
+(	O
+Fig	O
+.	O
+1D	O
+).	O
+The	O
+N	B-structure_element
+-	I-structure_element
+and	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+regions	I-structure_element
+form	O
+helical	B-structure_element
+structures	I-structure_element
+(	O
+α0	B-structure_element
+and	O
+α5	B-structure_element
+)	O
+stretching	O
+out	O
+from	O
+the	O
+central	O
+GCN5	B-structure_element
+-	I-structure_element
+domain	I-structure_element
+(	O
+Fig	O
+.	O
+1C	O
+).	O
+Interestingly	O
+,	O
+we	O
+found	O
+that	O
+the	O
+catalytic	O
+activity	O
+of	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+is	O
+much	O
+lower	O
+than	O
+that	O
+of	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+(	O
+Figure	O
+S1	O
+),	O
+indicating	O
+that	O
+residues	O
+200	B-residue_range
+–	I-residue_range
+242	I-residue_range
+may	O
+have	O
+some	O
+auto	O
+-	O
+inhibitory	O
+effect	O
+on	O
+the	O
+activity	O
+of	O
+the	O
+enzyme	O
+.	O
+However	O
+,	O
+since	O
+this	O
+region	O
+was	O
+not	O
+visible	O
+in	O
+the	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+crystal	B-evidence
+structure	I-evidence
+,	O
+we	O
+do	O
+not	O
+yet	O
+understand	O
+how	O
+this	O
+happens	O
+.	O
+Another	O
+possibility	O
+is	O
+that	O
+since	O
+hNaa60	B-protein
+is	O
+localized	O
+on	O
+Golgi	O
+apparatus	O
+,	O
+the	O
+observed	O
+low	O
+activity	O
+of	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+hNaa60	B-protein
+might	O
+be	O
+related	O
+to	O
+lack	O
+of	O
+Golgi	O
+localization	O
+of	O
+the	O
+enzyme	O
+in	O
+our	O
+in	O
+vitro	O
+studies	O
+.	O
+For	O
+the	O
+convenience	O
+of	O
+studying	O
+the	O
+kinetics	O
+of	O
+mutants	B-protein_state
+,	O
+the	O
+mutagenesis	B-experimental_method
+studies	I-experimental_method
+described	O
+hereafter	O
+were	O
+all	O
+based	O
+on	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+).	I-mutant
+An	O
+amphipathic	B-structure_element
+α	I-structure_element
+-	I-structure_element
+helix	I-structure_element
+in	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+may	O
+contribute	O
+to	O
+Golgi	O
+localization	O
+of	O
+hNaa60	B-protein
+There	O
+is	O
+one	O
+hNaa60	B-protein
+molecule	O
+in	O
+the	O
+asymmetric	O
+unit	O
+in	O
+the	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+structure	B-evidence
+.	O
+The	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+extended	O
+from	O
+the	O
+GCN5	B-structure_element
+-	I-structure_element
+domain	I-structure_element
+forms	O
+an	O
+amphipathic	B-structure_element
+helix	I-structure_element
+(	O
+α5	B-structure_element
+)	O
+and	O
+interacts	O
+with	O
+a	O
+molecule	O
+in	O
+a	O
+neighbor	O
+asymmetric	O
+unit	O
+through	O
+hydrophobic	B-bond_interaction
+interactions	I-bond_interaction
+between	O
+α5	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+and	O
+a	O
+hydrophobic	B-site
+groove	I-site
+between	O
+the	O
+N	O
+-	O
+terminal	O
+β1	B-structure_element
+and	O
+β3	B-structure_element
+strands	I-structure_element
+of	O
+the	O
+neighbor	O
+molecule	O
+(	O
+Fig	O
+.	O
+2A	O
+).	O
+The	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+extension	I-structure_element
+following	O
+α5	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+forms	O
+a	O
+β	B-structure_element
+-	I-structure_element
+turn	I-structure_element
+that	O
+wraps	O
+around	O
+and	O
+interacts	O
+with	O
+the	O
+neighbor	O
+protein	O
+molecule	O
+through	O
+hydrophobic	B-bond_interaction
+interactions	I-bond_interaction
+,	O
+too	O
+.	O
+In	O
+the	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+structure	B-evidence
+,	O
+a	O
+part	O
+of	O
+the	O
+α5	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+is	O
+deleted	O
+due	O
+to	O
+truncation	O
+of	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+(	O
+Fig	O
+.	O
+1B	O
+).	O
+Interestingly	O
+,	O
+the	O
+remaining	O
+residues	O
+in	O
+α5	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+still	O
+form	O
+an	O
+amphipathic	B-structure_element
+helix	I-structure_element
+although	O
+the	O
+hydrophobic	B-bond_interaction
+interaction	I-bond_interaction
+with	O
+the	O
+N	O
+-	O
+terminal	O
+hydrophobic	B-site
+groove	I-site
+of	O
+a	O
+neighbor	O
+molecule	O
+is	O
+abolished	O
+and	O
+the	O
+helix	B-structure_element
+is	O
+largely	O
+exposed	O
+in	O
+solvent	O
+due	O
+to	O
+different	O
+crystal	B-evidence
+packing	I-evidence
+(	O
+Fig	O
+.	O
+2B	O
+).	O
+A	O
+recent	O
+research	O
+showed	O
+that	O
+residues	O
+182	B-residue_range
+–	I-residue_range
+216	I-residue_range
+are	O
+important	O
+for	O
+the	O
+localization	O
+of	O
+hNaa60	B-protein
+on	O
+Golgi	O
+.	O
+According	O
+to	O
+our	O
+structure	B-evidence
+,	O
+the	O
+solvent	B-protein_state
+-	I-protein_state
+exposed	I-protein_state
+amphipathic	B-structure_element
+helix	I-structure_element
+(	O
+α5	B-structure_element
+)	O
+formed	O
+by	O
+residues	O
+190	B-residue_range
+-	I-residue_range
+202	I-residue_range
+with	O
+an	O
+array	O
+of	O
+hydrophobic	O
+residues	O
+located	O
+on	O
+one	O
+side	O
+(	O
+Ile	B-residue_name_number
+190	I-residue_name_number
+,	O
+Leu	B-residue_name_number
+191	I-residue_name_number
+,	O
+Ile	B-residue_name_number
+194	I-residue_name_number
+,	O
+Leu	B-residue_name_number
+197	I-residue_name_number
+and	O
+Leu	B-residue_name_number
+201	I-residue_name_number
+)	O
+and	O
+hydrophilic	O
+residues	O
+on	O
+the	O
+other	O
+side	O
+(	O
+Fig	O
+.	O
+S2	O
+)	O
+might	O
+account	O
+for	O
+interaction	O
+between	O
+hNaa60	B-protein
+and	O
+Golgi	O
+membrane	O
+,	O
+as	O
+it	O
+is	O
+a	O
+typical	O
+structure	O
+accounting	O
+for	O
+membrane	O
+association	O
+through	O
+immersing	O
+into	O
+the	O
+lipid	O
+bi	O
+-	O
+layer	O
+with	O
+its	O
+hydrophobic	O
+side	O
+as	O
+was	O
+observed	O
+with	O
+KalSec14	B-protein
+,	O
+Atg3	B-protein
+,	O
+PB1	B-protein
+-	I-protein
+F2	I-protein
+etc	O
+.	O
+The	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+showed	O
+alternative	O
+conformations	O
+in	O
+the	O
+hNaa60	B-protein
+crystal	B-evidence
+structures	I-evidence
+Superposition	B-experimental_method
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+,	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+and	O
+hNaa50	B-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+/	I-complex_assembly
+peptide	I-complex_assembly
+(	O
+PDB	O
+3TFY	O
+)	O
+revealed	O
+considerable	O
+difference	O
+in	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+region	O
+despite	O
+the	O
+overall	O
+stability	O
+and	O
+similarity	O
+of	O
+the	O
+GNAT	B-structure_element
+domain	I-structure_element
+(	O
+Fig	O
+.	O
+1D	O
+).	O
+In	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+),	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+is	O
+located	O
+in	O
+close	O
+proximity	O
+to	O
+the	O
+α1	B-structure_element
+-	I-structure_element
+α2	I-structure_element
+loop	I-structure_element
+,	O
+creating	O
+a	O
+more	O
+compact	O
+substrate	B-site
+binding	I-site
+site	I-site
+than	O
+that	O
+in	O
+hNaa50	B-protein
+,	O
+where	O
+this	O
+region	O
+adopts	O
+a	O
+more	O
+flexible	B-protein_state
+loop	B-structure_element
+conformation	O
+(	O
+β6	B-structure_element
+-	I-structure_element
+β7	I-structure_element
+loop	I-structure_element
+).	O
+Upon	O
+removing	B-experimental_method
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+hNaa60	B-protein
+,	O
+we	O
+observed	O
+that	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+molecules	O
+pack	O
+in	O
+a	O
+different	O
+way	O
+involving	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+in	O
+the	O
+crystal	B-evidence
+,	O
+leading	O
+to	O
+about	O
+50	O
+degree	O
+rotation	O
+of	O
+the	O
+hairpin	B-structure_element
+which	O
+moves	O
+away	O
+from	O
+the	O
+α1	B-structure_element
+-	I-structure_element
+α2	I-structure_element
+loop	I-structure_element
+(	O
+Figs	O
+1D	O
+and	O
+2C	O
+).	O
+This	O
+conformational	O
+change	O
+substantially	O
+altered	O
+the	O
+geometry	O
+of	O
+the	O
+substrate	B-site
+binding	I-site
+site	I-site
+,	O
+which	O
+could	O
+potentially	O
+change	O
+the	O
+way	O
+in	O
+which	O
+the	O
+substrate	O
+accesses	O
+the	O
+active	B-site
+site	I-site
+of	O
+the	O
+enzyme	O
+.	O
+In	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+),	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+covers	O
+the	O
+active	B-site
+site	I-site
+in	O
+a	O
+way	O
+similar	O
+to	O
+that	O
+observed	O
+in	O
+hNaa50	B-protein
+,	O
+presumably	O
+leaving	O
+only	O
+one	O
+way	O
+for	O
+the	O
+substrate	O
+to	O
+access	O
+the	O
+active	B-site
+site	I-site
+,	O
+i	O
+.	O
+e	O
+.	O
+to	O
+enter	O
+from	O
+the	O
+opposite	O
+end	O
+into	O
+the	O
+same	O
+tunnel	B-site
+where	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+/	O
+CoA	B-chemical
+binds	O
+(	O
+Fig	O
+.	O
+2D	O
+),	O
+which	O
+may	O
+accommodate	O
+access	O
+of	O
+a	O
+NAT	B-protein_type
+substrate	O
+only	O
+.	O
+KAT	B-protein_type
+activity	O
+of	O
+hNaa60	B-protein
+toward	O
+histone	B-protein_type
+H4	B-protein_type
+has	O
+been	O
+noted	O
+in	O
+previous	O
+study	O
+,	O
+and	O
+our	O
+enzyme	B-evidence
+kinetic	I-evidence
+data	I-evidence
+also	O
+indicated	O
+that	O
+hNaa60	B-protein
+can	O
+acetylate	O
+H3	B-complex_assembly
+-	I-complex_assembly
+H4	I-complex_assembly
+tetramer	B-oligomeric_state
+in	O
+vitro	O
+(	O
+Figure	O
+S3	O
+).	O
+Furthermore	O
+,	O
+we	O
+analyzed	O
+the	O
+acetylation	B-ptm
+status	O
+of	O
+histone	B-protein_type
+H3	B-complex_assembly
+-	I-complex_assembly
+H4	I-complex_assembly
+tetramer	B-oligomeric_state
+using	O
+mass	B-experimental_method
+spectrometry	I-experimental_method
+and	O
+observed	O
+that	O
+multiple	O
+lysine	B-residue_name
+residues	O
+in	O
+the	O
+protein	O
+showed	O
+significantly	O
+increased	O
+acetylation	B-ptm
+level	O
+and	O
+changed	O
+acetylation	B-ptm
+profile	O
+upon	O
+treatment	O
+with	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+(	O
+Figure	O
+S4	O
+).	O
+We	O
+also	O
+conducted	O
+liquid	B-experimental_method
+chromatography	I-experimental_method
+-	I-experimental_method
+tandem	I-experimental_method
+mass	I-experimental_method
+spectrometry	I-experimental_method
+(	O
+LC	B-experimental_method
+/	I-experimental_method
+MS	I-experimental_method
+/	I-experimental_method
+MS	I-experimental_method
+)	O
+analysis	O
+on	O
+a	O
+synthetic	O
+peptide	B-chemical
+(	O
+NH2	B-chemical
+-	I-chemical
+MKGKEEKEGGAR	I-chemical
+-	I-chemical
+COOH	I-chemical
+)	O
+after	O
+treatment	O
+with	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+),	I-mutant
+and	O
+the	O
+data	O
+confirmed	O
+that	O
+both	O
+the	O
+N	O
+-	O
+terminal	O
+α	O
+-	O
+amine	O
+and	O
+lysine	B-residue_name
+side	O
+-	O
+chain	O
+ε	O
+-	O
+amine	O
+were	O
+robustly	O
+acetylated	B-protein_state
+after	O
+the	O
+treatment	O
+(	O
+Table	O
+S1	O
+).	O
+Recent	O
+structural	B-experimental_method
+investigation	I-experimental_method
+of	O
+other	O
+NATs	B-protein_type
+proposed	O
+that	O
+the	O
+β6	B-structure_element
+-	I-structure_element
+β7	I-structure_element
+loop	I-structure_element
+,	O
+corresponding	O
+to	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+in	O
+hNaa60	B-protein
+,	O
+and	O
+the	O
+α1	B-structure_element
+-	I-structure_element
+α2	I-structure_element
+loop	I-structure_element
+flanking	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+site	I-site
+of	O
+NATs	B-protein_type
+,	O
+prevent	O
+the	O
+lysine	B-residue_name
+side	O
+-	O
+chain	O
+of	O
+the	O
+KAT	B-protein_type
+substrates	O
+from	O
+inserting	O
+into	O
+the	O
+active	B-site
+site	I-site
+.	O
+Indeed	O
+,	O
+superposition	B-experimental_method
+of	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+structure	B-evidence
+on	O
+that	O
+of	O
+Hat1p	B-protein
+,	O
+a	O
+typical	O
+KAT	B-protein_type
+,	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+a	O
+histone	B-protein_type
+H4	B-protein_type
+peptide	B-chemical
+revealed	O
+obvious	O
+overlapping	O
+/	O
+clashing	O
+of	O
+the	O
+H4	B-protein_type
+peptide	B-chemical
+(	O
+a	O
+KAT	B-protein_type
+substrate	O
+)	O
+with	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+of	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+(	O
+Fig	O
+.	O
+2D	O
+).	O
+Interestingly	O
+,	O
+in	O
+the	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+crystal	B-evidence
+structure	I-evidence
+,	O
+the	O
+displaced	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+opened	O
+a	O
+second	O
+way	O
+for	O
+the	O
+substrate	O
+to	O
+access	O
+the	O
+active	B-site
+center	I-site
+that	O
+would	O
+readily	O
+accommodate	O
+the	O
+binding	O
+of	O
+the	O
+H4	B-protein_type
+peptide	B-chemical
+(	O
+Fig	O
+.	O
+2E	O
+),	O
+thus	O
+implied	O
+a	O
+potential	O
+explanation	O
+for	O
+KAT	B-protein_type
+activity	O
+of	O
+this	O
+enzyme	O
+from	O
+a	O
+structural	O
+biological	O
+view	O
+.	O
+However	O
+,	O
+since	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+and	O
+hNaa60	B-protein
+(	O
+1	O
+-	O
+199	O
+)	O
+were	O
+crystallized	B-experimental_method
+in	O
+different	O
+crystal	B-evidence
+forms	I-evidence
+,	O
+the	O
+observed	O
+conformational	O
+change	O
+of	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+may	O
+simply	O
+be	O
+an	O
+artifact	O
+related	O
+to	O
+the	O
+different	O
+crystal	B-evidence
+packing	I-evidence
+.	O
+Whether	O
+the	O
+KAT	B-protein_type
+substrates	O
+bind	O
+to	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+displaced	O
+conformation	O
+of	O
+the	O
+enzyme	O
+needs	O
+to	O
+be	O
+verified	O
+by	O
+further	O
+structural	B-experimental_method
+and	I-experimental_method
+functional	I-experimental_method
+studies	I-experimental_method
+.	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+facilitates	O
+proper	O
+positioning	O
+of	O
+the	O
+cofactor	O
+for	O
+acetyl	B-chemical
+-	O
+transfer	O
+The	O
+electron	B-evidence
+density	I-evidence
+of	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+side	O
+-	O
+chain	O
+is	O
+well	O
+defined	O
+in	O
+the	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+structure	B-evidence
+,	O
+but	O
+becomes	O
+invisible	O
+in	O
+the	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+structure	B-evidence
+,	O
+indicating	O
+displacement	O
+of	O
+the	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+side	O
+-	O
+chain	O
+in	O
+the	O
+latter	O
+(	O
+Fig	O
+.	O
+3A	O
+,	O
+B	O
+).	O
+A	O
+solvent	O
+-	O
+derived	O
+malonate	B-chemical
+molecule	O
+is	O
+found	O
+beside	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+and	O
+the	O
+ethanethioate	B-chemical
+moiety	O
+of	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+in	O
+the	O
+high	O
+-	O
+resolution	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+structure	B-evidence
+(	O
+Fig	O
+.	O
+3A	O
+).	O
+Superposition	B-experimental_method
+of	O
+this	O
+structure	B-evidence
+on	O
+that	O
+of	O
+hNaa50p	B-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+/	I-complex_assembly
+peptide	I-complex_assembly
+shows	O
+that	O
+the	O
+malonate	B-chemical
+molecule	O
+overlaps	O
+well	O
+on	O
+the	O
+N	O
+-	O
+terminal	O
+methionine	B-residue_name
+of	O
+the	O
+substrate	O
+peptide	B-chemical
+and	O
+residue	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+in	O
+hNaa60	B-protein
+overlaps	O
+well	O
+on	O
+Phe	B-residue_name_number
+27	I-residue_name_number
+in	O
+hNaa50	B-protein
+(	O
+Fig	O
+.	O
+4A	O
+).	O
+Interestingly	O
+,	O
+in	O
+the	O
+structure	B-evidence
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+,	O
+the	O
+terminal	O
+thiol	O
+of	O
+CoA	B-chemical
+adopts	O
+alternative	O
+conformations	O
+.	O
+One	O
+is	O
+to	O
+approach	O
+the	O
+substrate	O
+amine	B-chemical
+(	O
+as	O
+indicated	O
+by	O
+the	O
+superimposed	B-experimental_method
+hNaa50	B-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+/	I-complex_assembly
+peptide	I-complex_assembly
+structure	B-evidence
+),	O
+similar	O
+to	O
+the	O
+terminal	O
+ethanethioate	B-chemical
+of	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+in	O
+the	O
+structure	B-evidence
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+;	O
+the	O
+other	O
+is	O
+to	O
+approach	O
+the	O
+α1	B-structure_element
+-	I-structure_element
+α2	I-structure_element
+loop	I-structure_element
+and	O
+away	O
+from	O
+the	O
+substrate	O
+amine	O
+(	O
+Fig	O
+.	O
+3B	O
+).	O
+To	O
+rule	O
+out	O
+the	O
+possibility	O
+that	O
+the	O
+electron	B-evidence
+density	I-evidence
+we	O
+define	O
+as	O
+the	O
+alternative	O
+conformation	O
+of	O
+the	O
+thiol	O
+terminus	O
+is	O
+residual	O
+electron	B-evidence
+density	I-evidence
+of	O
+the	O
+displaced	O
+side	O
+-	O
+chain	O
+of	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+,	O
+we	O
+solved	B-experimental_method
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)	I-complex_assembly
+F34A	I-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+.	O
+The	O
+structure	B-evidence
+of	O
+this	O
+mutant	B-protein_state
+is	O
+highly	O
+similar	O
+to	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+and	O
+there	O
+is	O
+essentially	O
+the	O
+same	O
+electron	B-evidence
+density	I-evidence
+corresponding	O
+to	O
+the	O
+alternative	O
+conformation	O
+of	O
+the	O
+thiol	O
+(	O
+Fig	O
+.	O
+3C	O
+).	O
+Phe	B-residue_name_number
+27	I-residue_name_number
+in	O
+hNaa50p	B-protein
+(	O
+equivalent	O
+to	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+in	O
+hNaa60	B-protein
+)	O
+has	O
+been	O
+implicated	O
+to	O
+facilitate	O
+the	O
+binding	O
+of	O
+N	O
+-	O
+terminal	O
+methionine	B-residue_name
+of	O
+the	O
+substrate	O
+peptide	B-chemical
+through	O
+hydrophobic	B-bond_interaction
+interaction	I-bond_interaction
+.	O
+However	O
+,	O
+in	O
+the	O
+hNaa60	B-complex_assembly
+/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+structure	B-evidence
+,	O
+a	O
+hydrophilic	O
+malonate	B-chemical
+molecule	O
+is	O
+found	O
+at	O
+the	O
+same	O
+location	O
+where	O
+the	O
+N	O
+-	O
+terminal	O
+methionine	B-residue_name
+should	O
+bind	O
+as	O
+is	O
+indicated	O
+by	O
+the	O
+superposition	B-experimental_method
+(	O
+Fig	O
+.	O
+3A	O
+),	O
+suggesting	O
+that	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+may	O
+accommodate	O
+binding	O
+of	O
+hydrophilic	O
+substrate	O
+,	O
+too	O
+.	O
+Moreover	O
+,	O
+orientation	O
+of	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+side	O
+-	O
+chain	O
+seems	O
+to	O
+be	O
+co	O
+-	O
+related	O
+to	O
+positioning	O
+of	O
+the	O
+terminus	O
+of	O
+the	O
+co	O
+-	O
+enzyme	O
+and	O
+important	O
+for	O
+placing	O
+it	O
+at	O
+a	O
+location	O
+in	O
+close	O
+proximity	O
+to	O
+the	O
+substrate	O
+amine	O
+.	O
+We	O
+hypothesize	O
+that	O
+if	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+only	O
+works	O
+to	O
+facilitate	O
+the	O
+binding	O
+of	O
+the	O
+hydrophobic	O
+N	O
+-	O
+terminal	O
+Met	B-residue_name
+residue	O
+,	O
+to	O
+mutate	B-experimental_method
+it	O
+from	O
+Phe	B-residue_name
+to	O
+Ala	B-residue_name
+would	O
+not	O
+abolish	O
+the	O
+catalytic	O
+activity	O
+of	O
+this	O
+enzyme	O
+,	O
+while	O
+if	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+also	O
+plays	O
+an	O
+essential	O
+role	O
+to	O
+position	O
+the	O
+ethanethioate	B-chemical
+moiety	O
+of	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+,	O
+the	O
+mutation	B-experimental_method
+would	O
+be	O
+expected	O
+to	O
+abrogate	O
+the	O
+activity	O
+of	O
+the	O
+enzyme	O
+.	O
+Indeed	O
+,	O
+our	O
+enzyme	B-evidence
+kinetic	I-evidence
+data	I-evidence
+showed	O
+that	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+F34A	B-mutant
+mutant	B-protein_state
+showed	O
+no	O
+detectable	O
+activity	O
+(	O
+Fig	O
+.	O
+5A	O
+).	O
+In	O
+order	O
+to	O
+rule	O
+out	O
+the	O
+possibility	O
+that	O
+the	O
+observed	O
+loss	O
+of	O
+activity	O
+may	O
+be	O
+related	O
+to	O
+bad	O
+folding	O
+of	O
+the	O
+mutant	B-protein_state
+protein	O
+,	O
+we	O
+studied	O
+the	O
+circular	B-experimental_method
+dichroism	I-experimental_method
+(	O
+CD	B-experimental_method
+)	O
+spectrum	B-evidence
+of	O
+the	O
+protein	O
+(	O
+Fig	O
+.	O
+5B	O
+)	O
+and	O
+determined	O
+its	O
+crystal	B-evidence
+structure	I-evidence
+(	O
+Fig	O
+.	O
+3C	O
+).	O
+Both	O
+studies	O
+proved	O
+that	O
+the	O
+F34A	B-mutant
+mutant	B-protein_state
+protein	O
+is	O
+well	B-protein_state
+-	I-protein_state
+folded	I-protein_state
+.	O
+Many	O
+studies	O
+have	O
+addressed	O
+the	O
+crucial	O
+effect	O
+of	O
+α1	B-structure_element
+-	I-structure_element
+α2	I-structure_element
+loop	I-structure_element
+on	O
+catalysis	O
+,	O
+showing	O
+that	O
+some	O
+residues	O
+located	O
+in	O
+this	O
+area	O
+are	O
+involved	O
+in	O
+the	O
+binding	O
+of	O
+substrates	O
+.	O
+We	O
+propose	O
+that	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+may	O
+play	O
+a	O
+dual	O
+role	O
+both	O
+in	O
+interacting	O
+with	O
+the	O
+peptide	B-chemical
+substrate	O
+(	O
+recognition	O
+)	O
+and	O
+in	O
+positioning	O
+of	O
+the	O
+ethanethioate	B-chemical
+moiety	O
+of	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+to	O
+the	O
+right	O
+location	O
+to	O
+facilitate	O
+acetyl	B-chemical
+-	O
+transfer	O
+.	O
+Structural	O
+basis	O
+for	O
+hNaa60	B-protein
+substrate	O
+binding	O
+Several	O
+studies	O
+have	O
+demonstrated	O
+that	O
+the	O
+substrate	O
+specificities	O
+of	O
+hNaa60	B-protein
+and	O
+hNaa50	B-protein
+are	O
+highly	O
+overlapped	O
+.	O
+The	O
+structure	B-evidence
+of	O
+hNaa50p	B-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+/	I-complex_assembly
+peptide	I-complex_assembly
+provides	O
+detailed	O
+information	O
+about	O
+the	O
+position	O
+of	O
+substrate	O
+N	O
+-	O
+terminal	O
+residues	O
+in	O
+the	O
+active	B-site
+site	I-site
+of	O
+hNaa50	B-protein
+.	O
+Comparing	O
+the	O
+active	B-site
+site	I-site
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+with	O
+hNaa50p	B-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+/	I-complex_assembly
+peptide	I-complex_assembly
+revealed	O
+that	O
+key	O
+catalytic	B-site
+and	I-site
+substrate	I-site
+binding	I-site
+residues	I-site
+are	O
+highly	B-protein_state
+conserved	I-protein_state
+in	O
+both	O
+proteins	O
+(	O
+Fig	O
+.	O
+4A	O
+).	O
+With	O
+respect	O
+to	O
+catalysis	O
+,	O
+hNaa50p	B-protein
+has	O
+been	O
+shown	O
+to	O
+employ	O
+residues	O
+Tyr	B-residue_name_number
+73	I-residue_name_number
+and	O
+His	B-residue_name_number
+112	I-residue_name_number
+to	O
+abstract	O
+proton	O
+from	O
+the	O
+α	O
+-	O
+amino	O
+group	O
+from	O
+the	O
+substrate	O
+’	O
+s	O
+first	O
+residue	O
+through	O
+a	O
+well	B-protein_state
+-	I-protein_state
+ordered	I-protein_state
+water	B-chemical
+.	O
+A	O
+well	B-protein_state
+-	I-protein_state
+ordered	I-protein_state
+water	B-chemical
+was	O
+also	O
+found	O
+between	O
+Tyr	B-residue_name_number
+97	I-residue_name_number
+and	O
+His	B-residue_name_number
+138	I-residue_name_number
+in	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+and	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+(	O
+Fig	O
+.	O
+4B	O
+).	O
+To	O
+determine	O
+the	O
+function	O
+of	O
+Tyr	B-residue_name_number
+97	I-residue_name_number
+and	O
+His	B-residue_name_number
+138	I-residue_name_number
+in	O
+hNaa60	B-protein
+catalysis	O
+,	O
+we	O
+mutated	B-experimental_method
+these	O
+residues	O
+to	O
+alanine	B-residue_name
+and	O
+phenylalanine	B-residue_name
+,	O
+respectively	O
+,	O
+and	O
+confirmed	O
+that	O
+all	O
+these	O
+mutants	B-protein_state
+used	O
+in	O
+our	O
+kinetic	B-experimental_method
+assays	I-experimental_method
+are	O
+well	B-protein_state
+-	I-protein_state
+folded	I-protein_state
+by	O
+CD	B-experimental_method
+spectra	B-evidence
+(	O
+Fig	O
+.	O
+5B	O
+).	O
+Purity	O
+of	O
+all	O
+proteins	O
+were	O
+also	O
+analyzed	O
+by	O
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+(	O
+Figure	O
+S5	O
+).	O
+As	O
+show	O
+in	O
+Fig	O
+.	O
+5A	O
+,	O
+the	O
+mutants	B-protein_state
+Y97A	B-mutant
+,	O
+Y97F	B-mutant
+,	O
+H138A	B-mutant
+and	O
+H138F	B-mutant
+abolished	B-protein_state
+the	I-protein_state
+activity	I-protein_state
+of	O
+hNaa60	B-protein
+.	O
+In	O
+contrast	O
+,	O
+to	O
+mutate	B-experimental_method
+the	O
+nearby	O
+solvent	B-protein_state
+exposed	I-protein_state
+residue	O
+Glu	B-residue_name_number
+37	I-residue_name_number
+to	O
+Ala	B-residue_name
+(	O
+E37A	B-mutant
+)	O
+has	O
+little	O
+impact	O
+on	O
+the	O
+activity	O
+of	O
+hNaa60	B-protein
+(	O
+Figs	O
+4B	O
+and	O
+5A	O
+).	O
+In	O
+conclusion	O
+,	O
+the	O
+structural	B-experimental_method
+and	I-experimental_method
+functional	I-experimental_method
+studies	I-experimental_method
+indicate	O
+that	O
+hNaa60	B-protein
+applies	O
+the	O
+same	O
+two	O
+base	O
+mechanism	O
+through	O
+Tyr	B-residue_name_number
+97	I-residue_name_number
+,	O
+His	B-residue_name_number
+138	I-residue_name_number
+and	O
+a	O
+well	B-protein_state
+-	I-protein_state
+ordered	I-protein_state
+water	B-chemical
+as	O
+was	O
+described	O
+for	O
+hNaa50	B-protein
+.	O
+The	O
+malonate	B-chemical
+molecule	O
+observed	O
+in	O
+the	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+crystal	B-evidence
+structure	I-evidence
+may	O
+be	O
+indicative	O
+of	O
+the	O
+substrate	O
+binding	O
+position	O
+of	O
+hNaa60	B-protein
+since	O
+it	O
+is	O
+located	O
+in	O
+the	O
+active	B-site
+site	I-site
+and	O
+overlaps	O
+the	O
+N	O
+-	O
+terminal	O
+Met	B-residue_name
+of	O
+the	O
+substrate	O
+peptide	B-chemical
+in	O
+the	O
+superposition	B-experimental_method
+with	O
+the	O
+hNaa50p	B-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+/	I-complex_assembly
+peptide	I-complex_assembly
+structure	B-evidence
+(	O
+Fig	O
+.	O
+4A	O
+).	O
+Residues	O
+Tyr	B-residue_name_number
+38	I-residue_name_number
+,	O
+Asn	B-residue_name_number
+143	I-residue_name_number
+and	O
+Tyr	B-residue_name_number
+165	I-residue_name_number
+are	O
+located	O
+around	O
+the	O
+malonate	B-chemical
+and	O
+interact	O
+with	O
+it	O
+through	O
+direct	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+or	O
+water	B-bond_interaction
+bridge	I-bond_interaction
+(	O
+Fig	O
+.	O
+4C	O
+).	O
+Although	O
+malonate	B-chemical
+is	O
+negatively	O
+charged	O
+,	O
+which	O
+is	O
+different	O
+from	O
+that	O
+of	O
+lysine	B-residue_name
+ε	O
+-	O
+amine	O
+or	O
+peptide	B-chemical
+N	O
+-	O
+terminal	O
+amine	O
+,	O
+similar	O
+hydrophilic	B-bond_interaction
+interactions	I-bond_interaction
+may	O
+take	O
+place	O
+when	O
+substrate	O
+amine	O
+presents	O
+in	O
+the	O
+same	O
+position	O
+,	O
+since	O
+Tyr	B-residue_name_number
+38	I-residue_name_number
+,	O
+Asn	B-residue_name_number
+143	I-residue_name_number
+and	O
+Tyr	B-residue_name_number
+165	I-residue_name_number
+are	O
+not	O
+positively	O
+or	O
+negatively	O
+charged	O
+.	O
+In	O
+agreement	O
+with	O
+this	O
+hypothesis	O
+,	O
+it	O
+was	O
+found	O
+that	O
+the	O
+Y38A	B-mutant
+,	O
+N143A	B-mutant
+and	O
+Y165A	B-mutant
+mutants	B-protein_state
+all	O
+showed	O
+remarkably	O
+reduced	O
+activities	O
+as	O
+compared	O
+to	O
+WT	B-protein_state
+,	O
+implying	O
+that	O
+these	O
+residues	O
+may	O
+be	O
+critical	O
+for	O
+substrate	O
+binding	O
+(	O
+Figs	O
+4C	O
+and	O
+5A	O
+).	O
+The	O
+β3	B-structure_element
+-	I-structure_element
+β4	I-structure_element
+loop	I-structure_element
+participates	O
+in	O
+the	O
+regulation	O
+of	O
+hNaa60	B-protein
+-	O
+activity	O
+Residues	O
+between	O
+β3	B-structure_element
+and	O
+β4	B-structure_element
+of	O
+hNaa60	B-protein
+form	O
+a	O
+unique	O
+20	B-structure_element
+-	I-structure_element
+residue	I-structure_element
+long	I-structure_element
+loop	I-structure_element
+(	O
+residues	O
+73	B-residue_range
+–	I-residue_range
+92	I-residue_range
+)	O
+that	O
+is	O
+a	O
+short	B-structure_element
+turn	I-structure_element
+in	O
+many	O
+other	O
+NAT	B-protein_type
+members	O
+(	O
+Fig	O
+.	O
+1D	O
+).	O
+Previous	O
+study	O
+indicated	O
+that	O
+auto	B-ptm
+-	I-ptm
+acetylation	I-ptm
+of	O
+hNaa60K79	B-protein
+could	O
+influence	O
+the	O
+activity	O
+of	O
+hNaa60	B-protein
+;	O
+however	O
+,	O
+we	O
+were	O
+not	O
+able	O
+to	O
+determine	O
+if	O
+Lys	B-residue_name_number
+79	I-residue_name_number
+is	O
+acetylated	B-protein_state
+in	O
+our	O
+crystal	B-evidence
+structures	I-evidence
+due	O
+to	O
+poor	O
+quality	O
+of	O
+the	O
+electron	B-evidence
+density	I-evidence
+of	O
+Lys	B-residue_name_number
+79	I-residue_name_number
+side	O
+-	O
+chain	O
+.	O
+We	O
+therefore	O
+used	O
+mass	B-experimental_method
+spectrometry	I-experimental_method
+to	O
+analyze	O
+if	O
+Lys	B-residue_name_number
+79	I-residue_name_number
+was	O
+acetylated	B-protein_state
+in	O
+our	O
+bacterially	O
+purified	O
+proteins	O
+,	O
+and	O
+observed	O
+no	O
+modification	O
+on	O
+this	O
+residue	O
+(	O
+Figure	O
+S6	O
+).	O
+To	O
+assess	O
+the	O
+impact	O
+of	O
+hNaa60K79	B-protein
+auto	B-ptm
+-	I-ptm
+acetylation	I-ptm
+,	O
+we	O
+studied	O
+the	O
+kinetics	O
+of	O
+K79R	B-mutant
+and	O
+K79Q	B-mutant
+mutants	B-protein_state
+which	O
+mimic	O
+the	O
+un	B-protein_state
+-	I-protein_state
+acetylated	I-protein_state
+and	O
+acetylated	B-protein_state
+form	O
+of	O
+Lys	B-residue_name_number
+79	I-residue_name_number
+,	O
+respectively	O
+.	O
+Interestingly	O
+,	O
+both	O
+K79R	B-mutant
+and	O
+K79Q	B-mutant
+mutants	B-protein_state
+led	O
+to	O
+an	O
+increase	O
+in	O
+the	O
+catalytic	O
+activity	O
+of	O
+hNaa60	B-protein
+,	O
+while	O
+K79A	B-mutant
+mutant	B-protein_state
+led	O
+to	O
+modest	O
+decrease	O
+of	O
+the	O
+activity	O
+(	O
+Fig	O
+.	O
+5A	O
+).	O
+These	O
+data	O
+indicate	O
+that	O
+the	O
+acetylation	B-ptm
+of	O
+Lys	B-residue_name_number
+79	I-residue_name_number
+is	O
+not	O
+required	O
+for	O
+optimal	O
+catalytic	O
+activity	O
+of	O
+hNaa60	B-protein
+in	O
+vitro	O
+.	O
+It	O
+is	O
+noted	O
+that	O
+the	O
+β3	B-structure_element
+-	I-structure_element
+β4	I-structure_element
+loop	I-structure_element
+of	O
+hNaa60	B-protein
+acts	O
+like	O
+a	O
+door	O
+leaf	O
+to	O
+partly	O
+cover	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+pathway	I-site
+.	O
+We	O
+hence	O
+hypothesize	O
+that	O
+the	O
+β3	B-structure_element
+-	I-structure_element
+β4	I-structure_element
+loop	I-structure_element
+may	O
+interfere	O
+with	O
+the	O
+access	O
+of	O
+the	O
+peptide	B-chemical
+substrates	O
+and	O
+that	O
+the	O
+solvent	B-protein_state
+-	I-protein_state
+exposing	I-protein_state
+Lys	B-residue_name_number
+79	I-residue_name_number
+may	O
+play	O
+a	O
+potential	O
+role	O
+to	O
+remove	O
+the	O
+door	O
+leaf	O
+when	O
+it	O
+hovers	O
+in	O
+solvent	O
+(	O
+Fig	O
+.	O
+4D	O
+).	O
+Acidic	O
+residues	O
+Glu	B-residue_name_number
+80	I-residue_name_number
+,	O
+Asp	B-residue_name_number
+81	I-residue_name_number
+and	O
+Asp	B-residue_name_number
+83	I-residue_name_number
+interact	O
+with	O
+His	B-residue_name_number
+138	I-residue_name_number
+,	O
+His	B-residue_name_number
+159	I-residue_name_number
+and	O
+His	B-residue_name_number
+158	I-residue_name_number
+to	O
+maintain	O
+the	O
+conformation	O
+of	O
+the	O
+β3	B-structure_element
+-	I-structure_element
+β4	I-structure_element
+loop	I-structure_element
+,	O
+thus	O
+contribute	O
+to	O
+control	O
+the	O
+substrate	O
+binding	O
+(	O
+Fig	O
+.	O
+4D	O
+).	O
+To	O
+verify	O
+this	O
+hypothesis	O
+,	O
+we	O
+mutated	B-experimental_method
+Glu	B-residue_name_number
+80	I-residue_name_number
+,	O
+Asp	B-residue_name_number
+81	I-residue_name_number
+and	O
+Asp	B-residue_name_number
+83	I-residue_name_number
+to	O
+Ala	B-residue_name
+respectively	O
+.	O
+In	O
+line	O
+with	O
+our	O
+hypothesis	O
+,	O
+E80A	B-mutant
+,	O
+D81A	B-mutant
+and	O
+D83A	B-mutant
+mutants	B-protein_state
+exhibit	O
+at	O
+least	O
+2	O
+-	O
+fold	O
+increase	O
+in	O
+hNaa60	B-protein
+-	O
+activity	O
+(	O
+Fig	O
+.	O
+5A	O
+).	O
+Interestingly	O
+,	O
+the	O
+structure	B-evidence
+of	O
+an	O
+ancestral	O
+NAT	B-protein_type
+from	O
+S	B-species
+.	I-species
+solfataricus	I-species
+also	O
+exhibits	O
+a	O
+10	B-structure_element
+-	I-structure_element
+residue	I-structure_element
+long	I-structure_element
+extension	I-structure_element
+between	O
+β3	B-structure_element
+and	O
+β4	B-structure_element
+,	O
+and	O
+the	O
+structure	B-experimental_method
+and	I-experimental_method
+biochemical	I-experimental_method
+studies	I-experimental_method
+showed	O
+that	O
+the	O
+extension	B-structure_element
+of	O
+SsNat	B-protein
+has	O
+the	O
+ability	O
+to	O
+stabilize	O
+structure	O
+of	O
+the	O
+active	B-site
+site	I-site
+and	O
+potentiate	O
+SsNat	B-protein
+-	O
+activity	O
+.	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+,	O
+which	O
+is	O
+carried	O
+out	O
+by	O
+the	O
+NAT	B-protein_type
+family	I-protein_type
+acetyltransferases	I-protein_type
+,	O
+is	O
+an	O
+ancient	O
+and	O
+essential	O
+modification	O
+of	O
+proteins	O
+.	O
+Although	O
+many	O
+NATs	B-protein_type
+are	O
+highly	B-protein_state
+conserved	I-protein_state
+from	O
+lower	B-taxonomy_domain
+to	O
+higher	B-taxonomy_domain
+eukaryotes	I-taxonomy_domain
+and	O
+the	O
+substrate	O
+bias	O
+of	O
+them	O
+appears	O
+to	O
+be	O
+partially	O
+overlapped	O
+,	O
+there	O
+is	O
+a	O
+significant	O
+increase	O
+in	O
+the	O
+overall	O
+level	O
+of	O
+N	B-ptm
+-	I-ptm
+terminal	I-ptm
+acetylation	I-ptm
+from	O
+lower	B-taxonomy_domain
+to	O
+higher	B-taxonomy_domain
+eukaryotes	I-taxonomy_domain
+.	O
+In	O
+this	O
+study	O
+we	O
+provide	O
+structural	O
+insights	O
+into	O
+Naa60	B-protein
+found	O
+only	O
+in	O
+multicellular	B-taxonomy_domain
+eukaryotes	I-taxonomy_domain
+.	O
+The	O
+N	O
+-	O
+terminus	O
+of	O
+hNaa60	B-protein
+harbors	O
+three	O
+hydrophobic	O
+residues	O
+(	O
+VVP	B-structure_element
+)	O
+that	O
+makes	O
+it	O
+very	O
+difficult	O
+to	O
+express	O
+and	O
+purify	O
+the	O
+protein	O
+.	O
+This	O
+problem	O
+was	O
+solved	O
+by	O
+replacing	B-experimental_method
+residues	O
+4	B-residue_range
+–	I-residue_range
+6	I-residue_range
+from	O
+VVP	B-structure_element
+to	O
+EER	B-structure_element
+that	O
+are	O
+found	O
+in	O
+Naa60	B-protein
+from	O
+Xenopus	B-species
+Laevis	I-species
+.	O
+Since	O
+Naa60	B-protein
+from	O
+human	B-species
+and	O
+from	O
+Xenopus	B-species
+Laevis	I-species
+are	O
+highly	B-protein_state
+homologous	I-protein_state
+(	O
+Fig	O
+.	O
+1A	O
+),	O
+we	O
+speculate	O
+that	O
+these	O
+two	O
+proteins	O
+should	O
+have	O
+the	O
+same	O
+biological	O
+function	O
+.	O
+Therefore	O
+it	O
+is	O
+deduced	O
+that	O
+the	O
+VVP	B-mutant
+to	I-mutant
+EER	I-mutant
+replacement	B-experimental_method
+on	O
+the	O
+N	O
+-	O
+terminus	O
+of	O
+hNaa60	B-protein
+may	O
+not	O
+interfere	O
+with	O
+its	O
+function	O
+.	O
+However	O
+,	O
+in	O
+the	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+structure	B-evidence
+the	O
+N	O
+-	O
+terminus	O
+adopts	O
+an	O
+α	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+structure	I-structure_element
+which	O
+will	O
+probably	O
+be	O
+kinked	O
+if	O
+residue	O
+6	B-residue_number
+is	O
+proline	B-residue_name
+(	O
+Fig	O
+.	O
+1C	O
+),	O
+and	O
+in	O
+the	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+structure	B-evidence
+the	O
+N	O
+-	O
+terminus	O
+adopts	O
+a	O
+different	O
+semi	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+structure	I-structure_element
+(	O
+Fig	O
+.	O
+1B	O
+)	O
+likely	O
+due	O
+to	O
+different	O
+crystal	B-evidence
+packing	I-evidence
+.	O
+Hence	O
+it	O
+is	O
+not	O
+clear	O
+if	O
+the	O
+N	O
+-	O
+terminal	O
+end	O
+of	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+hNaa60	B-protein
+is	O
+an	O
+α	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+,	O
+and	O
+what	O
+roles	O
+the	O
+hydrophobic	O
+residues	O
+4	B-residue_range
+–	I-residue_range
+6	I-residue_range
+play	O
+in	O
+structure	O
+and	O
+function	O
+of	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+hNaa60	B-protein
+.	O
+In	O
+addition	O
+to	O
+the	O
+three	O
+-	O
+residue	O
+mutation	B-experimental_method
+(	O
+VVP	B-structure_element
+to	O
+EER	B-structure_element
+),	O
+we	O
+also	O
+tried	O
+many	O
+other	O
+hNaa60	B-protein
+constructs	O
+,	O
+but	O
+only	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+protein	O
+and	O
+the	O
+truncated	B-protein_state
+variant	O
+1	B-residue_range
+-	I-residue_range
+199	I-residue_range
+behaved	O
+well	O
+.	O
+The	O
+finding	O
+that	O
+the	O
+catalytic	O
+activity	O
+of	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+is	O
+much	O
+lower	O
+than	O
+that	O
+of	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+is	O
+intriguing	O
+.	O
+We	O
+speculate	O
+that	O
+low	O
+activity	O
+of	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+hNaa60	B-protein
+might	O
+be	O
+related	O
+to	O
+lack	O
+of	O
+Golgi	O
+localization	O
+of	O
+the	O
+enzyme	O
+in	O
+our	O
+in	O
+vitro	O
+studies	O
+or	O
+there	O
+remains	O
+some	O
+undiscovered	O
+auto	O
+-	O
+inhibitory	O
+regulation	O
+in	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+protein	O
+.	O
+The	O
+hNaa60	B-protein
+protein	O
+was	O
+proven	O
+to	O
+be	O
+localized	O
+on	O
+Golgi	O
+apparatus	O
+.	O
+Aksnes	O
+and	O
+colleagues	O
+predicted	O
+putative	O
+transmembrane	B-structure_element
+domains	I-structure_element
+and	O
+two	O
+putative	O
+sites	O
+of	O
+S	B-ptm
+-	I-ptm
+palmitoylation	I-ptm
+,	O
+by	O
+bioinformatics	O
+means	O
+,	O
+to	O
+account	O
+for	O
+Golgi	O
+localization	O
+of	O
+the	O
+protein	O
+.	O
+They	O
+then	O
+mutated	B-experimental_method
+all	O
+five	O
+cysteine	B-residue_name
+residues	O
+of	O
+hNaa60	B-protein
+’	O
+s	O
+to	O
+serine	B-residue_name
+,	O
+including	O
+the	O
+two	O
+putative	O
+S	B-site
+-	I-site
+palmitoylation	I-site
+sites	I-site
+.	O
+However	O
+,	O
+these	O
+mutations	B-experimental_method
+did	O
+not	O
+abolish	O
+Naa60	B-protein
+membrane	O
+localization	O
+,	O
+indicating	O
+that	O
+S	B-ptm
+-	I-ptm
+palmitoylation	I-ptm
+is	O
+unlikely	O
+to	O
+(	O
+solely	O
+)	O
+account	O
+for	O
+targeting	O
+hNaa60	B-protein
+on	O
+Golgi	O
+.	O
+Furthermore	O
+,	O
+adding	B-experimental_method
+residues	O
+217	B-residue_range
+–	I-residue_range
+242	I-residue_range
+of	O
+hNaa60	B-protein
+(	O
+containing	O
+residues	O
+217	B-residue_range
+–	I-residue_range
+236	I-residue_range
+,	O
+one	O
+of	O
+the	O
+putative	O
+transmembrane	B-structure_element
+domains	I-structure_element
+)	O
+to	O
+the	O
+C	O
+terminus	O
+of	O
+eGFP	B-experimental_method
+were	O
+not	O
+sufficient	O
+to	O
+localize	O
+the	O
+protein	O
+on	O
+Golgi	O
+apparatus	O
+,	O
+while	O
+eGFP	B-experimental_method
+-	O
+hNaa60182	B-mutant
+-	I-mutant
+242	I-mutant
+was	O
+sufficient	O
+to	O
+,	O
+suggesting	O
+that	O
+residues	O
+182	B-residue_range
+–	I-residue_range
+216	I-residue_range
+are	O
+important	O
+for	O
+Golgi	O
+localization	O
+of	O
+hNaa60	B-protein
+.	O
+We	O
+found	O
+that	O
+residues	O
+190	B-residue_range
+–	I-residue_range
+202	I-residue_range
+formed	O
+an	O
+amphipathic	B-structure_element
+helix	I-structure_element
+with	O
+an	O
+array	O
+of	O
+hydrophobic	O
+residues	O
+located	O
+on	O
+one	O
+side	O
+.	O
+This	O
+observation	O
+is	O
+reminiscent	O
+of	O
+the	O
+protein	O
+/	O
+membrane	O
+interaction	O
+through	O
+amphipathic	B-structure_element
+helices	I-structure_element
+in	O
+the	O
+cases	O
+of	O
+KalSec14	B-protein
+,	O
+Atg3	B-protein
+,	O
+PB1	B-protein
+-	I-protein
+F2	I-protein
+etc	O
+.	O
+In	O
+this	O
+model	O
+an	O
+amphipathic	B-structure_element
+helix	I-structure_element
+can	O
+immerse	O
+its	O
+hydrophobic	O
+side	O
+into	O
+the	O
+lipid	O
+bilayer	O
+through	O
+hydrophobic	B-bond_interaction
+interactions	I-bond_interaction
+.	O
+Therefore	O
+we	O
+propose	O
+that	O
+the	O
+amphipathic	B-structure_element
+helix	I-structure_element
+α5	B-structure_element
+may	O
+contribute	O
+to	O
+Golgi	O
+localization	O
+of	O
+hNaa60	B-protein
+.	O
+Previous	O
+studies	O
+indicated	O
+that	O
+members	O
+of	O
+NAT	B-protein_type
+family	O
+are	O
+bi	O
+-	O
+functional	O
+NAT	B-protein_type
+and	O
+KAT	B-protein_type
+enzymes	O
+.	O
+However	O
+,	O
+known	O
+structures	B-evidence
+of	O
+NATs	B-protein_type
+do	O
+not	O
+well	O
+support	O
+this	O
+hypothesis	O
+,	O
+since	O
+the	O
+β6	B-structure_element
+-	I-structure_element
+β7	I-structure_element
+hairpin	I-structure_element
+/	O
+loop	B-structure_element
+of	O
+most	O
+of	O
+NATs	B-protein_type
+is	O
+involved	O
+in	O
+the	O
+formation	O
+of	O
+a	O
+tunnel	B-site
+-	I-site
+like	I-site
+substrate	I-site
+-	I-site
+binding	I-site
+site	I-site
+with	O
+the	O
+α1	B-structure_element
+-	I-structure_element
+α2	I-structure_element
+loop	I-structure_element
+,	O
+which	O
+would	O
+be	O
+good	O
+for	O
+the	O
+NAT	B-protein_type
+but	O
+not	O
+KAT	B-protein_type
+activity	O
+of	O
+the	O
+enzyme	O
+.	O
+Kinetic	B-experimental_method
+studies	I-experimental_method
+have	O
+been	O
+conducted	O
+to	O
+compare	O
+the	O
+NAT	B-protein_type
+and	O
+KAT	B-protein_type
+activity	O
+of	O
+hNaa50	B-protein
+in	O
+vitro	O
+,	O
+and	O
+indicate	O
+that	O
+the	O
+NAT	B-protein_type
+activity	O
+of	O
+Naa50	B-protein
+is	O
+much	O
+higher	O
+than	O
+KAT	B-protein_type
+activity	O
+.	O
+However	O
+,	O
+the	O
+substrate	O
+used	O
+in	O
+this	O
+study	O
+for	O
+assessing	O
+KAT	B-protein_type
+activity	O
+was	O
+a	O
+small	O
+peptide	B-chemical
+which	O
+could	O
+not	O
+really	O
+mimic	O
+the	O
+3D	B-evidence
+structure	I-evidence
+of	O
+a	O
+folded	B-protein_state
+protein	O
+substrate	O
+in	O
+vivo	O
+.	O
+Our	O
+mass	B-experimental_method
+spectrometry	I-experimental_method
+data	B-evidence
+indicated	O
+that	O
+there	O
+were	O
+robust	O
+acetylation	B-ptm
+of	O
+histone	B-protein_type
+H3	B-complex_assembly
+-	I-complex_assembly
+H4	I-complex_assembly
+tetramer	B-oligomeric_state
+lysines	B-residue_name
+and	O
+both	O
+N	B-ptm
+-	I-ptm
+terminal	I-ptm
+acetylation	I-ptm
+and	O
+lysine	B-ptm
+acetylation	I-ptm
+of	O
+the	O
+peptide	B-chemical
+used	O
+in	O
+the	O
+activity	B-experimental_method
+assay	I-experimental_method
+,	O
+thus	O
+confirmed	O
+the	O
+KAT	B-protein_type
+activity	O
+of	O
+this	O
+enzyme	O
+in	O
+vitro	O
+.	O
+Conformational	O
+change	O
+of	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+(	O
+corresponding	O
+to	O
+the	O
+β6	B-structure_element
+-	I-structure_element
+β7	I-structure_element
+loop	I-structure_element
+of	O
+other	O
+NATs	B-protein_type
+)	O
+is	O
+noted	O
+in	O
+our	O
+structures	B-evidence
+(	O
+Figs	O
+1D	O
+and	O
+2C	O
+),	O
+which	O
+might	O
+provide	O
+an	O
+explanation	O
+to	O
+the	O
+NAT	B-protein_type
+/	O
+KAT	B-protein_type
+dual	O
+-	O
+activity	O
+in	O
+a	O
+structural	O
+biological	O
+view	O
+,	O
+but	O
+we	O
+were	O
+unable	O
+to	O
+rule	O
+out	O
+the	O
+possibility	O
+that	O
+the	O
+observed	O
+conformational	O
+change	O
+of	O
+this	O
+hairpin	B-structure_element
+might	O
+be	O
+an	O
+artifact	O
+related	O
+to	O
+crystal	B-evidence
+packing	I-evidence
+or	O
+truncation	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+end	O
+of	O
+the	O
+protein	O
+.	O
+Further	O
+studies	O
+are	O
+therefore	O
+needed	O
+to	O
+reveal	O
+the	O
+mechanism	O
+for	O
+the	O
+KAT	B-protein_type
+activity	O
+of	O
+this	O
+enzyme	O
+.	O
+In	O
+early	O
+years	O
+,	O
+researchers	O
+found	O
+adjustment	O
+of	O
+GCN5	B-protein_type
+histone	I-protein_type
+acetyltransferase	I-protein_type
+structure	B-evidence
+when	O
+it	O
+binds	O
+CoA	B-chemical
+molecule	O
+.	O
+The	O
+complexed	B-protein_state
+form	O
+of	O
+NatA	B-complex_assembly
+is	O
+more	O
+suitable	O
+for	O
+catalytic	O
+activation	O
+,	O
+since	O
+the	O
+α1	B-structure_element
+-	I-structure_element
+α2	I-structure_element
+loop	I-structure_element
+undergoes	O
+a	O
+conformation	O
+change	O
+to	O
+participate	O
+in	O
+the	O
+formation	O
+of	O
+substrate	B-site
+-	I-site
+binding	I-site
+site	I-site
+when	O
+the	O
+auxiliary	O
+subunit	O
+Naa15	B-protein
+interacts	O
+with	O
+Naa10	B-protein
+(	O
+the	O
+catalytic	B-protein_state
+subunit	B-structure_element
+of	O
+NatA	B-complex_assembly
+).	O
+In	O
+the	O
+structure	B-evidence
+of	O
+hNaa50	B-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+/	I-complex_assembly
+peptide	I-complex_assembly
+,	O
+Phe	B-residue_name_number
+27	I-residue_name_number
+in	O
+the	O
+α1	B-structure_element
+-	I-structure_element
+α2	I-structure_element
+loop	I-structure_element
+appears	O
+to	O
+make	O
+hydrophobic	B-bond_interaction
+interaction	I-bond_interaction
+with	O
+the	O
+N	O
+-	O
+terminal	O
+Met	B-residue_name
+of	O
+substrate	O
+peptide	B-chemical
+.	O
+However	O
+,	O
+the	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+crystal	B-evidence
+structure	I-evidence
+indicated	O
+that	O
+its	O
+counterpart	O
+in	O
+hNaa60	B-protein
+,	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+,	O
+could	O
+also	O
+accommodate	O
+the	O
+binding	O
+of	O
+a	O
+hydrophilic	O
+malonate	B-chemical
+that	O
+occupied	O
+the	O
+substrate	B-site
+binding	I-site
+site	I-site
+although	O
+it	O
+maintained	O
+the	O
+same	O
+conformation	O
+as	O
+that	O
+observed	O
+in	O
+hNaa50	B-protein
+.	O
+Interestingly	O
+,	O
+the	O
+terminal	O
+thiol	B-chemical
+of	O
+CoA	B-chemical
+adopted	O
+alternative	O
+conformations	O
+in	O
+the	O
+structure	B-evidence
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+.	O
+One	O
+was	O
+to	O
+approach	O
+the	O
+substrate	O
+amine	O
+;	O
+the	O
+other	O
+was	O
+to	O
+approach	O
+the	O
+α1	B-structure_element
+-	I-structure_element
+α2	I-structure_element
+loop	I-structure_element
+and	O
+away	O
+from	O
+the	O
+substrate	O
+amine	O
+.	O
+Same	O
+alternative	O
+conformations	O
+of	O
+CoA	B-chemical
+were	O
+observed	O
+in	O
+the	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)(	I-mutant
+F34A	I-mutant
+)	I-mutant
+crystal	B-evidence
+structure	I-evidence
+,	O
+and	O
+our	O
+kinetic	B-evidence
+data	I-evidence
+showed	O
+that	O
+the	O
+F34A	B-mutant
+mutation	B-experimental_method
+abolished	O
+the	O
+activity	O
+of	O
+the	O
+enzyme	O
+.	O
+Taken	O
+together	O
+,	O
+our	O
+data	O
+indicated	O
+that	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+in	O
+hNaa60	B-protein
+may	O
+play	O
+a	O
+role	O
+in	O
+placing	O
+co	O
+-	O
+enzyme	O
+at	O
+the	O
+right	O
+location	O
+to	O
+facilitate	O
+the	O
+acetyl	B-chemical
+-	O
+transfer	O
+.	O
+However	O
+,	O
+these	O
+data	O
+did	O
+not	O
+rule	O
+out	O
+that	O
+possibility	O
+that	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+may	O
+coordinate	O
+the	O
+binding	O
+of	O
+the	O
+N	O
+-	O
+terminal	O
+Met	B-residue_name
+through	O
+hydrophobic	B-bond_interaction
+interaction	I-bond_interaction
+as	O
+was	O
+proposed	O
+by	O
+previous	O
+studies	O
+.	O
+Furthermore	O
+,	O
+we	O
+showed	O
+that	O
+hNaa60	B-protein
+adopts	O
+the	O
+classical	O
+two	O
+base	O
+mechanism	O
+to	O
+catalyze	O
+acetyl	B-chemical
+-	O
+transfer	O
+.	O
+Although	O
+sequence	O
+identity	O
+between	O
+hNaa60	B-protein
+and	O
+hNaa50	B-protein
+is	O
+low	O
+,	O
+key	O
+residues	O
+in	O
+the	O
+active	B-site
+site	I-site
+of	O
+both	O
+enzymes	O
+are	O
+highly	B-protein_state
+conserved	I-protein_state
+.	O
+This	O
+can	O
+reasonably	O
+explain	O
+the	O
+high	O
+overlapping	O
+substrates	O
+specificities	O
+between	O
+hNaa60	B-protein
+and	O
+hNaa50	B-protein
+.	O
+Another	O
+structural	O
+feature	O
+of	O
+hNaa60	B-protein
+that	O
+distinguishes	O
+it	O
+from	O
+other	O
+NATs	B-protein_type
+is	O
+the	O
+β3	B-structure_element
+-	I-structure_element
+β4	I-structure_element
+long	I-structure_element
+loop	I-structure_element
+which	O
+appears	O
+to	O
+inhibit	O
+the	O
+catalytic	O
+activity	O
+of	O
+hNaa60	B-protein
+.	O
+However	O
+,	O
+this	O
+loop	B-structure_element
+also	O
+seems	O
+to	O
+stabilize	O
+the	O
+whole	O
+hNaa60	B-protein
+structure	B-evidence
+,	O
+because	O
+deletion	B-experimental_method
+mutations	I-experimental_method
+of	O
+this	O
+region	O
+led	O
+to	O
+protein	O
+precipitation	O
+and	O
+aggregation	O
+(	O
+Figure	O
+S7	O
+).	O
+A	O
+previous	O
+study	O
+suggested	O
+that	O
+the	O
+auto	B-ptm
+-	I-ptm
+acetylation	I-ptm
+of	O
+Lys	B-residue_name_number
+79	I-residue_name_number
+was	O
+important	O
+for	O
+hNaa60	B-protein
+-	O
+activity	O
+,	O
+whereas	O
+the	O
+point	B-experimental_method
+mutation	I-experimental_method
+K79R	B-mutant
+did	O
+not	O
+decrease	O
+the	O
+activity	O
+of	O
+hNaa60	B-protein
+in	O
+our	O
+study	O
+.	O
+Meanwhile	O
+,	O
+no	O
+electron	B-evidence
+density	I-evidence
+of	O
+acetyl	B-chemical
+group	O
+was	O
+found	O
+on	O
+Lys	B-residue_name_number
+79	I-residue_name_number
+in	O
+our	O
+structures	B-evidence
+and	O
+mass	B-experimental_method
+spectrometry	I-experimental_method
+analysis	O
+.	O
+Hence	O
+,	O
+it	O
+appears	O
+that	O
+the	O
+auto	B-ptm
+-	I-ptm
+acetylation	I-ptm
+of	O
+hNaa60	B-protein
+is	O
+not	O
+an	O
+essential	O
+modification	O
+for	O
+its	O
+activity	O
+for	O
+the	O
+protein	O
+we	O
+used	O
+here	O
+.	O
+As	O
+for	O
+the	O
+reason	O
+why	O
+K79R	B-mutant
+in	O
+Yang	O
+’	O
+s	O
+previous	O
+studies	O
+reduced	O
+the	O
+activity	O
+of	O
+the	O
+enzyme	O
+,	O
+but	O
+in	O
+our	O
+studies	O
+it	O
+didn	O
+’	O
+t	O
+,	O
+we	O
+suspect	O
+that	O
+the	O
+stability	O
+of	O
+this	O
+mutant	B-protein_state
+may	O
+play	O
+some	O
+role	O
+.	O
+K79R	B-mutant
+is	O
+less	O
+stable	B-protein_state
+than	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+enzyme	O
+as	O
+was	O
+judged	O
+by	O
+its	O
+poorer	O
+gel	B-experimental_method
+-	I-experimental_method
+filtration	I-experimental_method
+behavior	O
+and	O
+tendency	O
+to	O
+precipitate	O
+.	O
+In	O
+our	O
+studies	O
+we	O
+have	O
+paid	O
+special	O
+attention	O
+and	O
+carefully	O
+handled	O
+this	O
+protein	O
+to	O
+ensure	O
+that	O
+we	O
+did	O
+get	O
+enough	O
+of	O
+the	O
+protein	O
+in	O
+good	O
+condition	O
+for	O
+kinetic	B-experimental_method
+assays	I-experimental_method
+.	O
+The	O
+intracellular	O
+environment	O
+is	O
+more	O
+complicated	O
+than	O
+our	O
+in	O
+vitro	O
+assay	O
+and	O
+the	O
+substrate	O
+specificity	O
+of	O
+hNaa60	B-protein
+most	O
+focuses	O
+on	O
+transmembrane	O
+proteins	O
+.	O
+The	O
+interaction	O
+between	O
+hNaa60	B-protein
+and	O
+its	O
+substrates	O
+may	O
+involve	O
+the	O
+protein	O
+-	O
+membrane	O
+interaction	O
+which	O
+would	O
+further	O
+increase	O
+the	O
+complexity	O
+.	O
+It	O
+is	O
+not	O
+clear	O
+if	O
+the	O
+structure	B-evidence
+of	O
+hNaa60	B-protein
+is	O
+different	O
+in	O
+vivo	O
+or	O
+if	O
+other	O
+potential	O
+partner	O
+proteins	O
+may	O
+help	O
+to	O
+regulate	O
+its	O
+activity	O
+.	O
+Nevertheless	O
+,	O
+our	O
+study	O
+may	O
+be	O
+an	O
+inspiration	O
+for	O
+further	O
+studies	O
+on	O
+the	O
+functions	O
+and	O
+regulation	O
+of	O
+this	O
+youngest	O
+member	O
+of	O
+the	O
+NAT	B-protein_type
+family	O
+.	O
+Overall	O
+structure	B-evidence
+of	O
+Naa60	B-protein
+.	O
+(	O
+A	O
+)	O
+Sequence	B-experimental_method
+alignment	I-experimental_method
+of	O
+Naa60	B-protein
+(	O
+NatF	B-complex_assembly
+,	O
+HAT4	B-protein
+)	O
+from	O
+different	O
+species	O
+including	O
+Homo	B-species
+sapiens	I-species
+(	O
+Homo	B-species
+),	O
+Bos	B-species
+mutus	I-species
+(	O
+Bos	B-species
+),	O
+Salmo	B-species
+salar	I-species
+(	O
+Salmo	B-species
+)	O
+and	O
+Xenopus	B-species
+(	O
+Silurana	B-species
+)	O
+tropicalis	B-species
+(	O
+Xenopus	B-species
+).	O
+Alignment	B-experimental_method
+was	O
+generated	O
+using	O
+NPS	O
+@	O
+and	O
+ESPript	O
+.	O
+3	O
+.	O
+0	O
+(	O
+http	O
+://	O
+espript	O
+.	O
+ibcp	O
+.	O
+fr	O
+/	O
+ESPript	O
+/	O
+ESPript	O
+/).	O
+Residues	O
+4	B-residue_range
+–	I-residue_range
+6	I-residue_range
+are	O
+highlighted	O
+in	O
+red	O
+box	O
+.	O
+(	O
+B	O
+)	O
+The	O
+structure	B-evidence
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+complex	O
+is	O
+shown	O
+as	O
+a	O
+yellow	O
+cartoon	O
+model	O
+.	O
+The	O
+CoA	B-chemical
+molecule	O
+is	O
+shown	O
+as	O
+sticks	O
+.	O
+(	O
+C	O
+)	O
+The	O
+structure	B-evidence
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+complex	O
+is	O
+presented	O
+as	O
+a	O
+cartoon	O
+model	O
+in	O
+cyan	O
+.	O
+The	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+and	O
+malonate	B-chemical
+molecules	O
+are	O
+shown	O
+as	O
+cyan	O
+and	O
+purple	O
+sticks	O
+,	O
+respectively	O
+.	O
+The	O
+secondary	O
+structures	O
+are	O
+labeled	O
+starting	O
+with	O
+α0	B-structure_element
+.	O
+(	O
+D	O
+)	O
+Superposition	B-experimental_method
+of	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+(	O
+cyan	O
+),	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+(	O
+yellow	O
+)	O
+and	O
+hNaa50	B-protein
+(	O
+pink	O
+,	O
+PDB	O
+3TFY	O
+).	O
+The	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+complex	O
+is	O
+represented	O
+as	O
+cyan	O
+sticks	O
+.	O
+Amphipathicity	B-protein_state
+of	O
+the	O
+α5	B-structure_element
+helix	I-structure_element
+and	O
+alternative	O
+conformations	O
+of	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+.	O
+(	O
+A	O
+)	O
+The	O
+α5	B-structure_element
+helix	I-structure_element
+of	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+in	O
+one	O
+asymmetric	O
+unit	O
+(	O
+slate	O
+)	O
+interacts	O
+with	O
+another	O
+hNaa60	B-protein
+molecule	O
+in	O
+a	O
+neighboring	O
+asymmetric	O
+unit	O
+(	O
+cyan	O
+).	O
+Side	O
+-	O
+chains	O
+of	O
+hydrophobic	O
+residues	O
+on	O
+α5	B-structure_element
+helix	I-structure_element
+and	O
+the	O
+neighboring	O
+molecule	O
+participating	O
+in	O
+the	O
+interaction	O
+are	O
+shown	O
+as	O
+yellow	O
+and	O
+green	O
+sticks	O
+,	O
+respectively	O
+.	O
+(	O
+B	O
+)	O
+The	O
+α5	B-structure_element
+helix	I-structure_element
+of	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+in	O
+one	O
+asymmetric	O
+unit	O
+(	O
+yellow	O
+)	O
+interacts	O
+with	O
+another	O
+hNaa60	B-protein
+molecule	O
+in	O
+the	O
+neighboring	O
+asymmetric	O
+units	O
+(	O
+green	O
+).	O
+Side	O
+-	O
+chains	O
+of	O
+hydrophobic	O
+residues	O
+on	O
+α5	B-structure_element
+helix	I-structure_element
+and	O
+the	O
+neighboring	O
+molecule	O
+(	O
+green	O
+)	O
+participating	O
+in	O
+the	O
+interaction	O
+are	O
+shown	O
+as	O
+yellow	O
+and	O
+green	O
+sticks	O
+,	O
+respectively	O
+.	O
+The	O
+third	O
+molecule	O
+(	O
+pink	O
+)	O
+does	O
+not	O
+directly	O
+interact	O
+with	O
+the	O
+α5	B-structure_element
+helix	I-structure_element
+.	O
+(	O
+C	O
+)	O
+Superposition	B-experimental_method
+of	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+(	O
+yellow	O
+)	O
+and	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+(	O
+cyan	O
+)	O
+showing	O
+conformational	O
+change	O
+of	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+in	O
+these	O
+two	O
+structures	B-evidence
+.	O
+(	O
+D	O
+,	O
+E	O
+)	O
+Superposition	B-experimental_method
+of	O
+Hat1p	B-protein
+/	O
+H4	B-protein_type
+(	O
+gray	O
+,	O
+drawn	O
+from	O
+PDB	O
+4PSW	O
+)	O
+with	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+(	O
+cyan	O
+,	O
+D	O
+)	O
+or	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+(	O
+yellow	O
+,	O
+E	O
+).	O
+The	O
+histone	B-protein_type
+H4	B-protein_type
+peptide	B-chemical
+(	O
+a	O
+KAT	B-protein_type
+substrate	O
+)	O
+bound	B-protein_state
+to	I-protein_state
+Hat1p	B-protein
+is	O
+shown	O
+in	O
+purple	O
+(	O
+D	O
+,	O
+E	O
+),	O
+while	O
+the	O
+peptide	B-chemical
+bound	B-protein_state
+to	I-protein_state
+hNaa50	B-protein
+(	O
+a	O
+NAT	B-protein_type
+substrate	O
+,	O
+drawn	O
+from	O
+PDB	O
+3TFY	O
+)	O
+is	O
+shown	O
+in	O
+orange	O
+(	O
+Nt	B-chemical
+-	I-chemical
+peptide	I-chemical
+)	O
+after	O
+superimposing	B-experimental_method
+hNaa50	B-protein
+(	O
+not	O
+shown	O
+in	O
+figure	O
+)	O
+on	O
+hNaa60	B-protein
+(	O
+D	O
+).	O
+The	O
+α	O
+-	O
+amine	O
+of	O
+the	O
+NAT	B-protein_type
+substrate	O
+and	O
+ε	O
+-	O
+amine	O
+of	O
+the	O
+KAT	B-protein_type
+substrate	O
+(	O
+along	O
+with	O
+the	O
+lysine	B-residue_name
+side	O
+-	O
+chain	O
+)	O
+subject	O
+to	O
+acetylation	B-ptm
+are	O
+shown	O
+as	O
+sticks	O
+.	O
+Electron	B-evidence
+density	I-evidence
+map	I-evidence
+of	O
+the	O
+active	B-site
+site	I-site
+.	O
+The	O
+2Fo	B-evidence
+-	I-evidence
+Fc	I-evidence
+maps	I-evidence
+contoured	O
+at	O
+1	O
+.	O
+0σ	O
+are	O
+shown	O
+for	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+(	O
+A	O
+),	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+(	O
+B	O
+)	O
+and	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)	I-complex_assembly
+F34A	I-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+(	O
+C	O
+).	O
+The	O
+putative	O
+substrate	B-site
+peptide	I-site
+binding	I-site
+site	I-site
+is	O
+indicated	O
+by	O
+the	O
+peptide	B-chemical
+(	O
+shown	O
+as	O
+pink	O
+sticks	O
+)	O
+from	O
+the	O
+hNaa50	B-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+/	I-complex_assembly
+peptide	I-complex_assembly
+complex	O
+structure	B-evidence
+after	O
+superimposing	B-experimental_method
+hNaa50	B-protein
+on	O
+the	O
+hNaa60	B-protein
+structures	B-evidence
+determined	O
+in	O
+this	O
+study	O
+.	O
+The	O
+black	O
+arrow	O
+indicates	O
+the	O
+α	O
+-	O
+amine	O
+of	O
+the	O
+first	B-residue_name_number
+Met	I-residue_name_number
+(	O
+M1	B-residue_name_number
+)	O
+(	O
+all	O
+panels	O
+).	O
+The	O
+purple	O
+arrow	O
+indicates	O
+the	O
+acetyl	B-chemical
+moiety	O
+of	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+(	O
+A	O
+).	O
+The	O
+red	O
+arrow	O
+indicates	O
+the	O
+alternative	O
+conformation	O
+of	O
+the	O
+thiol	O
+moiety	O
+of	O
+the	O
+co	O
+-	O
+enzyme	O
+when	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+side	O
+-	O
+chain	O
+is	O
+displaced	O
+(	O
+B	O
+)	O
+or	O
+mutated	B-experimental_method
+to	O
+Ala	B-residue_name
+(	O
+C	O
+).	O
+Structural	O
+basis	O
+for	O
+hNaa60	B-protein
+catalytic	O
+activity	O
+.	O
+(	O
+A	O
+)	O
+Superposition	B-experimental_method
+of	O
+hNaa60	B-protein
+active	B-site
+site	I-site
+(	O
+cyan	O
+)	O
+on	O
+that	O
+of	O
+hNaa50	B-protein
+(	O
+pink	O
+,	O
+PDB	O
+3TFY	O
+).	O
+Side	O
+-	O
+chains	O
+of	O
+key	O
+catalytic	B-site
+and	I-site
+substrate	I-site
+-	I-site
+binding	I-site
+residues	I-site
+are	O
+highlighted	O
+as	O
+sticks	O
+.	O
+The	O
+malonate	B-chemical
+molecule	O
+in	O
+the	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+structure	B-evidence
+and	O
+the	O
+peptide	B-chemical
+in	O
+the	O
+hNaa50	B-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+/	I-complex_assembly
+peptide	I-complex_assembly
+structure	B-evidence
+are	O
+shown	O
+as	O
+purple	O
+and	O
+yellow	O
+sticks	O
+respectively	O
+.	O
+(	O
+B	O
+)	O
+A	O
+close	O
+view	O
+of	O
+the	O
+active	B-site
+site	I-site
+of	O
+hNaa60	B-protein
+.	O
+Residues	O
+Glu	B-residue_name_number
+37	I-residue_name_number
+,	O
+Tyr	B-residue_name_number
+97	I-residue_name_number
+and	O
+His	B-residue_name_number
+138	I-residue_name_number
+in	O
+hNaa60	B-protein
+(	O
+cyan	O
+)	O
+and	O
+corresponding	O
+residues	O
+(	O
+Tyr	B-residue_name_number
+73	I-residue_name_number
+and	O
+His	B-residue_name_number
+112	I-residue_name_number
+)	O
+in	O
+hNaa50	B-protein
+(	O
+pink	O
+)	O
+as	O
+well	O
+as	O
+the	O
+side	O
+-	O
+chain	O
+of	O
+corresponding	O
+residues	O
+(	O
+Glu	B-residue_name_number
+24	I-residue_name_number
+,	O
+His	B-residue_name_number
+72	I-residue_name_number
+and	O
+His	B-residue_name_number
+111	I-residue_name_number
+)	O
+in	O
+complexed	B-protein_state
+formed	O
+hNaa10p	B-protein
+(	O
+warmpink	O
+)	O
+are	O
+highlighted	O
+as	O
+sticks	O
+.	O
+The	O
+water	B-chemical
+molecules	O
+participating	O
+in	O
+catalysis	O
+in	O
+the	O
+hNaa60	B-protein
+and	O
+hNaa50	B-protein
+structures	B-evidence
+are	O
+showed	O
+as	O
+green	O
+and	O
+red	O
+spheres	O
+,	O
+separately	O
+.	O
+(	O
+C	O
+)	O
+The	O
+interaction	O
+between	O
+the	O
+malonate	B-chemical
+molecule	O
+and	O
+surrounding	O
+residues	O
+observed	O
+in	O
+the	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+structure	B-evidence
+.	O
+The	O
+yellow	O
+dotted	O
+lines	O
+indicate	O
+the	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+.	O
+(	O
+D	O
+)	O
+A	O
+zoomed	O
+view	O
+of	O
+β3	B-structure_element
+-	I-structure_element
+β4	I-structure_element
+loop	I-structure_element
+of	O
+hNaa60	B-protein
+.	O
+Key	O
+residues	O
+discussed	O
+in	O
+the	O
+text	O
+(	O
+cyan	O
+),	O
+the	O
+malonate	B-chemical
+(	O
+purple	O
+)	O
+and	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+(	O
+gray	O
+)	O
+are	O
+shown	O
+as	O
+sticks	O
+.	O
+The	O
+yellow	O
+dotted	O
+lines	O
+indicate	O
+the	O
+salt	B-bond_interaction
+bridges	I-bond_interaction
+.	O
+Catalytic	O
+activity	O
+of	O
+hNaa60	B-protein
+and	O
+mutant	B-protein_state
+proteins	O
+.	O
+(	O
+A	O
+)	O
+Catalytic	B-evidence
+efficiency	I-evidence
+(	O
+shown	O
+as	O
+kcat	B-evidence
+/	O
+Km	B-evidence
+values	O
+)	O
+of	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+WT	B-protein_state
+and	O
+mutants	B-protein_state
+.	O
+(	O
+B	O
+)	O
+CD	B-experimental_method
+spectra	B-evidence
+of	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+mutant	B-protein_state
+proteins	O
+from	O
+250	O
+nm	O
+to	O
+190	O
+nm	O
+.	O
+The	O
+sample	O
+concentration	O
+was	O
+4	O
+.	O
+5	O
+μM	O
+in	O
+20	O
+mM	O
+Tris	O
+,	O
+pH	O
+8	O
+.	O
+0	O
+,	O
+150	O
+mM	O
+NaCl	O
+,	O
+1	O
+%	O
+glycerol	O
+and	O
+1	O
+mM	O
+TCEP	B-chemical
+at	O
+room	O
+temperature	O
+.	O
+Data	B-evidence
+collection	I-evidence
+and	I-evidence
+refinement	I-evidence
+statistics	I-evidence
+.	O
+Structure	O
+and	O
+PDB	O
+ID	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+5HGZ	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+5HH0	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)	I-complex_assembly
+F34A	I-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+5HH1	O
+Data	O
+collection	O
+*	O
+Space	O
+group	O
+P212121	O
+P21212	O
+P21212	O
+Cell	O
+dimensions	O
+a	O
+,	O
+b	O
+,	O
+c	O
+(	O
+Å	O
+)	O
+53	O
+.	O
+3	O
+,	O
+57	O
+.	O
+4	O
+,	O
+68	O
+.	O
+8	O
+67	O
+.	O
+8	O
+,	O
+73	O
+.	O
+8	O
+,	O
+43	O
+.	O
+2	O
+66	O
+.	O
+7	O
+,	O
+74	O
+.	O
+0	O
+,	O
+43	O
+.	O
+5	O
+α	O
+,	O
+β	O
+,	O
+γ	O
+(°)	O
+90	O
+.	O
+0	O
+,	O
+90	O
+.	O
+0	O
+,	O
+90	O
+.	O
+0	O
+90	O
+.	O
+0	O
+,	O
+90	O
+.	O
+0	O
+,	O
+90	O
+.	O
+0	O
+90	O
+.	O
+0	O
+,	O
+90	O
+.	O
+0	O
+,	O
+90	O
+.	O
+0	O
+Resolution	O
+(	O
+Å	O
+)	O
+50	O
+–	O
+1	O
+.	O
+38	O
+(	O
+1	O
+.	O
+42	O
+–	O
+1	O
+.	O
+38	O
+)	O
+50	O
+–	O
+1	O
+.	O
+60	O
+(	O
+1	O
+.	O
+66	O
+–	O
+1	O
+.	O
+60	O
+)	O
+50	O
+–	O
+1	O
+.	O
+80	O
+(	O
+1	O
+.	O
+86	O
+–	O
+1	O
+.	O
+80	O
+)	O
+Rp	O
+.	O
+i	O
+.	O
+m	O
+.(%)**	O
+3	O
+.	O
+0	O
+(	O
+34	O
+.	O
+4	O
+)	O
+2	O
+.	O
+1	O
+(	O
+32	O
+.	O
+5	O
+)	O
+2	O
+.	O
+6	O
+(	O
+47	O
+.	O
+8	O
+)	O
+I	O
+/	O
+σ	O
+21	O
+.	O
+5	O
+(	O
+2	O
+.	O
+0	O
+)	O
+31	O
+.	O
+8	O
+(	O
+2	O
+.	O
+0	O
+)	O
+28	O
+.	O
+0	O
+(	O
+2	O
+.	O
+4	O
+)	O
+Completeness	O
+(%)	O
+99	O
+.	O
+8	O
+(	O
+99	O
+.	O
+1	O
+)	O
+99	O
+.	O
+6	O
+(	O
+98	O
+.	O
+5	O
+)	O
+99	O
+.	O
+9	O
+(	O
+99	O
+.	O
+7	O
+)	O
+Redundancy	O
+6	O
+.	O
+9	O
+(	O
+5	O
+.	O
+0	O
+)	O
+6	O
+.	O
+9	O
+(	O
+6	O
+.	O
+2	O
+)	O
+6	O
+.	O
+3	O
+(	O
+5	O
+.	O
+9	O
+)	O
+Refinement	O
+Resolution	O
+(	O
+Å	O
+)	O
+25	O
+.	O
+81	O
+–	O
+1	O
+.	O
+38	O
+33	O
+.	O
+55	O
+–	O
+1	O
+.	O
+60	O
+43	O
+.	O
+52	O
+–	O
+1	O
+.	O
+80	O
+No	O
+.	O
+reflections	O
+43660	O
+28588	O
+20490	O
+Rwork	O
+/	O
+Rfree	O
+0	O
+.	O
+182	O
+/	O
+0	O
+.	O
+192	O
+0	O
+.	O
+181	O
+/	O
+0	O
+.	O
+184	O
+0	O
+.	O
+189	O
+/	O
+0	O
+.	O
+209	O
+No	O
+.	O
+atoms	O
+Protein	O
+1717	O
+1576	O
+1566	O
+Ligand	O
+/	O
+ion	O
+116	O
+96	O
+96	O
+Water	B-chemical
+289	O
+258	O
+168	O
+B	O
+-	O
+factors	O
+Protein	O
+23	O
+.	O
+8	O
+32	O
+.	O
+0	O
+37	O
+.	O
+4	O
+Ligand	O
+/	O
+ion	O
+22	O
+.	O
+2	O
+34	O
+.	O
+6	O
+43	O
+.	O
+7	O
+Water	B-chemical
+35	O
+.	O
+1	O
+46	O
+.	O
+4	O
+49	O
+.	O
+1	O
+R	O
+.	O
+m	O
+.	O
+s	O
+.	O
+One	O
+crystal	B-evidence
+was	O
+used	O
+for	O
+each	O
+data	O
+set	O
+.	O
+**	O
+Rp	O
+.	O
+i	O
+.	O
+m	O
+.,	O
+a	O
+redundancy	O
+-	O
+independent	O
+R	B-evidence
+factor	I-evidence
+was	O
+used	O
+to	O
+evaluate	O
+the	O
+diffraction	B-evidence
+data	I-evidence
+quality	O
+as	O
+was	O
+proposed	O
+by	O
+Evans	O
+.	O

annotation_IOB/PMC5012862.tsv ADDED Viewed

The diff for this file is too large to render. See raw diff

annotation_IOB/PMC5014086.tsv ADDED Viewed

	@@ -0,0 +1,4243 @@

+Structure	B-evidence
+of	O
+the	O
+Dual	O
+-	O
+Mode	O
+Wnt	B-protein_type
+Regulator	O
+Kremen1	B-protein
+and	O
+Insight	O
+into	O
+Ternary	O
+Complex	O
+Formation	O
+with	O
+LRP6	B-protein
+and	O
+Dickkopf	B-protein_type
+Kremen	B-protein_type
+1	I-protein_type
+and	I-protein_type
+2	I-protein_type
+have	O
+been	O
+identified	O
+as	O
+co	B-protein_type
+-	I-protein_type
+receptors	I-protein_type
+for	O
+Dickkopf	B-protein_type
+(	O
+Dkk	B-protein_type
+)	O
+proteins	O
+,	O
+hallmark	O
+secreted	O
+antagonists	O
+of	O
+canonical	O
+Wnt	B-protein_type
+signaling	O
+.	O
+We	O
+present	O
+here	O
+three	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+the	O
+ectodomain	B-structure_element
+of	O
+human	B-species
+Kremen1	B-protein
+(	O
+KRM1ECD	B-protein
+)	O
+at	O
+resolutions	O
+between	O
+1	O
+.	O
+9	O
+and	O
+3	O
+.	O
+2	O
+Å	O
+.	O
+KRM1ECD	B-protein
+emerges	O
+as	O
+a	O
+rigid	O
+molecule	O
+with	O
+tight	O
+interactions	O
+stabilizing	O
+a	O
+triangular	B-protein_state
+arrangement	I-protein_state
+of	O
+its	O
+Kringle	B-structure_element
+,	O
+WSC	B-structure_element
+,	O
+and	O
+CUB	B-structure_element
+structural	O
+domains	O
+.	O
+The	O
+structures	B-evidence
+reveal	O
+an	O
+unpredicted	O
+homology	O
+of	O
+the	O
+WSC	B-structure_element
+domain	O
+to	O
+hepatocyte	B-protein_type
+growth	I-protein_type
+factor	I-protein_type
+.	O
+We	O
+further	O
+report	O
+the	O
+general	O
+architecture	O
+of	O
+the	O
+ternary	O
+complex	O
+formed	O
+by	O
+the	O
+Wnt	B-protein_type
+co	B-protein_type
+-	I-protein_type
+receptor	I-protein_type
+Lrp5	B-protein_type
+/	I-protein_type
+6	I-protein_type
+,	O
+Dkk	B-protein_type
+,	O
+and	O
+Krm	B-protein_type
+,	O
+determined	O
+from	O
+a	O
+low	O
+-	O
+resolution	O
+complex	O
+crystal	B-evidence
+structure	I-evidence
+between	O
+β	B-structure_element
+-	I-structure_element
+propeller	I-structure_element
+/	I-structure_element
+EGF	I-structure_element
+repeats	I-structure_element
+(	I-structure_element
+PE	I-structure_element
+)	I-structure_element
+3	I-structure_element
+and	I-structure_element
+4	I-structure_element
+of	O
+the	O
+Wnt	B-protein_type
+co	B-protein_type
+-	I-protein_type
+receptor	I-protein_type
+LRP6	B-protein
+(	O
+LRP6PE3PE4	B-protein
+),	O
+the	O
+cysteine	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+domain	I-structure_element
+2	I-structure_element
+(	O
+CRD2	B-structure_element
+)	O
+of	O
+DKK1	B-protein
+,	O
+and	O
+KRM1ECD	B-protein
+.	O
+DKK1CRD2	B-protein
+is	O
+sandwiched	O
+between	O
+LRP6PE3	B-protein
+and	O
+KRM1Kringle	B-protein
+-	B-structure_element
+WSC	I-structure_element
+.	O
+Modeling	B-experimental_method
+studies	O
+supported	O
+by	O
+surface	B-experimental_method
+plasmon	I-experimental_method
+resonance	I-experimental_method
+suggest	O
+a	O
+direct	O
+interaction	B-site
+site	I-site
+between	O
+Krm1CUB	B-protein
+and	O
+Lrp6PE2	B-protein
+.	O
+The	O
+structure	B-evidence
+of	O
+the	O
+KREMEN	B-protein
+1	I-protein
+ectodomain	B-structure_element
+is	O
+solved	B-experimental_method
+from	O
+three	O
+crystal	B-evidence
+forms	I-evidence
+Kringle	B-structure_element
+,	O
+WSC	B-structure_element
+,	O
+and	O
+CUB	B-structure_element
+subdomains	O
+interact	O
+tightly	O
+to	O
+form	O
+a	O
+single	O
+structural	O
+unit	O
+The	O
+interface	B-site
+to	O
+DKKs	B-protein_type
+is	O
+formed	O
+from	O
+the	O
+Kringle	B-structure_element
+and	O
+WSC	B-structure_element
+domains	O
+The	O
+CUB	B-structure_element
+domain	O
+is	O
+found	O
+to	O
+interact	O
+directly	O
+with	O
+LRP6PE1PE2	B-protein
+Zebisch	O
+et	O
+al	O
+.	O
+describe	O
+the	O
+ectodomain	B-structure_element
+structure	B-evidence
+of	O
+KREMEN	B-protein
+1	I-protein
+,	O
+a	O
+receptor	B-protein_type
+for	O
+Wnt	B-protein_type
+antagonists	O
+of	O
+the	O
+DKK	B-protein_type
+family	O
+.	O
+Apo	B-protein_state
+structures	B-evidence
+and	O
+a	O
+complex	B-protein_state
+with	I-protein_state
+functional	B-protein_state
+fragments	I-protein_state
+of	O
+DKK1	B-protein
+and	O
+LRP6	B-protein
+shed	O
+light	O
+on	O
+the	O
+function	O
+of	O
+this	O
+dual	O
+-	O
+mode	O
+regulator	O
+of	O
+Wnt	B-protein_type
+signaling	O
+.	O
+Signaling	O
+by	O
+Wnt	B-protein_type
+morphogens	O
+is	O
+renowned	O
+for	O
+its	O
+fundamental	O
+roles	O
+in	O
+embryonic	O
+development	O
+,	O
+tissue	O
+homeostasis	O
+,	O
+and	O
+stem	O
+cell	O
+maintenance	O
+.	O
+Due	O
+to	O
+these	O
+functions	O
+,	O
+generation	O
+,	O
+delivery	O
+,	O
+and	O
+interpretation	O
+of	O
+Wnt	B-protein_type
+signals	O
+are	O
+all	O
+heavily	O
+regulated	O
+in	O
+the	O
+animal	O
+body	O
+.	O
+Vertebrate	B-taxonomy_domain
+Dickkopf	B-protein_type
+proteins	O
+(	O
+Dkk1	B-protein_type
+,	O
+2	B-protein_type
+,	O
+and	O
+4	B-protein_type
+)	O
+are	O
+one	O
+of	O
+many	O
+secreted	O
+antagonists	O
+of	O
+Wnt	B-protein_type
+and	O
+function	O
+by	O
+blocking	O
+access	O
+to	O
+the	O
+Wnt	B-protein_type
+co	B-protein_type
+-	I-protein_type
+receptor	I-protein_type
+LRP5	B-protein
+/	I-protein
+6	I-protein
+.	O
+Kremen	B-protein_type
+proteins	O
+(	O
+Krm1	B-protein_type
+and	O
+Krm2	B-protein_type
+)	O
+have	O
+been	O
+identified	O
+as	O
+additional	O
+high	O
+-	O
+affinity	O
+transmembrane	B-protein_type
+receptors	I-protein_type
+for	O
+Dkk	B-protein_type
+.	O
+Krm	B-protein_type
+and	O
+Dkk	B-protein_type
+synergize	O
+in	O
+Wnt	B-protein_type
+inhibition	O
+during	O
+Xenopus	B-taxonomy_domain
+embryogenesis	O
+to	O
+regulate	O
+anterior	O
+-	O
+posterior	O
+patterning	O
+.	O
+Mechanistically	O
+it	O
+is	O
+thought	O
+that	O
+,	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+Dkk	B-protein_type
+,	O
+Krm	B-protein_type
+forms	O
+a	O
+ternary	O
+complex	B-protein_state
+with	I-protein_state
+Lrp6	B-protein_type
+,	O
+which	O
+is	O
+then	O
+rapidly	O
+endocytosed	O
+.	O
+This	O
+amplifies	O
+the	O
+intrinsic	O
+Wnt	B-protein_type
+antagonistic	O
+activity	O
+of	O
+Dkk	B-protein_type
+by	O
+efficiently	O
+depleting	O
+the	O
+cell	O
+surface	O
+of	O
+the	O
+Wnt	B-protein_type
+co	B-protein_type
+-	I-protein_type
+receptor	I-protein_type
+.	O
+In	O
+accordance	O
+with	O
+this	O
+,	O
+Krm1	B-protein_type
+−/−	O
+and	O
+Krm2	B-protein_type
+−/−	O
+double	B-experimental_method
+knockout	I-experimental_method
+mice	B-taxonomy_domain
+show	O
+a	O
+high	O
+bone	O
+mass	O
+phenotype	O
+typical	O
+of	O
+increased	O
+Wnt	B-protein_type
+signaling	O
+,	O
+as	O
+well	O
+as	O
+growth	O
+of	O
+ectopic	O
+forelimb	O
+digits	O
+.	O
+Growth	O
+of	O
+ectopic	O
+digits	O
+is	O
+further	O
+enhanced	O
+upon	O
+additional	O
+loss	O
+of	O
+dkk	B-protein_type
+expression	O
+.	O
+The	O
+Wnt	B-protein_type
+antagonistic	O
+activity	O
+of	O
+Krm1	B-protein_type
+is	O
+also	O
+linked	O
+to	O
+its	O
+importance	O
+for	O
+correct	O
+thymus	O
+epithelium	O
+formation	O
+in	O
+mice	B-taxonomy_domain
+.	O
+The	O
+importance	O
+of	O
+intact	B-protein_state
+KRM1	B-protein
+for	O
+normal	O
+human	B-species
+development	O
+and	O
+health	O
+is	O
+highlighted	O
+by	O
+the	O
+recent	O
+finding	O
+that	O
+a	O
+homozygous	O
+mutation	O
+in	O
+the	O
+ectodomain	B-structure_element
+of	O
+KRM1	B-protein
+leads	O
+to	O
+severe	O
+ectodermal	O
+dysplasia	O
+including	O
+oligodontia	O
+.	O
+Interestingly	O
+,	O
+the	O
+Wnt	B-protein_type
+antagonistic	O
+activity	O
+of	O
+Krm	B-protein_type
+is	O
+context	O
+dependent	O
+,	O
+and	O
+Krm	B-protein_type
+proteins	O
+are	O
+actually	O
+dual	O
+-	O
+mode	O
+Wnt	B-protein_type
+regulators	O
+.	O
+In	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+Dkk	B-protein_type
+,	O
+Krm1	B-protein_type
+and	O
+2	B-protein_type
+change	O
+their	O
+function	O
+from	O
+inhibition	O
+to	O
+enhancement	O
+of	O
+Lrp6	B-protein_type
+-	O
+mediated	O
+signaling	O
+.	O
+By	O
+direct	O
+binding	O
+to	O
+Lrp6	B-protein_type
+via	O
+the	O
+ectodomains	B-structure_element
+,	O
+Krm	B-protein_type
+proteins	O
+promote	O
+Lrp6	B-protein_type
+cell	O
+-	O
+surface	O
+localization	O
+and	O
+hence	O
+increase	O
+receptor	O
+availability	O
+.	O
+Further	O
+increasing	O
+the	O
+complexity	O
+of	O
+Krm	B-protein_type
+functionality	O
+,	O
+it	O
+was	O
+recently	O
+found	O
+that	O
+Krm1	B-protein_type
+(	O
+but	O
+not	O
+Krm2	B-protein_type
+)	O
+can	O
+also	O
+act	O
+independently	O
+of	O
+LRP5	B-protein
+/	I-protein
+6	I-protein
+and	O
+Wnt	B-protein_type
+as	O
+a	O
+dependence	O
+receptor	O
+,	O
+triggering	O
+apoptosis	O
+unless	O
+bound	B-protein_state
+to	I-protein_state
+Dkk	B-protein_type
+.	O
+Structurally	O
+,	O
+Krm1	B-protein_type
+and	O
+2	B-protein_type
+are	O
+type	B-protein_type
+I	I-protein_type
+transmembrane	I-protein_type
+proteins	I-protein_type
+with	O
+a	O
+40	O
+kDa	O
+ectodomain	B-structure_element
+and	O
+a	O
+flexible	B-protein_state
+cytoplasmic	B-structure_element
+tail	I-structure_element
+consisting	O
+of	O
+60	B-residue_range
+–	O
+75	B-residue_range
+residues	O
+.	O
+The	O
+ectodomain	B-structure_element
+consists	O
+of	O
+three	O
+similarly	O
+sized	O
+structural	O
+domains	O
+of	O
+around	O
+10	O
+kDa	O
+each	O
+:	O
+the	O
+N	O
+-	O
+terminal	O
+Kringle	B-structure_element
+domain	O
+(	O
+KR	B-structure_element
+)	O
+is	O
+followed	O
+by	O
+a	O
+WSC	B-structure_element
+domain	O
+of	O
+unknown	O
+fold	O
+.	O
+The	O
+third	O
+structural	O
+domain	O
+is	O
+a	O
+CUB	B-structure_element
+domain	O
+.	O
+An	O
+approximately	B-residue_range
+70	I-residue_range
+-	I-residue_range
+residue	I-residue_range
+linker	B-structure_element
+connects	O
+the	O
+CUB	B-structure_element
+domain	O
+to	O
+the	O
+transmembrane	B-structure_element
+span	I-structure_element
+.	O
+An	O
+intact	B-protein_state
+KR	B-structure_element
+-	I-structure_element
+WSC	I-structure_element
+-	I-structure_element
+CUB	I-structure_element
+domain	O
+triplet	O
+and	O
+membrane	O
+attachment	O
+is	O
+required	O
+for	O
+Wnt	B-protein_type
+antagonism	O
+.	O
+The	O
+transmembrane	B-structure_element
+span	I-structure_element
+and	O
+cytoplasmic	B-structure_element
+tail	I-structure_element
+can	O
+be	O
+replaced	O
+with	O
+a	O
+GPI	B-structure_element
+linker	B-structure_element
+without	O
+impact	O
+on	O
+Wnt	B-protein_type
+antagonism	O
+.	O
+The	O
+structures	B-evidence
+presented	O
+here	O
+reveal	O
+the	O
+unknown	O
+fold	O
+of	O
+the	O
+WSC	B-structure_element
+domain	O
+and	O
+the	O
+tight	O
+interactions	O
+of	O
+all	O
+three	O
+domains	O
+.	O
+We	O
+further	O
+succeeded	O
+in	O
+determination	O
+of	O
+a	O
+low	O
+-	O
+resolution	O
+LRP6PE3PE4	B-complex_assembly
+-	I-complex_assembly
+DKK1CRD2	I-complex_assembly
+-	I-complex_assembly
+KRM1ECD	I-complex_assembly
+complex	O
+,	O
+defining	O
+the	O
+architecture	O
+of	O
+the	O
+Wnt	B-protein_type
+inhibitory	B-complex_assembly
+complex	I-complex_assembly
+that	O
+leads	O
+to	O
+Lrp6	B-protein
+cell	O
+-	O
+surface	O
+depletion	O
+.	O
+The	O
+recombinant	O
+production	O
+of	O
+the	O
+extracellular	B-structure_element
+domain	I-structure_element
+of	O
+Krm	B-protein_type
+for	O
+structural	B-experimental_method
+studies	I-experimental_method
+proved	O
+challenging	O
+(	O
+see	O
+Experimental	O
+Procedures	O
+).	O
+We	O
+succeeded	O
+in	O
+purifying	O
+KRM1ECD	B-protein
+complexes	B-protein_state
+with	I-protein_state
+DKK1fl	B-protein
+,	O
+DKK1Linker	B-protein
+-	B-structure_element
+CRD2	I-structure_element
+,	O
+and	O
+DKK1CRD2	B-protein
+that	O
+were	O
+monodisperse	O
+and	O
+stable	O
+in	O
+gel	B-experimental_method
+filtration	I-experimental_method
+,	O
+hence	O
+indicating	O
+at	O
+least	O
+micromolar	O
+affinity	O
+(	O
+data	O
+not	O
+shown	O
+).	O
+Several	O
+crystal	B-evidence
+forms	I-evidence
+were	O
+obtained	O
+from	O
+these	O
+complexes	O
+,	O
+however	O
+,	O
+crystals	B-evidence
+always	O
+contained	O
+only	O
+KRM1	B-protein
+protein	O
+.	O
+We	O
+solved	B-experimental_method
+the	O
+structure	B-evidence
+of	O
+KRM1ECD	B-protein
+in	O
+three	O
+crystal	O
+forms	O
+at	O
+1	O
+.	O
+9	O
+,	O
+2	O
+.	O
+8	O
+,	O
+and	O
+3	O
+.	O
+2	O
+Å	O
+resolution	O
+(	O
+Table	O
+1	O
+).	O
+The	O
+high	O
+-	O
+resolution	O
+structure	B-evidence
+is	O
+a	O
+near	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+model	O
+(	O
+Figure	O
+1	O
+).	O
+The	O
+small	B-protein_state
+,	O
+flexible	B-protein_state
+,	O
+and	O
+charged	B-protein_state
+98AEHED102	B-structure_element
+loop	I-structure_element
+could	O
+only	O
+be	O
+modeled	O
+in	O
+a	O
+slightly	O
+lower	O
+resolution	O
+structure	B-evidence
+and	O
+in	O
+crystal	O
+form	O
+III	O
+.	O
+The	O
+KR	B-structure_element
+,	O
+WSC	B-structure_element
+,	O
+and	O
+CUB	B-structure_element
+are	O
+arranged	O
+in	O
+a	O
+roughly	O
+triangular	O
+fashion	O
+with	O
+tight	O
+interactions	O
+between	O
+all	O
+three	O
+domains	O
+.	O
+The	O
+KR	B-structure_element
+domain	O
+,	O
+which	O
+bears	O
+two	O
+of	O
+the	O
+four	O
+glycosylation	B-site
+sites	I-site
+,	O
+contains	O
+the	O
+canonical	O
+three	O
+disulfide	B-ptm
+bridges	I-ptm
+(	O
+C32	B-residue_name_number
+-	O
+C114	B-residue_name_number
+,	O
+C55	B-residue_name_number
+-	O
+C95	B-residue_name_number
+,	O
+C84	B-residue_name_number
+-	O
+C109	B-residue_name_number
+)	O
+and	O
+,	O
+like	O
+other	O
+Kringle	B-structure_element
+domains	O
+,	O
+is	O
+low	O
+in	O
+secondary	O
+structure	O
+elements	O
+.	O
+The	O
+structurally	O
+most	O
+similar	O
+Kringle	B-structure_element
+domain	O
+is	O
+that	O
+of	O
+human	B-species
+plasminogen	B-protein
+(	O
+PDB	O
+:	O
+1PKR	O
+)	O
+with	O
+an	O
+root	B-evidence
+-	I-evidence
+mean	I-evidence
+-	I-evidence
+square	I-evidence
+deviation	I-evidence
+(	O
+RMSD	B-evidence
+)	O
+of	O
+1	O
+.	O
+7	O
+Å	O
+for	O
+73	O
+aligned	O
+Cα	O
+(	O
+Figure	O
+1B	O
+).	O
+The	O
+KRM1	B-protein
+structure	B-evidence
+reveals	O
+the	O
+fold	O
+of	O
+the	O
+WSC	B-structure_element
+domain	O
+for	O
+the	O
+first	O
+time	O
+.	O
+The	O
+structure	B-evidence
+is	O
+best	O
+described	O
+as	O
+a	O
+sandwich	B-structure_element
+of	O
+a	O
+β1	B-structure_element
+-	I-structure_element
+β5	I-structure_element
+-	I-structure_element
+β3	I-structure_element
+-	I-structure_element
+β4	I-structure_element
+-	I-structure_element
+β2	I-structure_element
+antiparallel	I-structure_element
+β	I-structure_element
+sheet	I-structure_element
+and	O
+a	O
+single	O
+α	B-structure_element
+helix	I-structure_element
+.	O
+The	O
+structure	B-evidence
+is	O
+also	O
+rich	O
+in	O
+loops	B-structure_element
+and	O
+is	O
+stabilized	O
+by	O
+four	O
+disulfide	B-ptm
+bridges	I-ptm
+(	O
+C122	B-residue_name_number
+-	O
+C186	B-residue_name_number
+,	O
+C147	B-residue_name_number
+-	O
+C167	B-residue_name_number
+,	O
+C151	B-residue_name_number
+-	O
+C169	B-residue_name_number
+,	O
+C190	B-residue_name_number
+-	O
+C198	B-residue_name_number
+).	O
+Using	O
+the	O
+PDBeFold	B-experimental_method
+server	I-experimental_method
+,	O
+we	O
+detected	O
+a	O
+surprising	O
+yet	O
+significant	O
+homology	O
+to	O
+PAN	B-structure_element
+module	I-structure_element
+domains	I-structure_element
+.	O
+The	O
+closest	O
+structural	O
+relative	O
+is	O
+hepatocyte	B-protein_type
+growth	I-protein_type
+factor	I-protein_type
+(	O
+HGF	B-protein_type
+,	O
+PDB	O
+:	O
+1GP9	O
+),	O
+which	O
+superposes	B-experimental_method
+with	O
+an	O
+RMSD	B-evidence
+of	O
+2	O
+.	O
+3	O
+Å	O
+for	O
+58	O
+aligned	O
+Cα	O
+(	O
+Figure	O
+1B	O
+).	O
+The	O
+CUB	B-structure_element
+domain	O
+bears	O
+two	O
+glycosylation	B-site
+sites	I-site
+.	O
+Although	O
+present	O
+,	O
+the	O
+quality	O
+of	O
+the	O
+electron	B-evidence
+density	I-evidence
+around	O
+N217	B-residue_name_number
+did	O
+not	O
+allow	O
+modeling	O
+of	O
+the	O
+sugar	O
+moiety	O
+.	O
+In	O
+crystal	B-evidence
+form	I-evidence
+I	I-evidence
+,	O
+a	O
+calcium	B-chemical
+ion	O
+is	O
+present	O
+at	O
+the	O
+canonical	O
+position	O
+coordinated	B-bond_interaction
+by	I-bond_interaction
+the	O
+carboxylates	O
+of	O
+D263	B-residue_name_number
+,	O
+D266	B-residue_name_number
+(	O
+bidentate	O
+),	O
+and	O
+D306	B-residue_name_number
+,	O
+as	O
+well	O
+as	O
+the	O
+carbonyl	O
+of	O
+N309	B-residue_name_number
+and	O
+a	O
+water	B-chemical
+molecule	O
+.	O
+The	O
+coordination	B-site
+sphere	I-site
+deviates	O
+significantly	O
+from	O
+perfectly	O
+octahedral	O
+(	O
+not	O
+shown	O
+).	O
+This	O
+might	O
+result	O
+in	O
+the	O
+site	O
+having	O
+a	O
+low	O
+affinity	O
+and	O
+may	O
+explain	O
+why	O
+calcium	B-chemical
+is	O
+not	O
+present	O
+in	O
+the	O
+two	O
+low	O
+-	O
+resolution	O
+crystal	B-evidence
+forms	I-evidence
+.	O
+Loss	B-protein_state
+of	I-protein_state
+calcium	B-chemical
+has	O
+led	O
+to	O
+loop	B-structure_element
+rearrangements	O
+and	O
+partial	O
+disorder	O
+in	O
+these	O
+crystal	B-evidence
+forms	I-evidence
+.	O
+The	O
+closest	O
+structural	O
+relative	O
+is	O
+the	O
+CUB_C	B-structure_element
+domain	O
+of	O
+Tsg	B-protein
+-	I-protein
+6	I-protein
+(	O
+PDB	O
+:	O
+2WNO	O
+),	O
+which	O
+superposes	B-experimental_method
+with	O
+KRMCUB	B-protein
+with	O
+an	O
+RMSD	B-evidence
+of	O
+1	O
+.	O
+6	O
+Å	O
+for	O
+104	O
+Cα	O
+(	O
+Figure	O
+1B	O
+).	O
+A	O
+superposition	B-experimental_method
+of	O
+the	O
+three	O
+KRM1	B-protein
+structures	B-evidence
+reveals	O
+no	O
+major	O
+structural	O
+differences	O
+(	O
+Figure	O
+1C	O
+)	O
+as	O
+anticipated	O
+from	O
+the	O
+plethora	O
+of	O
+interactions	O
+between	O
+the	O
+three	O
+domains	O
+.	O
+Minor	O
+differences	O
+are	O
+caused	O
+by	O
+the	O
+collapse	O
+of	O
+the	O
+Ca2	B-site
++	I-site
+binding	I-site
+site	I-site
+in	O
+crystal	B-evidence
+forms	I-evidence
+II	I-evidence
+and	I-evidence
+III	I-evidence
+and	O
+loop	B-structure_element
+flexibility	O
+in	O
+the	O
+KR	B-structure_element
+domain	O
+.	O
+The	O
+F207S	B-mutant
+mutation	O
+recently	O
+found	O
+to	O
+cause	O
+ectodermal	O
+dysplasia	O
+in	O
+Palestinian	O
+families	O
+maps	O
+to	O
+the	O
+hydrophobic	B-site
+core	I-site
+of	O
+the	O
+protein	O
+at	O
+the	O
+interface	B-site
+of	O
+the	O
+three	O
+subdomains	O
+(	O
+Figure	O
+1A	O
+).	O
+Such	O
+a	O
+mutation	O
+is	O
+bound	B-protein_state
+to	I-protein_state
+severely	O
+destabilize	O
+the	O
+protein	O
+structure	O
+of	O
+KRM1	B-protein
+,	O
+leading	O
+to	O
+disturbance	O
+of	O
+its	O
+Wnt	B-protein_type
+antagonistic	O
+,	O
+Wnt	B-protein_type
+stimulatory	O
+,	O
+and	O
+Wnt	B-protein_type
+independent	O
+activity	O
+.	O
+Co	B-experimental_method
+-	I-experimental_method
+crystallization	I-experimental_method
+of	O
+LRP6PE3PE4	B-protein
+with	O
+DKK1CRD2	B-protein
+,	O
+and	O
+LRP6PE1	B-protein
+with	O
+an	O
+N	O
+-	O
+terminal	O
+peptide	O
+of	O
+DKK1	B-protein
+has	O
+provided	O
+valuable	O
+structural	O
+insight	O
+into	O
+direct	O
+Wnt	B-protein_type
+inhibition	O
+by	O
+Dkk	B-protein_type
+ligands	O
+.	O
+One	O
+face	O
+of	O
+the	O
+rather	O
+flat	B-protein_state
+DKK1CRD2	B-protein
+fragment	O
+binds	B-protein_state
+to	I-protein_state
+the	O
+third	B-structure_element
+β	I-structure_element
+propeller	I-structure_element
+of	O
+LRP6	B-protein
+.	O
+Mutational	B-experimental_method
+analyses	I-experimental_method
+further	O
+implied	O
+that	O
+the	O
+LRP6PE3	B-protein
+-	O
+averted	O
+face	O
+of	O
+DKK1CRD2	B-protein
+bears	O
+the	O
+Krm	B-site
+binding	I-site
+site	I-site
+,	O
+hence	O
+suggesting	O
+how	O
+Dkk	B-protein_type
+can	O
+recruit	O
+both	O
+receptors	B-protein_type
+into	O
+a	O
+ternary	O
+complex	O
+.	O
+To	O
+obtain	O
+direct	O
+insight	O
+into	O
+ternary	O
+complex	O
+formation	O
+by	O
+Lrp5	B-protein_type
+/	I-protein_type
+6	I-protein_type
+,	O
+Dkk	B-protein_type
+,	O
+and	O
+Krm	B-protein_type
+,	O
+we	O
+subjected	O
+an	O
+LRP6PE3PE4	B-complex_assembly
+-	I-complex_assembly
+DKK1fl	I-complex_assembly
+-	I-complex_assembly
+KRM1ECD	I-complex_assembly
+complex	O
+to	O
+crystallization	B-experimental_method
+trials	I-experimental_method
+.	O
+Diffraction	B-evidence
+data	I-evidence
+collected	O
+from	O
+the	O
+resulting	O
+crystals	B-evidence
+were	O
+highly	O
+anisotropic	O
+with	O
+diffraction	O
+extending	O
+in	O
+the	O
+best	O
+directions	O
+to	O
+3	O
+.	O
+5	O
+Å	O
+and	O
+3	O
+.	O
+7	O
+Å	O
+but	O
+only	O
+to	O
+6	O
+.	O
+4	O
+Å	O
+in	O
+the	O
+third	O
+direction	O
+.	O
+Despite	O
+the	O
+lack	O
+of	O
+high	O
+-	O
+resolution	O
+diffraction	B-evidence
+,	O
+the	O
+general	O
+architecture	O
+of	O
+the	O
+ternary	O
+complex	O
+is	O
+revealed	O
+(	O
+Figure	O
+2A	O
+).	O
+DKK1CRD2	B-protein
+binds	B-protein_state
+to	I-protein_state
+the	O
+top	O
+face	O
+of	O
+the	O
+LRP6	B-protein
+PE3	B-structure_element
+β	B-structure_element
+propeller	I-structure_element
+as	O
+described	O
+earlier	O
+for	O
+the	O
+binary	O
+complex	O
+.	O
+KRM1ECD	B-protein
+does	O
+indeed	O
+bind	B-protein_state
+on	I-protein_state
+the	O
+opposite	O
+side	O
+of	O
+DKK1CRD2	B-protein
+with	O
+only	O
+its	O
+KR	B-structure_element
+and	O
+WSC	B-structure_element
+domains	O
+engaged	O
+in	O
+binding	O
+(	O
+Figure	O
+2A	O
+).	O
+Although	O
+present	O
+in	O
+the	O
+complex	O
+subjected	O
+to	O
+crystallization	B-experimental_method
+,	O
+we	O
+observe	O
+no	O
+density	B-evidence
+that	O
+could	O
+correspond	O
+to	O
+CRD1	B-structure_element
+or	O
+the	O
+domain	B-structure_element
+linker	I-structure_element
+(	O
+L	B-structure_element
+).	O
+We	O
+confirm	O
+that	O
+the	O
+CRD2	B-structure_element
+of	O
+DKK1	B-protein
+is	O
+required	O
+and	O
+sufficient	O
+for	O
+binding	O
+to	O
+KRM1	B-protein
+:	O
+In	O
+surface	B-experimental_method
+plasmon	I-experimental_method
+resonance	I-experimental_method
+(	O
+SPR	B-experimental_method
+),	O
+we	O
+measured	O
+low	O
+micromolar	O
+affinity	B-evidence
+between	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+DKK1	B-protein
+and	O
+immobilized	O
+KRM1ECD	B-protein
+(	O
+Figure	O
+2B	O
+).	O
+A	O
+SUMO	B-experimental_method
+fusion	I-experimental_method
+of	O
+DKK1L	B-structure_element
+-	I-structure_element
+CRD2	I-structure_element
+displayed	O
+a	O
+similar	O
+(	O
+slightly	O
+higher	O
+)	O
+affinity	B-evidence
+.	O
+In	O
+contrast	O
+,	O
+a	O
+SUMO	B-experimental_method
+fusion	I-experimental_method
+of	O
+DKK1CRD1	B-structure_element
+-	I-structure_element
+L	I-structure_element
+did	O
+not	O
+display	O
+binding	O
+for	O
+concentrations	O
+tested	O
+up	O
+to	O
+325	O
+μM	O
+(	O
+Figure	O
+2B	O
+).	O
+Overall	O
+,	O
+the	O
+DKK1	B-site
+-	I-site
+KRM1	I-site
+interface	I-site
+is	O
+characterized	O
+by	O
+a	O
+large	O
+number	O
+of	O
+polar	B-bond_interaction
+interactions	I-bond_interaction
+but	O
+only	O
+few	O
+hydrophobic	B-bond_interaction
+contacts	I-bond_interaction
+(	O
+Figure	O
+2C	O
+).	O
+The	O
+crystal	B-evidence
+structure	I-evidence
+gives	O
+an	O
+explanation	O
+for	O
+DKK1	B-protein
+loss	O
+-	O
+of	O
+-	O
+binding	O
+mutations	O
+identified	O
+previously	O
+:	O
+R191	B-residue_name_number
+of	O
+DKK1	B-protein
+forms	O
+a	O
+double	O
+salt	B-bond_interaction
+bridge	I-bond_interaction
+to	O
+D125	B-residue_name_number
+and	O
+E162	B-residue_name_number
+of	O
+KRM1	B-protein
+(	O
+Figure	O
+2C	O
+).	O
+A	O
+charge	B-experimental_method
+reversal	I-experimental_method
+as	O
+in	O
+the	O
+mouse	B-taxonomy_domain
+Dkk1	B-protein
+(	O
+mDkk1	B-protein
+)	O
+R197E	B-mutant
+variant	O
+would	O
+severely	O
+disrupt	O
+the	O
+binding	O
+.	O
+Similarly	O
+,	O
+the	O
+K226	B-residue_name_number
+side	O
+chain	O
+of	O
+DKK1	B-protein
+,	O
+which	O
+points	O
+to	O
+a	O
+small	O
+hydrophobic	B-site
+pocket	I-site
+on	O
+the	O
+surface	O
+of	O
+KRM1	B-protein
+formed	O
+by	O
+Y108	B-residue_name_number
+,	O
+W94	B-residue_name_number
+,	O
+and	O
+W106	B-residue_name_number
+,	O
+forms	O
+salt	B-bond_interaction
+bridges	I-bond_interaction
+with	O
+the	O
+side	O
+chains	O
+of	O
+KRM1	B-protein
+D88	B-residue_name_number
+and	O
+D90	B-residue_name_number
+.	O
+Again	O
+,	O
+a	O
+charge	B-experimental_method
+reversal	I-experimental_method
+as	O
+shown	O
+before	O
+for	O
+mDkk1	B-protein
+K232E	B-mutant
+would	O
+be	O
+incompatible	O
+with	O
+binding	O
+.	O
+The	O
+side	O
+chain	O
+of	O
+DKK1	B-protein
+S192	B-residue_name_number
+was	O
+also	O
+predicted	O
+to	O
+be	O
+involved	O
+in	O
+Krm	B-protein_type
+binding	O
+.	O
+Indeed	O
+,	O
+we	O
+found	O
+(	O
+Figure	O
+2C	O
+)	O
+that	O
+the	O
+side	O
+chain	O
+of	O
+D201	B-residue_name_number
+of	O
+KRM1	B-protein
+forms	O
+two	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+to	O
+the	O
+side	O
+-	O
+chain	O
+hydroxyl	O
+and	O
+the	O
+backbone	O
+amide	O
+of	O
+S192	B-residue_name_number
+(	O
+mouse	B-taxonomy_domain
+,	O
+S198	B-residue_name_number
+).	O
+Additional	O
+polar	B-bond_interaction
+interactions	I-bond_interaction
+are	O
+formed	O
+between	O
+the	O
+N140	B-residue_name_number
+,	O
+S163	B-residue_name_number
+,	O
+and	O
+Y165	B-residue_name_number
+side	O
+chains	O
+of	O
+KRM1	B-protein
+and	O
+DKK1	B-protein
+backbone	O
+carbonyls	O
+of	O
+W206	B-residue_name_number
+,	O
+L190	B-residue_name_number
+,	O
+and	O
+C189	B-residue_name_number
+,	O
+respectively	O
+.	O
+The	O
+carbonyl	O
+of	O
+DKK1	B-protein
+R224	B-residue_name_number
+is	O
+hydrogen	B-bond_interaction
+bonded	I-bond_interaction
+to	O
+Y105	B-residue_name_number
+and	O
+W106	B-residue_name_number
+of	O
+KRM1	B-protein
+.	O
+We	O
+suspect	O
+that	O
+the	O
+Dkk	B-protein_type
+charge	B-experimental_method
+reversal	I-experimental_method
+mutations	I-experimental_method
+performed	O
+in	O
+the	O
+murine	B-taxonomy_domain
+background	O
+and	O
+shown	O
+to	O
+diminish	O
+Krm	B-protein_type
+binding	O
+K211E	B-mutant
+and	O
+R203E	B-mutant
+(	O
+mouse	B-taxonomy_domain
+K217E	B-mutant
+and	O
+R209E	B-mutant
+)	O
+do	O
+so	O
+likely	O
+indirectly	O
+by	O
+disruption	O
+of	O
+the	O
+Dkk	B-protein_type
+fold	O
+.	O
+We	O
+further	O
+validated	O
+the	O
+DKK1	B-site
+binding	I-site
+site	I-site
+on	O
+KRM1	B-protein
+by	O
+introducing	B-experimental_method
+glycosylation	B-site
+sites	I-site
+at	O
+the	O
+KR	B-structure_element
+(	O
+90DVS92	B-mutant
+→	I-mutant
+NVS	I-mutant
+)	O
+and	O
+WSC	B-structure_element
+(	O
+189VCF191	B-mutant
+→	I-mutant
+NCS	I-mutant
+)	O
+domains	O
+pointing	O
+toward	O
+DKK	B-protein
+(	O
+Figures	O
+2A	O
+and	O
+2D	O
+).	O
+Introduction	O
+of	O
+N	B-ptm
+-	I-ptm
+linked	I-ptm
+glycans	I-ptm
+in	O
+protein	B-site
+-	I-site
+protein	I-site
+-	I-site
+binding	I-site
+sites	I-site
+is	O
+an	O
+established	O
+way	O
+of	O
+disrupting	O
+protein	B-site
+-	I-site
+binding	I-site
+interfaces	I-site
+.	O
+Both	O
+ectodomain	B-structure_element
+mutants	B-protein_state
+were	O
+secreted	O
+comparably	O
+with	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+,	O
+indicating	O
+correct	O
+folding	O
+,	O
+but	O
+failed	O
+to	O
+achieve	O
+any	O
+detectable	O
+binding	O
+in	O
+SPR	B-experimental_method
+using	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+DKK1	B-protein
+as	O
+analyte	O
+.	O
+In	O
+contrast	O
+,	O
+a	O
+mutant	B-protein_state
+carrying	O
+an	O
+additional	O
+N	B-ptm
+-	I-ptm
+glycan	I-ptm
+outside	O
+the	O
+interface	B-site
+at	O
+the	O
+CUB	B-structure_element
+domain	O
+(	O
+309NQA311	B-mutant
+→	I-mutant
+NQS	I-mutant
+),	O
+was	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+-	O
+like	O
+in	O
+DKK1	B-protein
+binding	O
+(	O
+Figure	O
+2D	O
+).	O
+Identification	O
+of	O
+a	O
+Direct	O
+LRP6	B-site
+-	I-site
+KRM1	I-site
+Binding	I-site
+Site	I-site
+The	O
+LRP6PE3PE4	B-complex_assembly
+-	I-complex_assembly
+DKK1CRD2	I-complex_assembly
+-	I-complex_assembly
+KRM1ECD	I-complex_assembly
+complex	O
+structure	B-evidence
+reveals	O
+no	O
+direct	O
+interactions	O
+between	O
+KRM1	B-protein
+and	O
+LRP6	B-protein
+.	O
+We	O
+constructed	O
+in	O
+silico	O
+a	O
+ternary	O
+complex	B-protein_state
+with	I-protein_state
+a	O
+close	O
+to	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+LRP6	B-protein
+ectodomain	B-structure_element
+(	O
+PE1PE2PE3PE4	B-structure_element
+horse	B-structure_element
+shoe	I-structure_element
+)	O
+similar	O
+to	O
+but	O
+without	O
+refinement	O
+against	O
+electron	B-experimental_method
+microscopy	I-experimental_method
+(	O
+EM	B-experimental_method
+)	O
+or	O
+small	B-experimental_method
+-	I-experimental_method
+angle	I-experimental_method
+X	I-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+scattering	I-experimental_method
+data	O
+.	O
+An	O
+auxiliary	O
+PE3PE4	B-structure_element
+fragment	O
+was	O
+superimposed	B-experimental_method
+via	O
+PE4	B-structure_element
+onto	O
+PE3	B-structure_element
+of	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+,	O
+and	O
+the	O
+LRP6PE1PE2	B-protein
+structure	B-evidence
+was	O
+superimposed	B-experimental_method
+via	O
+PE2	B-structure_element
+onto	O
+PE3	B-structure_element
+of	O
+this	O
+auxiliary	O
+fragment	O
+(	O
+Figure	O
+3A	O
+).	O
+For	O
+this	O
+crude	O
+approximation	O
+of	O
+a	O
+true	O
+ternary	O
+complex	O
+,	O
+we	O
+noted	O
+very	O
+close	O
+proximity	O
+between	O
+the	O
+Ca2	B-site
++-	I-site
+binding	I-site
+region	I-site
+of	O
+KRM1	B-protein
+and	O
+the	O
+top	O
+face	O
+of	O
+the	O
+PE2	B-structure_element
+β	B-structure_element
+propeller	I-structure_element
+of	O
+LRP6	B-protein
+.	O
+The	O
+solvent	B-protein_state
+-	I-protein_state
+exposed	I-protein_state
+residues	O
+R307	B-residue_name_number
+,	O
+I308	B-residue_name_number
+,	O
+and	O
+N309	B-residue_name_number
+of	O
+the	O
+central	O
+Ca2	B-structure_element
++-	I-structure_element
+binding	I-structure_element
+β	I-structure_element
+connection	I-structure_element
+loop	I-structure_element
+of	O
+KRM1	B-protein
+would	O
+be	O
+almost	O
+ideally	O
+positioned	O
+for	O
+binding	O
+to	O
+this	O
+face	O
+,	O
+which	O
+is	O
+commonly	O
+used	O
+as	O
+a	O
+binding	B-site
+site	I-site
+on	O
+β	B-structure_element
+propellers	I-structure_element
+.	O
+Peptides	O
+containing	O
+arginine	B-residue_name
+/	O
+lysine	B-residue_name
+,	O
+isoleucine	B-residue_name
+,	O
+and	O
+asparagine	B-residue_name
+(	O
+consensus	O
+sequence	O
+N	B-structure_element
+-	I-structure_element
+X	I-structure_element
+-	I-structure_element
+I	I-structure_element
+-(	I-structure_element
+G	I-structure_element
+)-	I-structure_element
+R	I-structure_element
+/	I-structure_element
+K	I-structure_element
+)	O
+are	O
+also	O
+employed	O
+by	O
+DKK1	B-protein
+and	O
+SOST	B-protein
+to	O
+bind	O
+to	O
+LRP6	B-protein
+(	O
+albeit	O
+to	O
+propeller	B-structure_element
+1	I-structure_element
+;	O
+Figure	O
+3B	O
+).	O
+To	O
+support	O
+the	O
+hypothesis	O
+that	O
+KRM1CUB	B-protein
+binds	B-protein_state
+to	I-protein_state
+LRP6PE2	B-protein
+,	O
+we	O
+used	O
+SPR	B-experimental_method
+and	O
+compared	O
+binding	O
+of	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+the	O
+GlycoCUB	B-protein_state
+mutant	I-protein_state
+of	O
+KRM1ECD	B-protein
+(	O
+bearing	O
+an	O
+N	B-site
+-	I-site
+glycosylation	I-site
+site	I-site
+at	O
+N309	B-residue_name_number
+)	O
+with	O
+a	O
+purified	O
+LRP6PE1PE2	B-protein
+fragment	O
+.	O
+Indeed	O
+,	O
+we	O
+found	O
+that	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+Dkk	B-protein_type
+,	O
+KRM1ECD	B-protein
+bound	B-protein_state
+with	O
+considerable	O
+affinity	O
+to	B-protein_state
+LRP6PE1PE2	B-protein
+(	O
+Figure	O
+3C	O
+).	O
+In	O
+contrast	O
+,	O
+no	O
+saturable	O
+binding	O
+was	O
+observed	O
+between	O
+KRM1	B-protein
+and	O
+LRP6PE3PE4	B-protein
+.	O
+Introduction	B-experimental_method
+of	I-experimental_method
+an	O
+N	B-site
+-	I-site
+glycosylation	I-site
+site	I-site
+at	O
+N309	B-residue_name_number
+in	O
+KRM1ECD	B-protein
+abolished	O
+LRP6PE1PE2	B-protein
+binding	O
+(	O
+Figure	O
+3C	O
+),	O
+while	O
+binding	O
+to	O
+DKK1	B-protein
+was	O
+unaffected	O
+(	O
+Figure	O
+2D	O
+).	O
+We	O
+conclude	O
+that	O
+the	O
+predicted	O
+binding	B-site
+site	I-site
+between	O
+KRM1CUB	B-protein
+and	O
+LRP6PE2	B-protein
+is	O
+a	O
+strong	O
+candidate	O
+for	O
+mediating	O
+the	O
+direct	O
+Lrp6	B-complex_assembly
+-	I-complex_assembly
+Krm	I-complex_assembly
+interaction	O
+,	O
+which	O
+is	O
+thought	O
+to	O
+increase	O
+Wnt	B-protein_type
+responsiveness	O
+by	O
+stabilizing	O
+Lrp6	B-protein
+at	O
+the	O
+cell	O
+surface	O
+.	O
+Further	O
+experiments	O
+are	O
+required	O
+to	O
+pinpoint	O
+the	O
+exact	O
+binding	B-site
+site	I-site
+.	O
+Although	O
+LRP6PE1	B-protein
+appears	O
+somewhat	O
+out	O
+of	O
+reach	O
+in	O
+the	O
+modeled	O
+ternary	O
+complex	O
+,	O
+it	O
+cannot	O
+be	O
+excluded	O
+as	O
+the	O
+Krm	B-site
+binding	I-site
+site	I-site
+in	O
+the	O
+ternary	O
+complex	O
+and	O
+LRP6	B-complex_assembly
+-	I-complex_assembly
+Krm	I-complex_assembly
+binary	O
+complex	O
+.	O
+The	O
+presence	B-protein_state
+of	I-protein_state
+DKK	B-protein
+may	O
+govern	O
+which	O
+propeller	B-structure_element
+(	O
+PE1	B-structure_element
+versus	O
+PE2	B-structure_element
+)	O
+of	O
+LRP6	B-protein
+is	O
+available	O
+for	O
+Krm	B-protein_type
+binding	O
+.	O
+Apparent	O
+binding	O
+across	O
+the	O
+proposed	O
+KRM1CUB	B-site
+-	I-site
+LRP6PE2	I-site
+interface	I-site
+is	O
+expected	O
+to	O
+be	O
+higher	O
+once	O
+Krm	B-protein_type
+is	O
+also	O
+cross	O
+-	O
+linked	O
+to	O
+LRP6PE3	B-protein
+via	O
+DKK1CRD2	B-protein
+(	O
+Figure	O
+3D	O
+).	O
+Low	O
+-	O
+resolution	O
+negative	B-experimental_method
+-	I-experimental_method
+stain	I-experimental_method
+EM	I-experimental_method
+and	O
+small	B-experimental_method
+-	I-experimental_method
+angle	I-experimental_method
+X	I-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+scattering	I-experimental_method
+studies	O
+of	O
+LRP6PE1PE2PE3PE4	B-protein
+,	O
+in	B-protein_state
+isolation	I-protein_state
+and	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+Dkk1	B-protein_type
+,	O
+plus	O
+negative	B-experimental_method
+-	I-experimental_method
+stain	I-experimental_method
+EM	I-experimental_method
+of	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+LRP6	B-protein
+ectodomain	B-structure_element
+,	O
+have	O
+indicated	O
+curved	B-protein_state
+,	O
+platform	B-protein_state
+-	I-protein_state
+like	I-protein_state
+conformations	O
+but	O
+also	O
+potential	O
+flexibility	O
+between	O
+PE2	B-structure_element
+and	O
+PE3	B-structure_element
+.	O
+It	O
+is	O
+therefore	O
+possible	O
+that	O
+the	O
+interplay	O
+of	O
+Krm	B-protein_type
+and	O
+Dkk	B-protein_type
+binding	O
+can	O
+promote	O
+changes	O
+in	O
+LRP6	B-protein
+ectodomain	B-structure_element
+conformation	O
+with	O
+functional	O
+consequences	O
+;	O
+however	O
+,	O
+such	O
+ideas	O
+await	O
+investigation	O
+.	O
+Taken	O
+together	O
+,	O
+the	O
+structural	B-experimental_method
+and	I-experimental_method
+biophysical	I-experimental_method
+studies	I-experimental_method
+we	O
+report	O
+here	O
+extend	O
+our	O
+mechanistic	O
+understanding	O
+of	O
+Wnt	B-protein_type
+signal	O
+regulation	O
+.	O
+We	O
+describe	O
+the	O
+ectodomain	B-structure_element
+structure	B-evidence
+of	O
+the	O
+dual	O
+Wnt	B-protein_type
+regulator	O
+Krm1	B-protein_type
+,	O
+providing	O
+an	O
+explanation	O
+for	O
+the	O
+detrimental	O
+effect	O
+on	O
+health	O
+and	O
+development	O
+of	O
+a	O
+homozygous	O
+KRM1	B-protein
+mutation	O
+.	O
+We	O
+also	O
+reveal	O
+the	O
+interaction	O
+mode	O
+of	O
+Krm	B-complex_assembly
+-	I-complex_assembly
+Dkk	I-complex_assembly
+and	O
+the	O
+architecture	O
+of	O
+the	O
+ternary	O
+complex	O
+formed	O
+by	O
+Lrp5	B-protein_type
+/	I-protein_type
+6	I-protein_type
+,	O
+Dkk	B-protein_type
+,	O
+and	O
+Krm	B-protein_type
+.	O
+Furthermore	O
+,	O
+the	O
+ternary	O
+crystal	B-evidence
+structure	I-evidence
+has	O
+guided	O
+in	B-experimental_method
+silico	I-experimental_method
+and	I-experimental_method
+biophysical	I-experimental_method
+analyses	I-experimental_method
+to	O
+suggest	O
+a	O
+direct	O
+LRP6	B-site
+-	I-site
+KRM1	I-site
+interaction	I-site
+site	I-site
+.	O
+Our	O
+findings	O
+provide	O
+a	O
+solid	O
+foundation	O
+for	O
+additional	O
+studies	O
+to	O
+probe	O
+how	O
+ternary	O
+complex	O
+formation	O
+triggers	O
+internalization	O
+,	O
+whereas	O
+Krm	B-protein_type
+binding	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+Dkk	B-protein_type
+stabilizes	O
+the	O
+Wnt	B-protein_type
+co	B-protein_type
+-	I-protein_type
+receptor	I-protein_type
+at	O
+the	O
+cell	O
+surface	O
+.	O
+Structure	B-evidence
+of	O
+Unliganded	B-protein_state
+KRM1ECD	B-protein
+(	O
+A	O
+)	O
+The	O
+KRM1ECD	B-protein
+fold	O
+(	O
+crystal	B-evidence
+form	I-evidence
+I	I-evidence
+)	O
+colored	O
+blue	O
+to	O
+red	O
+from	O
+the	O
+N	O
+to	O
+C	O
+terminus	O
+.	O
+Cysteines	B-residue_name
+as	O
+ball	O
+and	O
+sticks	O
+,	O
+glycosylation	B-site
+sites	I-site
+as	O
+sticks	O
+.	O
+The	O
+bound	O
+calcium	B-chemical
+is	O
+shown	O
+as	O
+a	O
+gray	O
+sphere	O
+.	O
+The	O
+site	O
+of	O
+the	O
+F207S	B-mutant
+mutation	O
+associated	O
+with	O
+ectodermal	O
+dysplasia	O
+in	O
+humans	B-species
+is	O
+shown	O
+as	O
+mesh	O
+.	O
+(	O
+B	O
+)	O
+Superposition	B-experimental_method
+of	O
+the	O
+three	O
+KRM1ECD	B-protein
+subdomains	O
+(	O
+solid	O
+)	O
+with	O
+their	O
+next	O
+structurally	O
+characterized	O
+homologs	O
+(	O
+half	O
+transparent	O
+).	O
+(	O
+C	O
+)	O
+Superposition	B-experimental_method
+of	O
+KRM1ECD	B-protein
+from	O
+the	O
+three	O
+crystal	B-evidence
+forms	I-evidence
+.	O
+Alignment	B-evidence
+scores	I-evidence
+for	O
+each	O
+pairing	O
+are	O
+indicated	O
+on	O
+the	O
+dashed	O
+triangle	O
+.	O
+(	O
+A	O
+)	O
+The	O
+structure	B-evidence
+of	O
+the	O
+ternary	O
+LRP6PE3PE4	B-complex_assembly
+-	I-complex_assembly
+DKK1CRD2	I-complex_assembly
+-	I-complex_assembly
+KRM1ECD	I-complex_assembly
+complex	O
+.	O
+DKK1	B-protein
+(	O
+orange	O
+)	O
+is	O
+sandwiched	O
+between	O
+the	O
+PE3	B-structure_element
+module	O
+of	O
+LRP6	B-protein
+(	O
+blue	O
+)	O
+and	O
+the	O
+KR	B-structure_element
+-	I-structure_element
+WSC	I-structure_element
+domain	O
+pair	O
+of	O
+KRM1	B-protein
+(	O
+green	O
+).	O
+Colored	O
+symbols	O
+indicate	O
+introduced	O
+N	B-site
+-	I-site
+glycan	I-site
+attachment	I-site
+sites	I-site
+(	O
+see	O
+D	O
+).	O
+(	O
+B	O
+)	O
+SPR	B-experimental_method
+data	O
+comparing	O
+binding	O
+of	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+DKK1	B-protein
+and	O
+SUMO	B-experimental_method
+fusions	I-experimental_method
+of	O
+DKK1	B-protein
+truncations	O
+for	O
+binding	O
+to	O
+immobilized	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+KRM1ECD	B-protein
+.	O
+(	O
+C	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+DKK1CRD2	B-site
+-	I-site
+KRM1ECD	I-site
+interface	I-site
+.	O
+Residues	O
+involved	O
+in	O
+interface	B-site
+formation	O
+are	O
+shown	O
+as	O
+sticks	O
+;	O
+those	O
+mentioned	O
+in	O
+the	O
+text	O
+are	O
+labeled	O
+.	O
+Salt	B-bond_interaction
+bridges	I-bond_interaction
+are	O
+in	O
+pink	O
+and	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+in	O
+black	O
+.	O
+(	O
+D	O
+)	O
+SPR	B-experimental_method
+binding	B-evidence
+data	I-evidence
+comparing	O
+DKK1	B-protein
+analyte	O
+binding	O
+with	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+KRM1ECD	B-protein
+and	O
+three	O
+variants	O
+bearing	O
+engineered	B-protein_state
+glycosylation	B-site
+sites	I-site
+on	O
+the	O
+KR	B-structure_element
+and	O
+WSC	B-structure_element
+domains	O
+(	O
+green	O
+and	O
+blue	O
+pointing	O
+to	O
+DKK1	B-protein
+)	O
+and	O
+on	O
+the	O
+CUB	B-structure_element
+domain	O
+(	O
+orange	O
+).	O
+LRP6	B-complex_assembly
+-	I-complex_assembly
+KRM1	I-complex_assembly
+Direct	O
+Interaction	O
+and	O
+Summary	O
+(	O
+A	O
+)	O
+In	O
+a	O
+construction	O
+of	O
+a	O
+ternary	O
+complex	B-protein_state
+with	I-protein_state
+all	O
+four	O
+β	B-structure_element
+propellers	I-structure_element
+of	O
+LRP6	B-protein
+intact	B-protein_state
+,	O
+the	O
+CUB	B-structure_element
+domain	O
+points	O
+via	O
+its	O
+Ca2	B-site
++-	I-site
+binding	I-site
+region	I-site
+toward	O
+the	O
+top	O
+face	O
+of	O
+the	O
+second	B-structure_element
+β	I-structure_element
+propeller	I-structure_element
+.	O
+(	O
+B	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+potential	O
+interaction	B-site
+site	I-site
+.	O
+In	O
+addition	O
+,	O
+LRP6PE2	B-protein
+has	O
+been	O
+superimposed	B-experimental_method
+with	O
+DKK1	B-protein
+(	O
+yellow	O
+)	O
+and	O
+SOST	B-protein
+(	O
+pink	O
+)	O
+peptide	O
+complexes	O
+of	O
+LRP6PE1	B-protein
+.	O
+(	O
+C	O
+)	O
+SPR	B-experimental_method
+measurements	I-experimental_method
+comparing	O
+LRP6PE1PE2	B-protein
+binding	O
+with	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+KRM1ECD	B-protein
+and	O
+the	O
+GlycoCUB	B-protein_state
+mutant	I-protein_state
+bearing	O
+an	O
+N	B-ptm
+-	I-ptm
+glycan	I-ptm
+at	O
+N309	B-residue_name_number
+.	O
+(	O
+D	O
+)	O
+Schematic	O
+representation	O
+of	O
+structural	O
+and	O
+biophysical	O
+findings	O
+and	O
+their	O
+implications	O
+for	O
+Wnt	B-protein_type
+-	O
+dependent	O
+(	O
+left	O
+,	O
+middle	O
+)	O
+and	O
+independent	O
+(	O
+right	O
+)	O
+signaling	O
+.	O
+Conformational	O
+differences	O
+in	O
+the	O
+depictions	O
+of	O
+LRP6	B-protein
+are	O
+included	O
+purely	O
+for	O
+ease	O
+of	O
+representation	O
+.	O
+Diffraction	B-evidence
+and	I-evidence
+Refinement	I-evidence
+Statistics	I-evidence
+KRM1ECD	B-protein
+KRM1ECD	B-protein
+KRM1ECD	B-protein
+KRM1ECD	B-protein
+LRP6PE3PE4	B-complex_assembly
+-	I-complex_assembly
+DKKCRD2	I-complex_assembly
+-	I-complex_assembly
+KRM1ECD	I-complex_assembly
+Crystal	O
+form	O
+I	O
+I	O
+II	O
+III	O
+I	O
+X	O
+-	O
+ray	O
+source	O
+Diamond	O
+i04	O
+Diamond	O
+i03	O
+Diamond	O
+i03	O
+Diamond	O
+i04	O
+Diamond	O
+i04	O
+Wavelength	O
+(	O
+Å	O
+)	O
+0	O
+.	O
+9793	O
+0	O
+.	O
+9700	O
+0	O
+.	O
+9700	O
+0	O
+.	O
+9795	O
+0	O
+.	O
+9795	O
+Space	O
+group	O
+P3121	O
+P3121	O
+P43	O
+P41212	O
+C2221	O
+Unit	O
+cell	O
+a	O
+/	O
+α	O
+(	O
+Å	O
+/°)	O
+50	O
+.	O
+9	O
+/	O
+90	O
+50	O
+.	O
+5	O
+/	O
+90	O
+65	O
+.	O
+8	O
+/	O
+90	O
+67	O
+.	O
+8	O
+/	O
+90	O
+86	O
+.	O
+9	O
+/	O
+90	O
+b	O
+/	O
+β	O
+(	O
+Å	O
+/°)	O
+50	O
+.	O
+9	O
+/	O
+90	O
+50	O
+.	O
+5	O
+/	O
+90	O
+65	O
+.	O
+8	O
+/	O
+90	O
+67	O
+.	O
+8	O
+/	O
+90	O
+100	O
+.	O
+1	O
+/	O
+90	O
+c	O
+/	O
+γ	O
+(	O
+Å	O
+/°)	O
+188	O
+.	O
+4	O
+/	O
+120	O
+187	O
+.	O
+4	O
+/	O
+120	O
+75	O
+.	O
+0	O
+/	O
+90	O
+198	O
+.	O
+2	O
+/	O
+90	O
+270	O
+.	O
+7	O
+/	O
+90	O
+Wilson	O
+B	O
+factor	O
+(	O
+Å2	O
+)	O
+31	O
+41	O
+76	O
+77	O
+NA	O
+Resolution	O
+range	O
+(	O
+Å	O
+)	O
+47	O
+.	O
+10	O
+–	O
+1	O
+.	O
+90	O
+(	O
+1	O
+.	O
+95	O
+–	O
+1	O
+.	O
+90	O
+)	O
+62	O
+.	O
+47	O
+–	O
+2	O
+.	O
+10	O
+(	O
+2	O
+.	O
+16	O
+–	O
+2	O
+.	O
+10	O
+)	O
+75	O
+.	O
+00	O
+–	O
+2	O
+.	O
+80	O
+(	O
+2	O
+.	O
+99	O
+–	O
+2	O
+.	O
+80	O
+)	O
+67	O
+.	O
+80	O
+–	O
+3	O
+.	O
+20	O
+(	O
+3	O
+.	O
+42	O
+–	O
+3	O
+.	O
+20	O
+)	O
+67	O
+.	O
+68	O
+–	O
+3	O
+.	O
+50	O
+(	O
+7	O
+.	O
+16	O
+–	O
+6	O
+.	O
+40	O
+,	O
+3	O
+.	O
+92	O
+–	O
+3	O
+.	O
+50	O
+)	O
+Unique	O
+reflections	O
+23	O
+,	O
+300	O
+(	O
+1	O
+,	O
+524	O
+)	O
+17	O
+,	O
+089	O
+(	O
+1	O
+,	O
+428	O
+)	O
+7	O
+,	O
+964	O
+(	O
+1	O
+,	O
+448	O
+)	O
+8	O
+,	O
+171	O
+(	O
+1	O
+,	O
+343	O
+)	O
+8	O
+,	O
+070	O
+(	O
+723	O
+,	O
+645	O
+)	O
+Average	O
+multiplicity	O
+9	O
+.	O
+1	O
+(	O
+9	O
+.	O
+2	O
+)	O
+5	O
+.	O
+2	O
+(	O
+5	O
+.	O
+3	O
+)	O
+3	O
+.	O
+7	O
+(	O
+3	O
+.	O
+7	O
+)	O
+22	O
+.	O
+7	O
+(	O
+12	O
+.	O
+6	O
+)	O
+3	O
+.	O
+8	O
+(	O
+3	O
+.	O
+5	O
+,	O
+4	O
+.	O
+4	O
+)	O
+Completeness	O
+(%)	O
+99	O
+.	O
+8	O
+(	O
+98	O
+.	O
+5	O
+)	O
+100	O
+(	O
+100	O
+)	O
+99	O
+.	O
+8	O
+(	O
+100	O
+)	O
+98	O
+.	O
+8	O
+(	O
+93	O
+.	O
+4	O
+)	O
+51	O
+.	O
+6	O
+(	O
+98	O
+.	O
+5	O
+,	O
+14	O
+.	O
+1	O
+)	O
+<	O
+I	O
+/	O
+σI	O
+>	O
+11	O
+.	O
+4	O
+(	O
+1	O
+.	O
+7	O
+)	O
+12	O
+.	O
+0	O
+(	O
+1	O
+.	O
+7	O
+)	O
+14	O
+.	O
+9	O
+(	O
+1	O
+.	O
+5	O
+)	O
+13	O
+.	O
+1	O
+(	O
+1	O
+.	O
+9	O
+)	O
+4	O
+.	O
+6	O
+(	O
+4	O
+.	O
+1	O
+,	O
+2	O
+.	O
+2	O
+)	O
+Rmerge	O
+(%)	O
+14	O
+.	O
+8	O
+(	O
+158	O
+.	O
+3	O
+)	O
+9	O
+.	O
+3	O
+(	O
+98	O
+.	O
+0	O
+)	O
+6	O
+.	O
+2	O
+(	O
+98	O
+.	O
+9	O
+)	O
+29	O
+.	O
+8	O
+(	O
+142	O
+.	O
+2	O
+)	O
+44	O
+.	O
+9	O
+(	O
+40	O
+.	O
+5	O
+,	O
+114	O
+.	O
+2	O
+)	O
+Rpim	O
+(%)	O
+15	O
+.	O
+7	O
+(	O
+55	O
+.	O
+3	O
+)	O
+10	O
+.	O
+3	O
+(	O
+109	O
+.	O
+0	O
+)	O
+3	O
+.	O
+7	O
+(	O
+53	O
+.	O
+8	O
+)	O
+6	O
+.	O
+3	O
+(	O
+40	O
+.	O
+0	O
+)	O
+24	O
+.	O
+7	O
+(	O
+23	O
+.	O
+9	O
+,	O
+59	O
+.	O
+9	O
+)	O
+Refinement	O
+Rwork	O
+(%)	O
+17	O
+.	O
+9	O
+18	O
+.	O
+4	O
+21	O
+.	O
+6	O
+20	O
+.	O
+2	O
+32	O
+.	O
+1	O
+Rfree	O
+(%)	O
+22	O
+.	O
+7	O
+23	O
+.	O
+2	O
+30	O
+.	O
+7	O
+27	O
+.	O
+1	O
+35	O
+.	O
+5	O
+No	O
+.	O
+of	O
+Non	O
+-	O
+Hydrogen	O
+Atoms	O
+Protein	O
+2	O
+,	O
+260	O
+2	O
+,	O
+301	O
+2	O
+,	O
+102	O
+2	O
+,	O
+305	O
+7	O
+,	O
+730	O
+N	O
+-	O
+glycans	O
+42	O
+42	O
+28	O
+28	O
+0	O
+Water	B-chemical
+79	O
+54	O
+0	O
+2	O
+0	O
+Ligands	O
+6	O
+6	O
+2	O
+5	O
+0	O
+Average	O
+B	O
+factor	O
+(	O
+Å2	O
+)	O
+Protein	O
+63	O
+65	O
+108	O
+84	O
+–	O
+N	O
+-	O
+glycans	O
+35	O
+46	O
+102	O
+18	O
+–	O
+Water	B-chemical
+68	O
+85	O
+–	O
+75	O
+–	O
+Ligands	O
+36	O
+47	O
+91	O
+75	O
+66	O
+RMSD	B-evidence
+from	O
+Ideality	O
+Bond	O
+lengths	O
+(	O
+Å	O
+)	O
+0	O
+.	O
+020	O
+0	O
+.	O
+016	O
+0	O
+.	O
+019	O
+0	O
+.	O
+016	O
+0	O
+.	O
+004	O
+Bond	O
+angles	O
+(°)	O
+2	O
+.	O
+050	O
+1	O
+.	O
+748	O
+1	O
+.	O
+952	O
+1	O
+.	O
+796	O
+0	O
+.	O
+770	O
+Ramachandran	O
+Plot	O
+Favored	O
+(%)	O
+96	O
+.	O
+8	O
+95	O
+.	O
+5	O
+96	O
+.	O
+9	O
+94	O
+.	O
+9	O
+92	O
+.	O
+3	O
+Allowed	O
+(%)	O
+99	O
+.	O
+7	O
+100	O
+.	O
+0	O
+100	O
+.	O
+0	O
+99	O
+.	O
+7	O
+99	O
+.	O
+8	O
+Number	O
+of	O
+outliers	O
+1	O
+0	O
+0	O
+1	O
+2	O
+PDB	O
+code	O
+5FWS	O
+5FWT	O
+5FWU	O
+5FWV	O
+5FWW	O
+An	O
+additional	O
+shell	O
+given	O
+for	O
+the	O
+ternary	O
+complex	O
+corresponds	O
+to	O
+the	O
+last	O
+shell	O
+with	O
+near	O
+-	O
+complete	O
+diffraction	B-evidence
+data	I-evidence
+.	O

annotation_IOB/PMC5063996.tsv ADDED Viewed

	@@ -0,0 +1,7314 @@

+The	O
+Mechanism	O
+by	O
+Which	O
+Arabinoxylanases	B-protein_type
+Can	O
+Recognize	O
+Highly	B-protein_state
+Decorated	I-protein_state
+Xylans	B-chemical
+*	O
+The	O
+enzymatic	O
+degradation	O
+of	O
+plant	B-taxonomy_domain
+cell	O
+walls	O
+is	O
+an	O
+important	O
+biological	O
+process	O
+of	O
+increasing	O
+environmental	O
+and	O
+industrial	O
+significance	O
+.	O
+Xylan	B-chemical
+,	O
+a	O
+major	O
+component	O
+of	O
+the	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+,	O
+consists	O
+of	O
+a	O
+backbone	O
+of	O
+β	B-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+xylose	I-chemical
+(	O
+Xylp	B-chemical
+)	O
+units	O
+that	O
+are	O
+often	O
+decorated	O
+with	O
+arabinofuranose	B-chemical
+(	O
+Araf	B-chemical
+)	O
+side	O
+chains	O
+.	O
+A	O
+large	O
+penta	B-protein_type
+-	I-protein_type
+modular	I-protein_type
+enzyme	I-protein_type
+,	O
+CtXyl5A	B-protein
+,	O
+was	O
+shown	O
+previously	O
+to	O
+specifically	O
+target	O
+arabinoxylans	B-chemical
+.	O
+Here	O
+we	O
+report	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+arabinoxylanase	B-protein_type
+and	O
+the	O
+enzyme	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+ligands	B-chemical
+.	O
+The	O
+data	O
+showed	O
+that	O
+four	O
+of	O
+the	O
+protein	O
+modules	O
+adopt	O
+a	O
+rigid	O
+structure	O
+,	O
+which	O
+stabilizes	O
+the	O
+catalytic	B-structure_element
+domain	I-structure_element
+.	O
+The	O
+C	O
+-	O
+terminal	O
+non	B-structure_element
+-	I-structure_element
+catalytic	I-structure_element
+carbohydrate	I-structure_element
+binding	I-structure_element
+module	I-structure_element
+could	O
+not	O
+be	O
+observed	O
+in	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+,	O
+suggesting	O
+positional	O
+flexibility	O
+.	O
+The	O
+structure	B-evidence
+of	O
+the	O
+enzyme	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+Xylp	B-chemical
+-	I-chemical
+β	I-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+Xylp	I-chemical
+-	I-chemical
+β	I-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+Xylp	I-chemical
+-[	I-chemical
+α	I-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+Araf	I-chemical
+]-	I-chemical
+β	I-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+Xylp	I-chemical
+showed	O
+that	O
+the	O
+Araf	B-chemical
+decoration	O
+linked	O
+O3	O
+to	O
+the	O
+xylose	B-chemical
+in	O
+the	O
+active	B-site
+site	I-site
+is	O
+located	O
+in	O
+the	O
+pocket	B-site
+(−	O
+2	B-site
+*	I-site
+subsite	I-site
+)	O
+that	O
+abuts	O
+onto	O
+the	O
+catalytic	B-site
+center	I-site
+.	O
+The	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+can	O
+also	O
+bind	O
+to	O
+Xylp	B-chemical
+and	O
+Arap	B-chemical
+,	O
+explaining	O
+why	O
+the	O
+enzyme	O
+can	O
+utilize	O
+xylose	B-chemical
+and	O
+arabinose	B-chemical
+as	O
+specificity	O
+determinants	O
+.	O
+Alanine	B-experimental_method
+substitution	I-experimental_method
+of	O
+Glu68	B-residue_name_number
+,	O
+Tyr92	B-residue_name_number
+,	O
+or	O
+Asn139	B-residue_name_number
+,	O
+which	O
+interact	O
+with	O
+arabinose	B-chemical
+and	O
+xylose	B-chemical
+side	O
+chains	O
+at	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+,	O
+abrogates	O
+catalytic	O
+activity	O
+.	O
+Distal	O
+to	O
+the	O
+active	B-site
+site	I-site
+,	O
+the	O
+xylan	B-chemical
+backbone	O
+makes	O
+limited	O
+apolar	O
+contacts	O
+with	O
+the	O
+enzyme	O
+,	O
+and	O
+the	O
+hydroxyls	O
+are	O
+solvent	B-protein_state
+-	I-protein_state
+exposed	I-protein_state
+.	O
+This	O
+explains	O
+why	O
+CtXyl5A	B-protein
+is	O
+capable	O
+of	O
+hydrolyzing	O
+xylans	B-chemical
+that	O
+are	O
+extensively	O
+decorated	O
+and	O
+that	O
+are	O
+recalcitrant	O
+to	O
+classic	O
+endo	B-protein_type
+-	I-protein_type
+xylanase	I-protein_type
+attack	O
+.	O
+The	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+is	O
+an	O
+important	O
+biological	O
+substrate	O
+.	O
+This	O
+complex	O
+composite	O
+structure	O
+is	O
+depolymerized	O
+by	O
+microorganisms	B-taxonomy_domain
+that	O
+occupy	O
+important	O
+highly	O
+competitive	O
+ecological	O
+niches	O
+,	O
+whereas	O
+the	O
+process	O
+makes	O
+an	O
+important	O
+contribution	O
+to	O
+the	O
+carbon	O
+cycle	O
+.	O
+Given	O
+that	O
+the	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+is	O
+the	O
+most	O
+abundant	O
+source	O
+of	O
+renewable	O
+organic	O
+carbon	O
+on	O
+the	O
+planet	O
+,	O
+this	O
+macromolecular	O
+substrate	O
+has	O
+substantial	O
+industrial	O
+potential	O
+.	O
+An	O
+example	O
+of	O
+the	O
+chemical	O
+complexity	O
+of	O
+the	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+is	O
+provided	O
+by	O
+xylan	B-chemical
+,	O
+which	O
+is	O
+the	O
+major	O
+hemicellulosic	O
+component	O
+.	O
+This	O
+polysaccharide	B-chemical
+comprises	O
+a	O
+backbone	O
+of	O
+β	B-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+d	I-chemical
+-	I-chemical
+xylose	I-chemical
+residues	O
+in	O
+their	O
+pyranose	B-chemical
+configuration	O
+(	O
+Xylp	B-chemical
+)	O
+that	O
+are	O
+decorated	O
+at	O
+O2	O
+with	O
+4	B-chemical
+-	I-chemical
+O	I-chemical
+-	I-chemical
+methyl	I-chemical
+-	I-chemical
+d	I-chemical
+-	I-chemical
+glucuronic	I-chemical
+acid	I-chemical
+(	O
+GlcA	B-chemical
+)	O
+and	O
+at	O
+O2	O
+and	O
+/	O
+or	O
+O3	O
+with	O
+α	B-chemical
+-	I-chemical
+l	I-chemical
+-	I-chemical
+arabinofuranose	I-chemical
+(	O
+Araf	B-chemical
+)	O
+residues	O
+,	O
+whereas	O
+the	O
+polysaccharide	B-chemical
+can	O
+also	O
+be	O
+extensively	O
+acetylated	O
+.	O
+In	O
+addition	O
+,	O
+the	O
+Araf	B-chemical
+side	O
+chain	O
+decorations	O
+can	O
+also	O
+be	O
+esterified	O
+to	O
+ferulic	B-chemical
+acid	I-chemical
+that	O
+,	O
+in	O
+some	O
+species	O
+,	O
+provide	O
+a	O
+chemical	O
+link	O
+between	O
+hemicellulose	B-chemical
+and	O
+lignin	B-chemical
+.	O
+The	O
+precise	O
+structure	O
+of	O
+xylans	B-chemical
+varies	O
+between	O
+plant	B-taxonomy_domain
+species	O
+,	O
+in	O
+particular	O
+in	O
+different	O
+tissues	O
+and	O
+during	O
+cellular	O
+differentiation	O
+.	O
+In	O
+specialized	O
+plant	B-taxonomy_domain
+tissues	O
+,	O
+such	O
+as	O
+the	O
+outer	O
+layer	O
+of	O
+cereal	B-taxonomy_domain
+grains	O
+,	O
+xylans	B-chemical
+are	O
+extremely	O
+complex	O
+,	O
+and	O
+side	O
+chains	O
+may	O
+comprise	O
+a	O
+range	O
+of	O
+other	O
+sugars	B-chemical
+including	O
+l	B-chemical
+-	I-chemical
+and	I-chemical
+d	I-chemical
+-	I-chemical
+galactose	I-chemical
+and	O
+β	B-chemical
+-	I-chemical
+and	I-chemical
+α	I-chemical
+-	I-chemical
+Xylp	I-chemical
+units	O
+.	O
+Indeed	O
+,	O
+in	O
+these	O
+cereal	B-taxonomy_domain
+brans	O
+,	O
+xylans	B-chemical
+have	O
+very	O
+few	O
+backbone	O
+Xylp	B-chemical
+units	O
+that	O
+are	O
+undecorated	O
+,	O
+and	O
+the	O
+side	O
+chains	O
+can	O
+contain	O
+up	O
+to	O
+six	O
+sugars	B-chemical
+.	O
+Reflecting	O
+the	O
+chemical	O
+and	O
+physical	O
+complexity	O
+of	O
+the	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+,	O
+microorganisms	B-taxonomy_domain
+that	O
+utilize	O
+these	O
+composite	O
+structures	O
+express	O
+a	O
+large	O
+number	O
+of	O
+polysaccharide	B-protein_type
+-	I-protein_type
+degrading	I-protein_type
+enzymes	I-protein_type
+,	O
+primarily	O
+glycoside	B-protein_type
+hydrolases	I-protein_type
+,	O
+but	O
+also	O
+polysaccharide	B-protein_type
+lyases	I-protein_type
+,	O
+carbohydrate	B-protein_type
+esterases	I-protein_type
+,	O
+and	O
+lytic	B-protein_type
+polysaccharide	I-protein_type
+monooxygenases	I-protein_type
+.	O
+These	O
+carbohydrate	B-protein_type
+active	I-protein_type
+enzymes	I-protein_type
+are	O
+grouped	O
+into	O
+sequence	O
+-	O
+based	O
+families	O
+in	O
+the	O
+CAZy	O
+database	O
+.	O
+With	O
+respect	O
+to	O
+xylan	B-chemical
+degradation	O
+,	O
+the	O
+backbone	O
+of	O
+simple	O
+xylans	B-chemical
+is	O
+hydrolyzed	O
+by	O
+endo	B-protein_type
+-	I-protein_type
+acting	I-protein_type
+xylanases	I-protein_type
+,	O
+the	O
+majority	O
+of	O
+which	O
+are	O
+located	O
+in	O
+glycoside	B-protein_type
+hydrolase	I-protein_type
+(	O
+GH	B-protein_type
+)	O
+5	B-protein_type
+families	O
+GH10	B-protein_type
+and	O
+GH11	B-protein_type
+,	O
+although	O
+they	O
+are	O
+also	O
+present	O
+in	O
+GH8	B-protein_type
+.	O
+The	O
+extensive	O
+decoration	O
+of	O
+the	O
+xylan	B-chemical
+backbone	O
+generally	O
+restricts	O
+the	O
+capacity	O
+of	O
+these	O
+enzymes	O
+to	O
+attack	O
+the	O
+polysaccharide	B-chemical
+prior	O
+to	O
+removal	O
+of	O
+the	O
+side	O
+chains	O
+by	O
+a	O
+range	O
+of	O
+α	B-protein_type
+-	I-protein_type
+glucuronidases	I-protein_type
+,	O
+α	B-protein_type
+-	I-protein_type
+arabinofuranosidases	I-protein_type
+,	O
+and	O
+esterases	B-protein_type
+.	O
+Two	O
+xylanases	B-protein_type
+,	O
+however	O
+,	O
+utilize	O
+the	O
+side	O
+chains	O
+as	O
+essential	O
+specificity	O
+determinants	O
+and	O
+thus	O
+target	O
+decorated	O
+forms	O
+of	O
+the	O
+hemicellulose	B-chemical
+.	O
+The	O
+GH30	B-protein_type
+glucuronoxylanases	B-protein_type
+require	O
+the	O
+Xylp	B-chemical
+bound	B-protein_state
+at	I-protein_state
+the	O
+−	B-site
+2	I-site
+to	O
+contain	O
+a	O
+GlcA	B-chemical
+side	O
+chain	O
+(	O
+the	O
+scissile	O
+bond	O
+targeted	O
+by	O
+glycoside	B-protein_type
+hydrolases	I-protein_type
+is	O
+between	O
+subsites	B-site
+−	I-site
+1	I-site
+and	I-site
++	I-site
+1	I-site
+,	O
+and	O
+subsites	B-site
+that	O
+extend	O
+toward	O
+the	O
+non	O
+-	O
+reducing	O
+and	O
+reducing	O
+ends	O
+of	O
+the	O
+substrate	O
+are	O
+assigned	O
+increasing	O
+negative	O
+and	O
+positive	O
+numbers	O
+,	O
+respectively	O
+).	O
+The	O
+GH5	B-protein_type
+arabinoxylanase	B-protein_type
+(	O
+CtXyl5A	B-protein
+)	O
+derived	O
+from	O
+Clostridium	B-species
+thermocellum	I-species
+displays	O
+an	O
+absolute	O
+requirement	O
+for	O
+xylans	B-chemical
+that	O
+contain	O
+Araf	B-chemical
+side	O
+chains	O
+.	O
+In	O
+this	O
+enzyme	O
+,	O
+the	O
+key	O
+specificity	O
+determinant	O
+is	O
+the	O
+Araf	B-chemical
+appended	O
+to	O
+O3	O
+of	O
+the	O
+Xylp	B-chemical
+bound	B-protein_state
+in	I-protein_state
+the	O
+active	B-site
+site	I-site
+(−	O
+1	B-site
+subsite	I-site
+).	O
+The	O
+reaction	O
+products	O
+generated	O
+from	O
+arabinoxylans	B-chemical
+,	O
+however	O
+,	O
+suggest	O
+that	O
+Araf	B-chemical
+can	O
+be	O
+accommodated	O
+at	O
+subsites	B-site
+distal	O
+to	O
+the	O
+active	B-site
+site	I-site
+.	O
+CtXyl5A	B-protein
+is	O
+a	O
+multimodular	O
+enzyme	O
+containing	O
+,	O
+in	O
+addition	O
+to	O
+the	O
+GH5	B-protein_type
+catalytic	B-structure_element
+module	I-structure_element
+(	O
+CtGH5	B-structure_element
+);	O
+three	O
+non	B-structure_element
+-	I-structure_element
+catalytic	I-structure_element
+carbohydrate	I-structure_element
+binding	I-structure_element
+modules	I-structure_element
+(	O
+CBMs	B-structure_element
+)	O
+belonging	O
+to	O
+families	O
+6	B-protein_type
+(	O
+CtCBM6	B-structure_element
+),	O
+13	B-protein_type
+(	O
+CtCBM13	B-structure_element
+),	O
+and	O
+62	B-protein_type
+(	O
+CtCBM62	B-structure_element
+);	O
+fibronectin	B-protein_type
+type	I-protein_type
+3	I-protein_type
+(	O
+Fn3	B-structure_element
+)	O
+domain	O
+;	O
+and	O
+a	O
+C	O
+-	O
+terminal	O
+dockerin	B-structure_element
+domain	O
+Fig	O
+.	O
+1	O
+.	O
+Previous	O
+studies	O
+of	O
+Fn3	B-structure_element
+domains	O
+have	O
+indicated	O
+that	O
+they	O
+might	O
+function	O
+as	O
+ligand	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+modules	I-structure_element
+,	O
+as	O
+a	O
+compact	O
+form	O
+of	O
+peptide	O
+linkers	O
+or	O
+spacers	O
+between	O
+other	O
+domains	O
+,	O
+as	O
+cellulose	B-structure_element
+-	I-structure_element
+disrupting	I-structure_element
+modules	I-structure_element
+,	O
+or	O
+as	O
+proteins	O
+that	O
+help	O
+large	O
+enzyme	O
+complexes	O
+remain	O
+soluble	O
+.	O
+The	O
+dockerin	B-structure_element
+domain	O
+recruits	O
+the	O
+enzyme	O
+into	O
+the	O
+cellulosome	B-complex_assembly
+,	O
+a	O
+multienzyme	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+degrading	O
+complex	O
+presented	O
+on	O
+the	O
+surface	O
+of	O
+C	B-species
+.	I-species
+thermocellum	I-species
+.	O
+CtCBM6	B-structure_element
+stabilizes	O
+CtGH5	B-structure_element
+,	O
+and	O
+CtCBM62	B-structure_element
+binds	O
+to	O
+d	B-chemical
+-	I-chemical
+galactopyranose	I-chemical
+and	O
+l	B-chemical
+-	I-chemical
+arabinopyranose	I-chemical
+.	O
+The	O
+function	O
+of	O
+the	O
+CtCBM13	B-structure_element
+and	O
+Fn3	B-structure_element
+modules	O
+remains	O
+unclear	O
+.	O
+This	O
+report	O
+exploits	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+mature	B-protein_state
+CtXyl5A	B-protein
+lacking	B-protein_state
+its	O
+C	O
+-	O
+terminal	O
+dockerin	B-structure_element
+domain	O
+(	O
+CtXyl5A	B-mutant
+-	I-mutant
+Doc	I-mutant
+),	O
+and	O
+the	O
+enzyme	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+ligands	B-chemical
+,	O
+to	O
+explore	O
+the	O
+mechanism	O
+of	O
+substrate	O
+specificity	O
+.	O
+The	O
+data	O
+show	O
+that	O
+the	O
+plasticity	O
+in	O
+substrate	O
+recognition	O
+enables	O
+the	O
+enzyme	O
+to	O
+hydrolyze	O
+highly	O
+complex	O
+xylans	B-chemical
+that	O
+are	O
+not	O
+accessible	O
+to	O
+classical	O
+GH10	B-protein_type
+and	O
+GH11	B-protein_type
+endo	B-protein_type
+-	I-protein_type
+xylanases	I-protein_type
+.	O
+Molecular	O
+architecture	O
+of	O
+GH5_34	B-protein_type
+enzymes	O
+.	O
+Modules	O
+prefaced	O
+by	O
+GH	B-structure_element
+,	O
+CBM	B-structure_element
+,	O
+or	O
+CE	B-structure_element
+are	O
+modules	O
+in	O
+the	O
+indicated	O
+glycoside	B-protein_type
+hydrolase	I-protein_type
+,	O
+carbohydrate	B-structure_element
+binding	I-structure_element
+module	I-structure_element
+,	O
+or	O
+carbohydrate	B-protein_type
+esterase	I-protein_type
+families	O
+,	O
+respectively	O
+.	O
+Laminin_3_G	B-structure_element
+domain	O
+belongs	O
+to	O
+the	O
+concanavalin	B-protein_type
+A	I-protein_type
+lectin	I-protein_type
+superfamily	I-protein_type
+,	O
+and	O
+FN3	B-structure_element
+denotes	O
+a	O
+fibronectin	B-structure_element
+type	I-structure_element
+3	I-structure_element
+domain	I-structure_element
+.	O
+Segments	O
+labeled	O
+D	O
+are	O
+dockerin	B-structure_element
+domains	O
+.	O
+Substrate	O
+Specificity	O
+of	O
+CtXyl5A	B-protein
+Previous	O
+studies	O
+showed	O
+that	O
+CtXyl5A	B-protein
+is	O
+an	O
+arabinoxylan	B-protein_type
+-	I-protein_type
+specific	I-protein_type
+xylanase	I-protein_type
+that	O
+generates	O
+xylooligosaccharides	B-chemical
+with	O
+an	O
+arabinose	B-chemical
+linked	O
+O3	O
+to	O
+the	O
+reducing	O
+end	O
+xylose	B-chemical
+.	O
+The	O
+enzyme	O
+is	O
+active	O
+against	O
+both	O
+wheat	B-taxonomy_domain
+and	O
+rye	B-taxonomy_domain
+arabinoxylans	B-chemical
+(	O
+abbreviated	O
+as	O
+WAX	B-chemical
+and	O
+RAX	B-chemical
+,	O
+respectively	O
+).	O
+It	O
+was	O
+proposed	O
+that	O
+arabinose	B-chemical
+decorations	O
+make	O
+productive	O
+interactions	O
+with	O
+a	O
+pocket	B-site
+(−	O
+2	B-site
+*)	I-site
+that	O
+is	O
+abutted	O
+onto	O
+the	O
+active	B-site
+site	I-site
+or	O
+−	B-site
+1	I-site
+subsite	I-site
+.	O
+Arabinose	B-chemical
+side	O
+chains	O
+of	O
+the	O
+other	O
+backbone	O
+xylose	B-chemical
+units	O
+in	O
+the	O
+oligosaccharides	B-chemical
+generated	O
+by	O
+CtXyl5A	B-protein
+were	O
+essentially	O
+random	O
+.	O
+These	O
+data	O
+suggest	O
+that	O
+O3	O
+,	O
+and	O
+possibly	O
+O2	O
+,	O
+on	O
+the	O
+xylose	B-chemical
+residues	O
+at	O
+subsites	B-site
+distal	O
+to	O
+the	O
+active	B-site
+site	I-site
+and	O
+−	B-site
+2	I-site
+*	I-site
+pocket	I-site
+are	O
+solvent	B-protein_state
+-	I-protein_state
+exposed	I-protein_state
+,	O
+implying	O
+that	O
+the	O
+enzyme	O
+can	O
+access	O
+highly	O
+decorated	O
+xylans	B-chemical
+.	O
+To	O
+test	O
+this	O
+hypothesis	O
+,	O
+the	O
+activity	O
+of	O
+CtXyl5A	B-protein
+against	O
+xylans	B-chemical
+from	O
+cereal	B-taxonomy_domain
+brans	O
+was	O
+assessed	O
+.	O
+CtXyl5a	B-protein
+was	O
+incubated	B-experimental_method
+with	O
+a	O
+range	O
+of	O
+xylans	B-chemical
+for	O
+16	O
+h	O
+at	O
+60	O
+°	O
+C	O
+,	O
+and	O
+the	O
+limit	O
+products	O
+were	O
+visualized	O
+by	O
+TLC	B-experimental_method
+.	O
+These	O
+xylans	B-chemical
+are	O
+highly	O
+decorated	O
+not	O
+only	O
+with	O
+Araf	B-chemical
+and	O
+GlcA	B-chemical
+units	O
+but	O
+also	O
+with	O
+l	B-chemical
+-	I-chemical
+Gal	I-chemical
+,	O
+d	B-chemical
+-	I-chemical
+Gal	I-chemical
+,	O
+and	O
+d	B-chemical
+-	I-chemical
+Xyl	I-chemical
+.	O
+Indeed	O
+,	O
+very	O
+few	O
+xylose	B-chemical
+units	O
+in	O
+the	O
+backbone	O
+of	O
+bran	O
+xylans	B-chemical
+lack	O
+side	O
+chains	O
+.	O
+The	O
+data	O
+presented	O
+in	O
+Table	O
+1	O
+showed	O
+that	O
+CtXyl5A	B-protein
+was	O
+active	O
+against	O
+corn	B-taxonomy_domain
+bran	O
+xylan	B-chemical
+(	O
+CX	B-chemical
+).	O
+In	O
+contrast	O
+typical	O
+endo	B-protein_type
+-	I-protein_type
+xylanases	I-protein_type
+from	O
+GH10	B-protein_type
+and	O
+GH11	B-protein_type
+were	O
+unable	O
+to	O
+attack	O
+CX	B-chemical
+,	O
+reflecting	O
+the	O
+lack	B-protein_state
+of	I-protein_state
+undecorated	O
+xylose	B-chemical
+units	O
+in	O
+the	O
+backbone	O
+(	O
+the	O
+active	B-site
+site	I-site
+of	O
+these	O
+enzymes	O
+can	O
+only	O
+bind	B-protein_state
+to	I-protein_state
+non	O
+-	O
+substituted	O
+xylose	B-chemical
+residues	O
+).	O
+The	O
+limit	O
+products	O
+generated	O
+by	O
+CtXyl5A	B-protein
+from	O
+CX	B-chemical
+consisted	O
+of	O
+an	O
+extensive	O
+range	O
+of	O
+oligosaccharides	B-chemical
+.	O
+These	O
+data	O
+support	O
+the	O
+view	O
+that	O
+in	O
+subsites	B-site
+out	O
+with	O
+the	O
+active	B-site
+site	I-site
+the	O
+O2	O
+and	O
+O3	O
+groups	O
+of	O
+the	O
+bound	O
+xylose	B-chemical
+units	O
+are	O
+solvent	B-protein_state
+-	I-protein_state
+exposed	I-protein_state
+and	O
+will	O
+thus	O
+tolerate	O
+decoration	O
+.	O
+Kinetics	B-evidence
+of	O
+GH5_34	B-protein_type
+arabinoxylanases	B-protein_type
+Enzyme	O
+Variant	O
+kcat	B-evidence
+/	O
+Km	B-evidence
+WAX	B-chemical
+RAX	B-chemical
+CX	B-chemical
+min	O
+−	O
+1mg	O
+−	O
+1ml	O
+CtXyl5A	B-protein
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+-	I-structure_element
+CBM13	I-structure_element
+-	I-structure_element
+Fn3	I-structure_element
+-	I-structure_element
+CBM62	I-structure_element
+800	O
+ND	O
+460	O
+CtXyl5A	B-protein
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+-	I-structure_element
+CBM13	I-structure_element
+-	I-structure_element
+Fn3	I-structure_element
+1	O
+,	O
+232	O
+ND	O
+659	O
+CtXyl5A	B-protein
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+-	I-structure_element
+CBM13	I-structure_element
+1	O
+,	O
+307	O
+ND	O
+620	O
+CtXyl5A	B-protein
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+488	O
+ND	O
+102	O
+CtXyl5A	B-protein
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+:	O
+E68A	B-mutant
+NA	O
+NA	O
+NA	O
+CtXyl5A	B-protein
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+:	O
+Y92A	B-mutant
+NA	O
+NA	O
+NA	O
+CtXyl5A	B-protein
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+:	O
+N135A	B-mutant
+260	O
+ND	O
+ND	O
+CtXyl5A	B-protein
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+:	O
+N139A	B-mutant
+NA	O
+NA	O
+NA	O
+AcGH5	B-protein
+Wild	B-protein_state
+type	I-protein_state
+628	O
+1	O
+,	O
+641	O
+289	O
+GpGH5	B-protein
+Wild	B-protein_state
+type	I-protein_state
+2	O
+,	O
+600	O
+9	O
+,	O
+986	O
+314	O
+VbGH5	B-protein
+Wild	B-protein_state
+type	I-protein_state
+ND	O
+ND	O
+ND	O
+VbGH5	B-protein
+D45A	B-mutant
+102	O
+203	O
+23	O
+To	O
+explore	O
+whether	O
+substrate	O
+bound	B-protein_state
+only	I-protein_state
+at	I-protein_state
+−	B-site
+2	I-site
+*	I-site
+and	O
+−	B-site
+1	I-site
+in	O
+the	O
+negative	B-site
+subsites	I-site
+was	O
+hydrolyzed	O
+by	O
+CtXyl5A	B-protein
+,	O
+the	O
+limit	O
+products	O
+of	O
+CX	B-chemical
+digested	O
+by	O
+the	O
+arabinoxylanase	B-protein_type
+were	O
+subjected	O
+to	O
+size	B-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+using	O
+a	O
+Bio	O
+-	O
+Gel	O
+P	O
+-	O
+2	O
+,	O
+and	O
+the	O
+smallest	O
+oligosaccharides	B-chemical
+(	O
+largest	O
+elution	O
+volume	O
+)	O
+were	O
+chosen	O
+for	O
+further	O
+study	O
+.	O
+HPAEC	B-experimental_method
+analysis	O
+of	O
+the	O
+smallest	O
+oligosaccharide	B-chemical
+fraction	O
+(	O
+pool	O
+4	O
+)	O
+contained	O
+two	O
+species	O
+with	O
+retention	O
+times	O
+of	O
+14	O
+.	O
+0	O
+min	O
+(	O
+oligosaccharide	B-chemical
+1	O
+)	O
+and	O
+20	O
+.	O
+8	O
+min	O
+(	O
+oligosaccharide	B-chemical
+2	O
+)	O
+(	O
+Fig	O
+.	O
+2	O
+).	O
+Positive	B-experimental_method
+mode	I-experimental_method
+electrospray	I-experimental_method
+mass	I-experimental_method
+spectrometry	I-experimental_method
+showed	O
+that	O
+pool	O
+4	O
+contained	O
+exclusively	O
+molecular	O
+ions	O
+with	O
+a	O
+m	O
+/	O
+z	O
+=	O
+305	O
+[	O
+M	O
++	O
+Na	O
+]+,	O
+which	O
+corresponds	O
+to	O
+a	O
+pentose	B-chemical
+-	O
+pentose	B-chemical
+disaccharide	B-chemical
+(	O
+molecular	O
+mass	O
+=	O
+282	O
+Da	O
+)	O
+as	O
+a	O
+sodium	O
+ion	O
+adduct	O
+,	O
+whereas	O
+a	O
+dimer	O
+of	O
+the	O
+disaccharide	B-chemical
+with	O
+a	O
+sodium	O
+adduct	O
+(	O
+m	O
+/	O
+z	O
+=	O
+587	O
+[	O
+2M	O
++	O
+Na	O
+]+)	O
+was	O
+also	O
+evident	O
+.	O
+The	O
+monosaccharide	O
+composition	O
+of	O
+pool	O
+4	O
+determined	O
+by	O
+TFA	B-experimental_method
+hydrolysis	I-experimental_method
+contained	O
+xylose	B-chemical
+and	O
+arabinose	B-chemical
+in	O
+a	O
+3	O
+:	O
+1	O
+ratio	O
+.	O
+This	O
+suggests	O
+that	O
+the	O
+two	O
+oligosaccharides	B-chemical
+consist	O
+of	O
+two	O
+disaccharides	B-chemical
+:	O
+one	O
+consisting	O
+of	O
+two	O
+xylose	B-chemical
+residues	O
+and	O
+the	O
+other	O
+consisting	O
+of	O
+an	O
+arabinose	B-chemical
+linked	O
+to	O
+a	O
+xylose	B-chemical
+.	O
+Treatment	O
+of	O
+pool	O
+4	O
+with	O
+the	O
+nonspecific	B-protein_type
+arabinofuranosidase	I-protein_type
+,	O
+CjAbf51A	B-protein
+,	O
+resulted	O
+in	O
+the	O
+loss	O
+of	O
+oligosaccharide	B-chemical
+2	O
+and	O
+the	O
+production	O
+of	O
+both	O
+xylose	B-chemical
+and	O
+arabinose	B-chemical
+,	O
+indicative	O
+of	O
+a	O
+disaccharide	B-chemical
+of	O
+xylose	B-chemical
+and	O
+arabinose	B-chemical
+.	O
+Incubation	O
+of	O
+pool	O
+4	O
+with	O
+a	O
+β	B-protein_type
+-	I-protein_type
+1	I-protein_type
+,	I-protein_type
+3	I-protein_type
+-	I-protein_type
+xylosidase	I-protein_type
+(	O
+XynB	B-protein
+)	O
+converted	O
+oligosaccharide	B-chemical
+1	O
+into	O
+xylose	B-chemical
+,	O
+demonstrating	O
+that	O
+this	O
+molecule	O
+is	O
+the	O
+disaccharide	B-chemical
+β	B-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+xylobiose	I-chemical
+.	O
+This	O
+view	O
+is	O
+supported	O
+by	O
+the	O
+inability	O
+of	O
+a	O
+β	B-protein_type
+-	I-protein_type
+1	I-protein_type
+,	I-protein_type
+4	I-protein_type
+-	I-protein_type
+specific	I-protein_type
+xylosidase	I-protein_type
+to	O
+hydrolyze	O
+oligosaccharide	B-chemical
+1	O
+or	O
+oligosaccharide	B-chemical
+2	O
+(	O
+data	O
+not	O
+shown	O
+).	O
+The	O
+crucial	O
+importance	O
+of	O
+occupancy	O
+of	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+pocket	I-site
+for	O
+catalytic	O
+competence	O
+is	O
+illustrated	O
+by	O
+the	O
+inability	O
+of	O
+the	O
+enzyme	O
+to	O
+hydrolyze	O
+linear	O
+β	B-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+xylooligosaccharides	I-chemical
+.	O
+The	O
+generation	O
+of	O
+Araf	B-chemical
+-	I-chemical
+Xylp	I-chemical
+and	O
+Xyl	B-chemical
+-	I-chemical
+β	I-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+Xyl	I-chemical
+as	O
+reaction	O
+products	O
+demonstrates	O
+that	O
+occupancy	O
+of	O
+the	O
+−	B-site
+2	I-site
+subsite	I-site
+is	O
+not	O
+essential	O
+for	O
+catalytic	O
+activity	O
+,	O
+which	O
+is	O
+in	O
+contrast	O
+to	O
+all	O
+endo	B-protein_type
+-	I-protein_type
+acting	I-protein_type
+xylanases	I-protein_type
+where	O
+this	O
+subsite	B-site
+plays	O
+a	O
+critical	O
+role	O
+in	O
+enzyme	O
+activity	O
+.	O
+Indeed	O
+,	O
+the	O
+data	O
+demonstrate	O
+that	O
+−	B-site
+2	I-site
+*	I-site
+plays	O
+a	O
+more	O
+important	O
+role	O
+in	O
+productive	O
+substrate	O
+binding	O
+than	O
+the	O
+−	B-site
+2	I-site
+subsite	I-site
+.	O
+Unfortunately	O
+,	O
+the	O
+inability	O
+to	O
+generate	O
+highly	O
+purified	O
+(	B-chemical
+Xyl	I-chemical
+-	I-chemical
+β	I-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+4	I-chemical
+)	I-chemical
+n	I-chemical
+-[	I-chemical
+β	I-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+Xyl	I-chemical
+/	I-chemical
+Ara	I-chemical
+]-	I-chemical
+Xyl	I-chemical
+oligosaccharides	B-chemical
+from	O
+arabinoxylans	B-chemical
+prevented	O
+the	O
+precise	O
+binding	O
+energies	O
+at	O
+the	O
+negative	O
+subsites	O
+to	O
+be	O
+determined	O
+.	O
+Identification	O
+of	O
+the	O
+disaccharide	B-chemical
+reaction	O
+products	O
+generated	O
+from	O
+CX	B-chemical
+.	O
+The	O
+smallest	O
+reaction	O
+products	O
+were	O
+purified	O
+by	O
+size	B-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+and	O
+analyzed	O
+by	O
+HPAEC	B-experimental_method
+(	O
+A	O
+)	O
+and	O
+positive	O
+mode	O
+ESI	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+(	O
+B	O
+),	O
+respectively	O
+.	O
+The	O
+samples	O
+were	O
+treated	O
+with	O
+a	O
+nonspecific	B-protein_type
+arabinofuranosidase	I-protein_type
+(	O
+CjAbf51A	B-protein
+)	O
+and	O
+a	O
+GH3	B-protein_type
+xylosidase	I-protein_type
+(	O
+XynB	B-protein
+)	O
+that	O
+targeted	O
+β	O
+-	O
+1	O
+,	O
+3	O
+-	O
+xylosidic	O
+bonds	O
+.	O
+X	O
+,	O
+xylose	B-chemical
+;	O
+A	O
+,	O
+arabinose	B-chemical
+.	O
+The	O
+m	O
+/	O
+z	O
+=	O
+305	O
+species	O
+denotes	O
+a	O
+pentose	B-chemical
+disaccharide	B-chemical
+as	O
+a	O
+sodium	O
+adduct	O
+[	O
+M	O
++	O
+Na	O
+]+,	O
+whereas	O
+the	O
+m	O
+/	O
+z	O
+=	O
+587	O
+signal	O
+corresponds	O
+to	O
+an	O
+ESI	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+dimer	O
+of	O
+the	O
+pentose	B-chemical
+disaccharide	B-chemical
+also	O
+as	O
+a	O
+sodium	O
+adduct	O
+[	O
+2M	O
++	O
+Na	O
+]+.	O
+Crystal	B-evidence
+Structure	I-evidence
+of	O
+the	O
+Catalytic	B-structure_element
+Module	I-structure_element
+of	O
+CtXyl5A	B-protein
+in	B-protein_state
+Complex	I-protein_state
+with	I-protein_state
+Ligands	B-chemical
+To	O
+understand	O
+the	O
+structural	O
+basis	O
+for	O
+the	O
+biochemical	O
+properties	O
+of	O
+CtXyl5A	B-protein
+,	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+enzyme	O
+with	O
+ligands	O
+that	O
+occupy	O
+the	O
+substrate	B-site
+binding	I-site
+cleft	I-site
+and	O
+the	O
+critical	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+were	O
+sought	O
+.	O
+The	O
+data	O
+presented	O
+in	O
+Fig	O
+.	O
+3A	O
+show	O
+the	O
+structure	B-evidence
+of	O
+the	O
+CtXyl5A	B-protein
+derivative	O
+CtGH5	B-structure_element
+-	I-structure_element
+CtCBM6	I-structure_element
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+arabinose	B-chemical
+bound	B-protein_state
+in	I-protein_state
+the	O
+−	B-site
+2	I-site
+*	I-site
+pocket	I-site
+.	O
+Interestingly	O
+,	O
+the	O
+bound	B-protein_state
+arabinose	B-chemical
+was	O
+in	O
+the	O
+pyranose	B-chemical
+conformation	O
+rather	O
+than	O
+in	O
+its	O
+furanose	B-chemical
+form	O
+found	O
+in	O
+arabinoxylans	B-chemical
+.	O
+O1	O
+was	O
+facing	O
+toward	O
+the	O
+active	B-site
+site	I-site
+−	B-site
+1	I-site
+subsite	I-site
+,	O
+indicative	O
+of	O
+the	O
+bound	B-protein_state
+arabinose	B-chemical
+being	O
+in	O
+the	O
+right	O
+orientation	O
+to	O
+be	O
+linked	O
+to	O
+the	O
+xylan	B-chemical
+backbone	O
+via	O
+an	O
+α	O
+-	O
+1	O
+,	O
+3	O
+linkage	O
+.	O
+As	O
+discussed	O
+on	O
+below	O
+,	O
+the	O
+axial	O
+O4	O
+of	O
+the	O
+Arap	B-chemical
+did	O
+not	O
+interact	O
+with	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+,	O
+suggesting	O
+that	O
+the	O
+pocket	B-site
+might	O
+be	O
+capable	O
+of	O
+binding	O
+a	O
+xylose	B-chemical
+molecule	O
+.	O
+Indeed	O
+,	O
+soaking	B-experimental_method
+apo	B-protein_state
+crystals	B-evidence
+with	O
+xylose	B-chemical
+showed	O
+that	O
+the	O
+pentose	B-chemical
+sugar	B-chemical
+also	O
+bound	B-protein_state
+in	I-protein_state
+the	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+in	O
+its	O
+pyranose	B-chemical
+conformation	O
+(	O
+Fig	O
+.	O
+3B	O
+).	O
+These	O
+crystal	B-evidence
+structures	I-evidence
+support	O
+the	O
+biochemical	O
+data	O
+presented	O
+above	O
+showing	O
+that	O
+the	O
+enzyme	O
+generated	O
+β	B-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+xylobiose	I-chemical
+from	O
+CX	B-chemical
+,	O
+which	O
+would	O
+require	O
+the	O
+disaccharide	B-chemical
+to	O
+bind	O
+at	O
+the	O
+−	B-site
+1	I-site
+and	I-site
+−	I-site
+2	I-site
+*	I-site
+subsites	I-site
+.	O
+A	O
+third	O
+product	O
+complex	O
+was	O
+generated	O
+by	O
+co	B-experimental_method
+-	I-experimental_method
+crystallizing	I-experimental_method
+the	O
+nucleophile	B-protein_state
+inactive	I-protein_state
+mutant	B-protein_state
+CtGH5E279S	B-mutant
+-	O
+CtCBM6	B-structure_element
+with	O
+a	O
+WAX	B-chemical
+-	O
+derived	O
+oligosaccharide	B-chemical
+(	O
+Fig	O
+.	O
+3C	O
+).	O
+The	O
+data	O
+revealed	O
+a	O
+pentasaccharide	B-chemical
+bound	B-protein_state
+to	I-protein_state
+the	O
+enzyme	O
+,	O
+comprising	O
+β	B-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+xylotetraose	I-chemical
+with	O
+an	O
+Araf	B-chemical
+linked	O
+α	O
+-	O
+1	O
+,	O
+3	O
+to	O
+the	O
+reducing	O
+end	O
+xylose	B-chemical
+.	O
+The	O
+xylotetraose	B-chemical
+was	O
+positioned	O
+in	O
+subsites	B-site
+−	I-site
+1	I-site
+to	I-site
+−	I-site
+4	I-site
+and	O
+the	O
+Araf	B-chemical
+in	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+pocket	I-site
+.	O
+Analysis	O
+of	O
+the	O
+three	O
+structures	B-evidence
+showed	O
+that	O
+O1	O
+,	O
+O2	O
+,	O
+O3	O
+,	O
+and	O
+the	O
+endocyclic	O
+oxygen	O
+occupied	O
+identical	O
+positions	O
+in	O
+the	O
+Arap	B-chemical
+,	O
+Araf	B-chemical
+,	O
+and	O
+Xylp	B-chemical
+ligands	O
+bound	B-protein_state
+in	I-protein_state
+the	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+and	O
+thus	O
+made	O
+identical	O
+interactions	O
+with	O
+the	O
+pocket	B-site
+.	O
+O1	O
+makes	O
+a	O
+polar	B-bond_interaction
+contact	I-bond_interaction
+with	O
+Nδ2	O
+of	O
+Asn139	B-residue_name_number
+,	O
+O2	O
+is	O
+within	O
+hydrogen	B-bond_interaction
+bonding	I-bond_interaction
+distance	O
+with	O
+Oδ1	O
+of	O
+Asn139	B-residue_name_number
+and	O
+the	O
+backbone	O
+N	O
+of	O
+Asn135	B-residue_name_number
+,	O
+and	O
+O3	O
+interacts	O
+with	O
+the	O
+N	O
+of	O
+Gly136	B-residue_name_number
+and	O
+Oϵ2	O
+of	O
+Glu68	B-residue_name_number
+.	O
+Although	O
+O4	O
+of	O
+Arap	B-chemical
+does	O
+not	O
+make	O
+a	O
+direct	O
+interaction	O
+with	O
+the	O
+enzyme	O
+,	O
+O4	O
+and	O
+O5	O
+of	O
+Xylp	B-chemical
+and	O
+Araf	B-chemical
+,	O
+respectively	O
+,	O
+form	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+with	O
+Oϵ1	O
+of	O
+Glu68	B-residue_name_number
+.	O
+Finally	O
+Tyr92	B-residue_name_number
+makes	O
+apolar	O
+parallel	B-bond_interaction
+interactions	I-bond_interaction
+with	O
+the	O
+pyranose	B-chemical
+or	O
+furanose	B-chemical
+rings	O
+of	O
+the	O
+three	O
+sugars	O
+.	O
+Representation	O
+of	O
+the	O
+residues	O
+involved	O
+in	O
+the	O
+ligands	O
+recognition	O
+at	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+.	O
+Interacting	O
+residues	O
+are	O
+represented	O
+as	O
+stick	O
+in	O
+blue	O
+,	O
+and	O
+the	O
+catalytic	B-site
+residues	I-site
+and	O
+the	O
+mutated	B-experimental_method
+glutamate	B-residue_name
+(	O
+into	O
+a	O
+serine	B-residue_name
+)	O
+are	O
+in	O
+magenta	O
+.	O
+A	O
+,	O
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+an	O
+arabinopyranose	B-chemical
+.	O
+B	O
+,	O
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+a	O
+xylopyranose	B-chemical
+.	O
+C	O
+,	O
+CtGH5E279S	B-mutant
+-	O
+CBM6	B-structure_element
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+a	O
+pentasaccharide	B-chemical
+(	O
+β1	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+xylotetraose	I-chemical
+with	O
+an	O
+l	B-chemical
+-	I-chemical
+Araf	I-chemical
+linked	O
+α1	O
+,	O
+3	O
+to	O
+the	O
+reducing	O
+end	O
+xylose	B-chemical
+).	O
+The	O
+xylan	B-chemical
+backbone	O
+is	O
+shown	O
+transparently	O
+for	O
+more	O
+clarity	O
+.	O
+Densities	B-evidence
+shown	O
+in	O
+blue	O
+are	O
+RefMac	O
+maximum	B-evidence
+-	I-evidence
+likelihood	I-evidence
+σA	I-evidence
+-	I-evidence
+weighted	I-evidence
+2Fo	I-evidence
+−	I-evidence
+Fc	I-evidence
+at	I-evidence
+1	I-evidence
+.	I-evidence
+5	I-evidence
+σ	I-evidence
+.	O
+The	O
+importance	O
+of	O
+the	O
+interactions	O
+between	O
+the	O
+ligands	O
+and	O
+the	O
+side	O
+chains	O
+of	O
+the	O
+residues	O
+in	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+pocket	I-site
+were	O
+evaluated	O
+by	O
+alanine	B-experimental_method
+substitution	I-experimental_method
+of	O
+these	O
+amino	O
+acids	O
+.	O
+The	O
+mutants	B-protein_state
+E68A	B-mutant
+,	O
+Y92A	B-mutant
+,	O
+and	O
+N139A	B-mutant
+were	O
+all	O
+inactive	B-protein_state
+(	O
+Table	O
+1	O
+),	O
+demonstrating	O
+the	O
+importance	O
+of	O
+the	O
+interactions	O
+of	O
+these	O
+residues	O
+with	O
+the	O
+substrate	O
+and	O
+reinforcing	O
+the	O
+critical	O
+role	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+plays	O
+in	O
+the	O
+activity	O
+of	O
+the	O
+enzyme	O
+.	O
+N135A	B-mutant
+retained	O
+wild	B-protein_state
+type	I-protein_state
+activity	O
+because	O
+the	O
+O2	O
+of	O
+the	O
+sugars	O
+interacts	O
+with	O
+the	O
+backbone	O
+N	O
+of	O
+Asn135	B-residue_name_number
+and	O
+not	O
+with	O
+the	O
+side	O
+chain	O
+.	O
+Because	O
+the	O
+hydroxyls	O
+of	O
+Xylp	B-chemical
+or	O
+Araf	B-chemical
+in	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+pocket	I-site
+are	O
+not	O
+solvent	B-protein_state
+-	I-protein_state
+exposed	I-protein_state
+,	O
+the	O
+active	B-site
+site	I-site
+of	O
+the	O
+arabinoxylanase	B-protein_type
+can	O
+only	O
+bind	O
+to	O
+xylose	B-chemical
+residues	O
+that	O
+contain	O
+a	O
+single	O
+xylose	B-chemical
+or	O
+arabinose	B-chemical
+O3	O
+decoration	O
+.	O
+This	O
+may	O
+explain	O
+why	O
+the	O
+kcat	B-evidence
+/	O
+Km	B-evidence
+for	O
+CtXyl5A	B-protein
+against	O
+WAX	B-chemical
+was	O
+2	O
+-	O
+fold	O
+higher	O
+than	O
+against	O
+CX	B-chemical
+(	O
+Table	O
+1	O
+).	O
+WAX	B-chemical
+is	O
+likely	O
+to	O
+have	O
+a	O
+higher	O
+concentration	O
+of	O
+single	O
+Araf	B-chemical
+decorations	O
+compared	O
+with	O
+CX	B-chemical
+and	O
+thus	O
+contain	O
+more	O
+substrate	O
+available	O
+to	O
+the	O
+arabinoxylanase	B-protein_type
+.	O
+In	O
+the	O
+active	B-site
+site	I-site
+of	O
+CtXyl5A	B-protein
+the	O
+α	B-chemical
+-	I-chemical
+d	I-chemical
+-	I-chemical
+Xylp	I-chemical
+,	O
+which	O
+is	O
+in	O
+its	O
+relaxed	O
+4C1	O
+conformation	O
+,	O
+makes	O
+the	O
+following	O
+interactions	O
+with	O
+the	O
+enzyme	O
+(	O
+Fig	O
+.	O
+4	O
+,	O
+A	O
+–	O
+C	O
+):	O
+O1	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+with	O
+the	O
+Nδ1	O
+of	O
+His253	B-residue_name_number
+and	O
+Oϵ2	O
+of	O
+Glu171	B-residue_name_number
+(	O
+catalytic	O
+acid	O
+-	O
+base	O
+)	O
+and	O
+makes	O
+a	O
+possible	O
+weak	O
+polar	B-bond_interaction
+contact	I-bond_interaction
+with	O
+the	O
+OH	O
+of	O
+Tyr255	B-residue_name_number
+and	O
+Oγ	O
+of	O
+Ser279	B-residue_name_number
+(	O
+mutation	O
+of	O
+the	O
+catalytic	O
+nucleophile	O
+);	O
+O2	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+with	O
+Nδ2	O
+of	O
+Asn170	B-residue_name_number
+and	O
+OH	O
+of	O
+Tyr92	B-residue_name_number
+.	O
+O3	O
+(	O
+O1	O
+of	O
+the	O
+Araf	B-chemical
+at	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+)	O
+makes	O
+a	O
+polar	B-bond_interaction
+contact	I-bond_interaction
+with	O
+Nδ2	O
+of	O
+Asn139	B-residue_name_number
+;	O
+the	O
+endocyclic	O
+oxygen	O
+hydrogens	B-bond_interaction
+bonds	I-bond_interaction
+with	O
+the	O
+OH	O
+of	O
+Tyr255	B-residue_name_number
+.	O
+The	O
+Xylp	B-chemical
+in	O
+the	O
+active	B-site
+site	I-site
+makes	O
+strong	O
+parallel	B-bond_interaction
+apolar	I-bond_interaction
+interactions	I-bond_interaction
+with	O
+Phe310	B-residue_name_number
+.	O
+Substrate	O
+recognition	O
+in	O
+the	O
+active	B-site
+site	I-site
+is	O
+conserved	B-protein_state
+between	O
+CtXyl5A	B-protein
+and	O
+the	O
+closest	O
+GH5	B-protein_type
+structural	O
+homolog	O
+,	O
+the	O
+endoglucanase	B-protein_type
+BaCel5A	B-protein
+(	O
+PDB	O
+code	O
+1qi2	O
+)	O
+as	O
+noted	O
+previously	O
+.	O
+Comparison	O
+of	O
+the	O
+ligand	O
+recognition	O
+at	O
+the	O
+distal	O
+negative	B-site
+subsites	I-site
+between	O
+CtGH5E279S	B-mutant
+-	O
+CBM6	B-structure_element
+,	O
+the	O
+cellulase	B-protein_type
+BaCel5A	B-protein
+,	O
+and	O
+the	O
+xylanase	B-protein_type
+GH10	B-protein_type
+.	O
+A	O
+–	O
+C	O
+show	O
+CtGH5E279S	B-mutant
+-	O
+CBM6	O
+is	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+a	O
+pentasaccharide	B-chemical
+(	O
+β1	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+xylotetraose	I-chemical
+with	O
+an	O
+l	B-chemical
+-	I-chemical
+Araf	I-chemical
+linked	O
+α1	O
+,	O
+3	O
+to	O
+the	O
+reducing	O
+end	O
+xylose	B-chemical
+).	O
+A	O
+,	O
+Poseview	O
+representation	O
+highlighting	O
+the	O
+hydrogen	B-bond_interaction
+bonding	I-bond_interaction
+and	O
+the	O
+hydrophobic	B-bond_interaction
+interactions	I-bond_interaction
+that	O
+occur	O
+in	O
+the	O
+negative	B-site
+subsites	I-site
+.	O
+C	O
+,	O
+density	B-evidence
+of	O
+the	O
+ligand	O
+shown	O
+in	O
+blue	O
+is	O
+RefMac	O
+maximum	B-evidence
+-	I-evidence
+likelihood	I-evidence
+σA	I-evidence
+-	I-evidence
+weighted	I-evidence
+2Fo	I-evidence
+−	I-evidence
+Fc	I-evidence
+at	I-evidence
+1	I-evidence
+.	I-evidence
+5	I-evidence
+σ	I-evidence
+.	O
+D	O
+and	O
+E	O
+display	O
+BaCel5A	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+deoxy	B-chemical
+-	I-chemical
+2	I-chemical
+-	I-chemical
+fluoro	I-chemical
+-	I-chemical
+β	I-chemical
+-	I-chemical
+d	I-chemical
+-	I-chemical
+cellotrioside	I-chemical
+(	O
+PDB	O
+code	O
+1qi2	O
+),	O
+and	O
+F	O
+and	O
+G	O
+show	O
+CmXyn10B	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+a	O
+xylotriose	B-chemical
+(	O
+PDB	O
+code	O
+1uqy	O
+).	O
+B	O
+,	O
+D	O
+,	O
+and	O
+F	O
+are	O
+surface	O
+representations	O
+(	O
+CtGH5E279S	B-mutant
+-	O
+CBM6	O
+in	O
+gray	O
+,	O
+BaCel5A	B-protein
+in	O
+cyan	O
+,	O
+and	O
+the	O
+xylanase	B-protein_type
+GH10	B-protein_type
+in	O
+light	O
+brown	O
+).	O
+The	O
+black	O
+dashes	O
+represent	O
+the	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+.	O
+The	O
+capacity	O
+of	O
+CtXyl5A	B-protein
+to	O
+act	O
+on	O
+the	O
+highly	O
+decorated	O
+xylan	B-chemical
+CX	B-chemical
+indicates	O
+that	O
+O3	O
+and	O
+possibly	O
+O2	O
+of	O
+the	O
+backbone	O
+Xylp	B-chemical
+units	O
+are	O
+solvent	B-protein_state
+-	I-protein_state
+exposed	I-protein_state
+.	O
+This	O
+is	O
+consistent	O
+with	O
+the	O
+interaction	O
+of	O
+the	O
+xylotetraose	B-chemical
+backbone	O
+with	O
+the	O
+enzyme	O
+distal	O
+to	O
+the	O
+active	B-site
+site	I-site
+.	O
+A	O
+surface	O
+representation	O
+of	O
+the	O
+enzyme	O
+(	O
+Fig	O
+.	O
+4B	O
+)	O
+shows	O
+that	O
+O3	O
+and	O
+O2	O
+of	O
+xylose	B-chemical
+units	O
+at	O
+subsites	B-site
+−	I-site
+2	I-site
+to	I-site
+−	I-site
+4	I-site
+are	O
+solvent	B-protein_state
+-	I-protein_state
+exposed	I-protein_state
+and	O
+are	O
+thus	O
+available	O
+for	O
+decoration	O
+.	O
+Indeed	O
+,	O
+these	O
+pyranose	B-chemical
+sugars	B-chemical
+make	O
+very	O
+weak	O
+apolar	B-bond_interaction
+interactions	I-bond_interaction
+with	O
+the	O
+arabinoxylanase	B-protein_type
+.	O
+At	O
+−	B-site
+2	I-site
+,	O
+Xylp	B-chemical
+makes	O
+planar	B-bond_interaction
+apolar	I-bond_interaction
+interactions	I-bond_interaction
+with	O
+the	O
+Araf	B-chemical
+bound	B-protein_state
+to	I-protein_state
+the	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+(	O
+Fig	O
+.	O
+4C	O
+).	O
+Xylp	B-chemical
+at	O
+subsites	B-site
+−	I-site
+2	I-site
+and	I-site
+−	I-site
+3	I-site
+,	O
+respectively	O
+,	O
+make	O
+weak	O
+hydrophobic	B-bond_interaction
+contact	I-bond_interaction
+with	O
+Val318	B-residue_name_number
+,	O
+the	O
+−	B-site
+3	I-site
+Xylp	B-chemical
+makes	O
+planar	B-bond_interaction
+apolar	I-bond_interaction
+interactions	I-bond_interaction
+with	O
+Ala137	B-residue_name_number
+,	O
+whereas	O
+the	O
+xylose	B-chemical
+at	O
+−	B-site
+4	I-site
+forms	O
+parallel	B-bond_interaction
+apolar	I-bond_interaction
+contacts	I-bond_interaction
+with	O
+Trp69	B-residue_name_number
+.	O
+Comparison	O
+of	O
+the	O
+distal	O
+negative	B-site
+subsites	I-site
+of	O
+CtXyl5A	B-protein
+with	O
+BaCel5A	B-protein
+and	O
+a	O
+typical	O
+GH10	B-protein_type
+xylanase	B-protein_type
+(	O
+CmXyn10B	B-protein
+,	O
+PDB	O
+code	O
+1uqy	O
+)	O
+highlights	O
+the	O
+paucity	O
+of	O
+interactions	O
+between	O
+the	O
+arabinoxylanase	B-protein_type
+and	O
+its	O
+substrate	O
+out	O
+with	O
+the	O
+active	B-site
+site	I-site
+(	O
+Fig	O
+.	O
+4	O
+).	O
+Thus	O
+,	O
+the	O
+cellulase	B-protein_type
+contains	O
+three	O
+negative	B-site
+subsites	I-site
+and	O
+the	O
+sugars	B-chemical
+bound	B-protein_state
+in	I-protein_state
+the	O
+−	B-site
+2	I-site
+and	I-site
+−	I-site
+3	I-site
+subsites	I-site
+make	O
+a	O
+total	O
+of	O
+9	O
+polar	B-bond_interaction
+interactions	I-bond_interaction
+with	O
+the	O
+enzyme	O
+(	O
+Fig	O
+.	O
+4	O
+,	O
+D	O
+and	O
+E	O
+).	O
+The	O
+GH10	B-protein_type
+xylanase	B-protein_type
+also	O
+contains	O
+a	O
+−	B-site
+2	I-site
+subsite	I-site
+that	O
+,	O
+similar	O
+to	O
+the	O
+cellulase	B-protein_type
+,	O
+makes	O
+numerous	O
+interactions	O
+with	O
+the	O
+substrate	O
+(	O
+Fig	O
+.	O
+4	O
+,	O
+F	O
+and	O
+G	O
+).	O
+The	O
+Influence	O
+of	O
+the	O
+Modular	O
+Architecture	O
+of	O
+CtXyl5A	B-protein
+on	O
+Catalytic	O
+Activity	O
+CtXyl5A	B-protein
+,	O
+in	O
+addition	O
+to	O
+its	O
+catalytic	B-structure_element
+module	I-structure_element
+,	O
+contains	O
+three	O
+CBMs	B-structure_element
+(	O
+CtCBM6	B-structure_element
+,	O
+CtCBM13	B-structure_element
+,	O
+and	O
+CtCBM62	B-structure_element
+)	O
+and	O
+a	O
+fibronectin	B-structure_element
+domain	I-structure_element
+(	O
+CtFn3	B-structure_element
+).	O
+A	O
+previous	O
+study	O
+showed	O
+that	O
+although	O
+the	O
+CBM6	B-structure_element
+bound	B-protein_state
+in	I-protein_state
+an	O
+exo	B-protein_state
+-	I-protein_state
+mode	I-protein_state
+to	O
+xylo	B-chemical
+-	I-chemical
+and	I-chemical
+cellulooligosaccharides	I-chemical
+,	O
+the	O
+primary	O
+role	O
+of	O
+this	O
+module	O
+was	O
+to	O
+stabilize	O
+the	O
+structure	O
+of	O
+the	O
+GH5	B-protein_type
+catalytic	B-structure_element
+module	I-structure_element
+.	O
+To	O
+explore	O
+the	O
+contribution	O
+of	O
+the	O
+other	O
+non	B-structure_element
+-	I-structure_element
+catalytic	I-structure_element
+modules	I-structure_element
+to	O
+CtXyl5A	B-protein
+function	O
+,	O
+the	O
+activity	O
+of	O
+a	O
+series	O
+of	O
+truncated	B-protein_state
+derivatives	O
+of	O
+the	O
+arabinoxylanase	B-protein_type
+were	O
+assessed	O
+.	O
+The	O
+data	O
+in	O
+Table	O
+1	O
+show	O
+that	O
+removal	B-experimental_method
+of	I-experimental_method
+CtCBM62	B-structure_element
+caused	O
+a	O
+modest	O
+increase	O
+in	O
+activity	O
+against	O
+both	O
+WAX	B-chemical
+and	O
+CX	B-chemical
+,	O
+whereas	O
+deletion	B-experimental_method
+of	I-experimental_method
+the	O
+Fn3	B-structure_element
+domain	O
+had	O
+no	O
+further	O
+impact	O
+on	O
+catalytic	O
+performance	O
+.	O
+Truncation	B-experimental_method
+of	O
+CtCBM13	B-structure_element
+,	O
+however	O
+,	O
+caused	O
+a	O
+4	O
+–	O
+5	O
+-	O
+fold	O
+reduction	O
+in	O
+activity	O
+against	O
+both	O
+substrates	O
+.	O
+Members	O
+of	O
+CBM13	B-structure_element
+have	O
+been	O
+shown	O
+to	O
+bind	O
+to	O
+xylans	B-chemical
+,	O
+mannose	B-chemical
+,	O
+and	O
+galactose	B-chemical
+residues	O
+in	O
+complex	B-chemical
+glycans	I-chemical
+,	O
+hinting	O
+that	O
+the	O
+function	O
+of	O
+CtCBM13	B-structure_element
+is	O
+to	O
+increase	O
+the	O
+proximity	O
+of	O
+substrate	O
+to	O
+the	O
+catalytic	B-structure_element
+module	I-structure_element
+of	O
+CtXyl5A	B-protein
+.	O
+Binding	B-experimental_method
+studies	I-experimental_method
+,	O
+however	O
+,	O
+showed	O
+that	O
+CtCBM13	B-structure_element
+displayed	O
+no	O
+affinity	O
+for	O
+a	O
+range	O
+of	O
+relevant	O
+glycans	B-chemical
+including	O
+WAX	B-chemical
+,	O
+CX	B-chemical
+,	O
+xylose	B-chemical
+,	O
+mannose	B-chemical
+,	O
+galactose	B-chemical
+,	O
+and	O
+birchwood	B-chemical
+xylan	I-chemical
+(	O
+BX	B-chemical
+)	O
+(	O
+data	O
+not	O
+shown	O
+).	O
+It	O
+would	O
+appear	O
+,	O
+therefore	O
+,	O
+that	O
+CtCBM13	B-structure_element
+makes	O
+a	O
+structural	O
+contribution	O
+to	O
+the	O
+function	O
+of	O
+CtXyl5A	B-protein
+.	O
+Crystal	B-evidence
+Structure	I-evidence
+of	O
+CtXyl5A	B-mutant
+-	I-mutant
+D	I-mutant
+To	O
+explore	O
+further	O
+the	O
+role	O
+of	O
+the	O
+non	B-structure_element
+-	I-structure_element
+catalytic	I-structure_element
+modules	I-structure_element
+in	O
+CtXyl5A	B-protein
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+CtXyl5A	B-protein
+extending	O
+from	O
+CtGH5	B-structure_element
+to	O
+CtCBM62	B-structure_element
+was	O
+sought	O
+.	O
+To	O
+obtain	O
+a	O
+construct	O
+that	O
+could	O
+potentially	O
+be	O
+crystallized	B-experimental_method
+,	O
+the	O
+protein	O
+was	O
+generated	O
+without	B-protein_state
+the	O
+C	O
+-	O
+terminal	O
+dockerin	B-structure_element
+domain	O
+because	O
+it	O
+is	O
+known	O
+to	O
+be	O
+unstable	O
+and	O
+prone	O
+to	O
+cleavage	O
+.	O
+Using	O
+this	O
+construct	O
+(	O
+CtXyl5A	B-mutant
+-	I-mutant
+D	I-mutant
+)	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+arabinoxylanase	B-protein_type
+was	O
+determined	O
+by	O
+molecular	B-experimental_method
+replacement	I-experimental_method
+to	O
+a	O
+resolution	O
+of	O
+2	O
+.	O
+64	O
+Å	O
+with	O
+Rwork	B-evidence
+and	O
+Rfree	B-evidence
+at	O
+23	O
+.	O
+7	O
+%	O
+and	O
+27	O
+.	O
+8	O
+%,	O
+respectively	O
+.	O
+The	O
+structure	B-evidence
+comprises	O
+a	O
+continuous	O
+polypeptide	O
+extending	O
+from	O
+Ala36	B-residue_range
+to	I-residue_range
+Trp742	I-residue_range
+displaying	O
+four	O
+modules	O
+GH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+-	I-structure_element
+CBM13	I-structure_element
+-	I-structure_element
+Fn3	I-structure_element
+.	O
+Although	O
+there	O
+was	O
+some	O
+electron	B-evidence
+density	I-evidence
+for	O
+CtCBM62	B-structure_element
+,	O
+it	O
+was	O
+not	O
+sufficient	O
+to	O
+confidently	O
+build	O
+the	O
+module	O
+(	O
+Fig	O
+.	O
+5	O
+).	O
+Further	O
+investigation	O
+of	O
+the	O
+crystal	B-evidence
+packing	I-evidence
+revealed	O
+a	O
+large	O
+solvent	B-site
+channel	I-site
+adjacent	O
+to	O
+the	O
+area	O
+the	O
+CBM62	B-structure_element
+occupies	O
+.	O
+We	O
+postulate	O
+that	O
+the	O
+reason	O
+for	O
+the	O
+poor	O
+electron	B-evidence
+density	I-evidence
+is	O
+due	O
+to	O
+the	O
+CtCBM62	B-structure_element
+being	O
+mobile	B-protein_state
+compared	O
+with	O
+the	O
+rest	O
+of	O
+the	O
+protein	O
+.	O
+The	O
+structures	B-evidence
+of	O
+CtGH5	B-structure_element
+and	O
+CtCBM6	B-structure_element
+have	O
+been	O
+described	O
+previously	O
+.	O
+Surface	O
+representation	O
+of	O
+the	O
+tetra	O
+-	O
+modular	O
+arabinoxylanase	B-protein_type
+and	O
+zoom	O
+view	O
+on	O
+the	O
+CtGH5	B-structure_element
+loop	B-structure_element
+.	O
+The	O
+blue	O
+module	O
+is	O
+the	O
+CtGH5	B-structure_element
+catalytic	B-structure_element
+domain	I-structure_element
+,	O
+the	O
+green	O
+module	O
+corresponds	O
+to	O
+the	O
+CtCBM6	B-structure_element
+,	O
+the	O
+yellow	O
+module	O
+is	O
+the	O
+CtCBM13	B-structure_element
+,	O
+and	O
+the	O
+salmon	O
+module	O
+is	O
+the	O
+fibronectin	B-structure_element
+domain	I-structure_element
+.	O
+The	O
+CtGH5	B-structure_element
+loop	B-structure_element
+is	O
+stabilized	O
+between	O
+the	O
+CtCBM6	B-structure_element
+and	O
+the	O
+CtCBM13	B-structure_element
+modules	O
+.	O
+CtCBM13	B-structure_element
+extends	O
+from	O
+Gly567	B-residue_range
+to	I-residue_range
+Pro648	I-residue_range
+.	O
+Typical	O
+of	O
+CBM13	B-protein_type
+proteins	O
+CtCBM13	B-structure_element
+displays	O
+a	O
+β	B-structure_element
+-	I-structure_element
+trefoil	I-structure_element
+fold	I-structure_element
+comprising	O
+the	O
+canonical	O
+pseudo	O
+3	O
+-	O
+fold	O
+symmetry	O
+with	O
+a	O
+3	B-structure_element
+-	I-structure_element
+fold	I-structure_element
+repeating	I-structure_element
+unit	I-structure_element
+of	O
+40	B-residue_range
+–	I-residue_range
+50	I-residue_range
+amino	I-residue_range
+acid	I-residue_range
+residues	O
+characteristic	O
+of	O
+the	O
+Ricin	B-protein_type
+superfamily	I-protein_type
+.	O
+Each	O
+repeat	B-structure_element
+contains	O
+two	O
+pairs	O
+of	O
+antiparallel	B-structure_element
+β	I-structure_element
+-	I-structure_element
+strands	I-structure_element
+.	O
+A	O
+Dali	B-experimental_method
+search	I-experimental_method
+revealed	O
+structural	O
+homologs	O
+from	O
+the	O
+CBM13	B-protein_type
+family	O
+with	O
+an	O
+root	B-evidence
+mean	I-evidence
+square	I-evidence
+deviation	I-evidence
+less	O
+than	O
+2	O
+.	O
+0	O
+Å	O
+and	O
+sequence	O
+identities	O
+of	O
+less	O
+than	O
+20	O
+%	O
+that	O
+include	O
+the	O
+functionally	O
+relevant	O
+homologs	O
+C	B-species
+.	I-species
+thermocellum	I-species
+exo	B-protein_type
+-	I-protein_type
+β	I-protein_type
+-	I-protein_type
+1	I-protein_type
+,	I-protein_type
+3	I-protein_type
+-	I-protein_type
+galactanase	I-protein_type
+(	O
+PDB	O
+code	O
+3vsz	O
+),	O
+Streptomyces	B-species
+avermitilis	I-species
+β	B-protein_type
+-	I-protein_type
+l	I-protein_type
+-	I-protein_type
+arabinopyranosidase	I-protein_type
+(	O
+PDB	O
+code	O
+3a21	O
+),	O
+Streptomyces	B-species
+lividans	I-species
+xylanase	B-protein
+10A	I-protein
+(	O
+PDB	O
+code	O
+,	O
+1mc9	O
+),	O
+and	O
+Streptomyces	B-species
+olivaceoviridis	I-species
+E	I-species
+-	I-species
+86	I-species
+xylanase	B-protein
+10A	I-protein
+(	O
+PDB	O
+code	O
+1v6v	O
+).	O
+The	O
+Fn3	B-structure_element
+module	O
+displays	O
+a	O
+typical	O
+β	B-structure_element
+-	I-structure_element
+sandwich	I-structure_element
+fold	I-structure_element
+with	O
+the	O
+two	O
+sheets	B-structure_element
+comprising	O
+,	O
+primarily	O
+,	O
+three	O
+antiparallel	B-structure_element
+strands	I-structure_element
+in	O
+the	O
+order	O
+β1	B-structure_element
+-	I-structure_element
+β2	I-structure_element
+-	I-structure_element
+β5	I-structure_element
+in	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+1	I-structure_element
+and	O
+β4	B-structure_element
+-	I-structure_element
+β3	I-structure_element
+-	I-structure_element
+β6	I-structure_element
+in	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+2	I-structure_element
+.	O
+Although	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+2	I-structure_element
+presents	O
+a	O
+cleft	B-site
+-	O
+like	O
+topology	O
+,	O
+typical	O
+of	O
+endo	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+CBMs	I-protein_type
+,	O
+the	O
+surface	O
+lacks	O
+aromatic	O
+residues	O
+that	O
+play	O
+a	O
+key	O
+role	O
+in	O
+ligand	O
+recognition	O
+,	O
+and	O
+in	O
+the	O
+context	O
+of	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+enzyme	B-protein
+,	O
+the	O
+cleft	B-site
+abuts	O
+into	O
+CtCBM13	B-structure_element
+and	O
+thus	O
+would	O
+not	O
+be	O
+able	O
+to	O
+accommodate	O
+an	O
+extended	O
+polysaccharide	B-chemical
+chain	O
+(	O
+see	O
+below	O
+).	O
+In	O
+the	O
+structure	B-evidence
+of	O
+CtXyl5A	B-mutant
+-	I-mutant
+D	I-mutant
+,	O
+the	O
+four	O
+modules	B-structure_element
+form	O
+a	O
+three	O
+-	O
+leaf	O
+clover	O
+-	O
+like	O
+structure	O
+(	O
+Fig	O
+.	O
+5	O
+).	O
+Between	O
+the	O
+interfaces	B-site
+of	O
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+-	I-structure_element
+CBM13	I-structure_element
+there	O
+are	O
+a	O
+number	O
+of	O
+interactions	O
+that	O
+maintain	O
+the	O
+modules	O
+in	O
+a	O
+fixed	O
+position	O
+relative	O
+to	O
+each	O
+other	O
+.	O
+The	O
+interaction	O
+of	O
+CtGH5	B-structure_element
+and	O
+CtCBM6	B-structure_element
+,	O
+which	O
+buries	O
+a	O
+substantial	O
+apolar	B-site
+solvent	I-site
+-	I-site
+exposed	I-site
+surface	I-site
+of	O
+the	O
+two	O
+modules	O
+,	O
+has	O
+been	O
+described	O
+previously	O
+.	O
+The	O
+polar	B-bond_interaction
+interactions	I-bond_interaction
+between	O
+these	O
+two	O
+modules	O
+comprise	O
+14	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+and	O
+5	O
+salt	B-bond_interaction
+bridges	I-bond_interaction
+.	O
+The	O
+apolar	B-bond_interaction
+and	I-bond_interaction
+polar	I-bond_interaction
+interactions	I-bond_interaction
+between	O
+these	O
+two	O
+modules	O
+likely	O
+explaining	O
+why	O
+they	O
+do	O
+not	O
+fold	O
+independently	O
+compared	O
+with	O
+other	O
+glycoside	B-protein_type
+hydrolases	I-protein_type
+that	O
+contain	O
+CBMs	B-structure_element
+.	O
+CtCBM13	B-structure_element
+acts	O
+as	O
+the	O
+central	B-structure_element
+domain	I-structure_element
+,	O
+which	O
+interacts	B-protein_state
+with	I-protein_state
+CtGH5	B-structure_element
+,	O
+CtCBM6	B-structure_element
+,	O
+and	O
+CtFn3	B-structure_element
+via	O
+2	O
+,	O
+5	O
+,	O
+and	O
+4	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+,	O
+respectively	O
+,	O
+burying	O
+a	O
+surface	O
+area	O
+of	O
+∼	O
+450	O
+,	O
+350	O
+,	O
+and	O
+500	O
+Å2	O
+,	O
+respectively	O
+,	O
+to	O
+form	O
+a	O
+compact	B-protein_state
+heterotetramer	B-oligomeric_state
+.	O
+With	O
+respect	O
+to	O
+the	O
+CtCBM6	B-site
+-	I-site
+CBM13	I-site
+interface	I-site
+,	O
+the	O
+linker	B-structure_element
+(	O
+SPISTGTIP	B-structure_element
+)	O
+between	O
+the	O
+two	O
+modules	B-structure_element
+,	O
+extending	O
+from	O
+Ser514	B-residue_name_number
+to	O
+Pro522	B-residue_name_number
+,	O
+adopts	O
+a	O
+fixed	B-protein_state
+conformation	I-protein_state
+.	O
+Such	O
+sequences	O
+are	O
+normally	O
+extremely	O
+flexible	O
+;	O
+however	O
+,	O
+the	O
+two	O
+Ile	B-residue_name
+residues	O
+make	O
+extensive	O
+apolar	B-bond_interaction
+contacts	I-bond_interaction
+within	O
+the	O
+linker	B-structure_element
+and	O
+with	O
+the	O
+two	O
+CBMs	B-structure_element
+,	O
+leading	O
+to	O
+conformational	O
+stabilization	O
+.	O
+The	O
+interactions	O
+between	O
+CtGH5	B-structure_element
+and	O
+the	O
+two	O
+CBMs	B-structure_element
+,	O
+which	O
+are	O
+mediated	O
+by	O
+the	O
+tip	O
+of	O
+the	O
+loop	B-structure_element
+between	O
+β	B-structure_element
+-	I-structure_element
+7	I-structure_element
+and	O
+α	B-structure_element
+-	I-structure_element
+7	I-structure_element
+(	O
+loop	B-structure_element
+7	I-structure_element
+)	O
+of	O
+CtGH5	B-structure_element
+,	O
+not	O
+only	O
+stabilize	O
+the	O
+trimodular	B-structure_element
+clover	I-structure_element
+-	O
+like	O
+structure	O
+but	O
+also	O
+make	O
+a	O
+contribution	O
+to	O
+catalytic	O
+function	O
+.	O
+Central	O
+to	O
+the	O
+interactions	O
+between	O
+the	O
+three	O
+modules	B-structure_element
+is	O
+Trp285	B-residue_name_number
+,	O
+which	O
+is	O
+intercalated	B-bond_interaction
+between	I-bond_interaction
+the	O
+two	O
+CBMs	B-structure_element
+.	O
+The	O
+Nϵ	O
+of	O
+this	O
+aromatic	O
+residue	O
+makes	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+with	O
+the	O
+backbone	O
+carbonyl	O
+of	O
+Val615	B-residue_name_number
+and	O
+Gly616	B-residue_name_number
+in	O
+CtCBM13	B-structure_element
+,	O
+and	O
+the	O
+indole	O
+ring	O
+makes	O
+several	O
+apolar	B-bond_interaction
+contacts	I-bond_interaction
+with	O
+CtCBM6	B-structure_element
+(	O
+Pro440	B-residue_name_number
+,	O
+Phe489	B-residue_name_number
+,	O
+Gly491	B-residue_name_number
+,	O
+and	O
+Ala492	B-residue_name_number
+)	O
+(	O
+Fig	O
+.	O
+5	O
+).	O
+Indeed	O
+,	O
+loop	B-structure_element
+7	I-structure_element
+is	O
+completely	B-protein_state
+disordered	I-protein_state
+in	O
+the	O
+truncated	B-protein_state
+derivative	O
+of	O
+CtXyl5A	B-protein
+comprising	O
+CtGH5	B-structure_element
+and	O
+CtCBM6	B-structure_element
+,	O
+demonstrating	O
+that	O
+the	O
+interactions	O
+with	O
+CtCBM13	B-structure_element
+stabilize	O
+the	O
+conformation	O
+of	O
+this	O
+loop	B-structure_element
+.	O
+Although	O
+the	O
+tip	O
+of	O
+loop	B-structure_element
+7	I-structure_element
+does	O
+not	O
+directly	O
+contribute	O
+to	O
+the	O
+topology	O
+of	O
+the	O
+active	B-site
+site	I-site
+,	O
+it	O
+is	O
+only	O
+∼	O
+12	O
+Å	O
+from	O
+the	O
+catalytic	O
+nucleophile	O
+Glu279	B-residue_name_number
+.	O
+Thus	O
+,	O
+any	O
+perturbation	O
+of	O
+the	O
+loop	B-structure_element
+(	O
+through	O
+the	O
+removal	B-experimental_method
+of	O
+CtCBM13	B-structure_element
+)	O
+is	O
+likely	O
+to	O
+influence	O
+the	O
+electrostatic	O
+and	O
+apolar	O
+environment	O
+of	O
+the	O
+catalytic	O
+apparatus	O
+,	O
+which	O
+could	O
+explain	O
+the	O
+reduction	O
+in	O
+activity	O
+associated	O
+with	O
+the	O
+deletion	B-experimental_method
+of	O
+CtCBM13	B-structure_element
+.	O
+Similar	O
+to	O
+the	O
+interactions	O
+between	O
+CtCBM6	B-structure_element
+and	O
+CtCBM13	B-structure_element
+,	O
+there	O
+are	O
+extensive	O
+hydrophobic	B-bond_interaction
+interactions	I-bond_interaction
+between	O
+CtCBM13	B-structure_element
+and	O
+CtFn3	B-structure_element
+,	O
+resulting	O
+in	O
+very	O
+little	O
+flexibility	O
+between	O
+these	O
+modules	B-structure_element
+.	O
+As	O
+stated	O
+above	O
+,	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+CtCBM62	B-structure_element
+in	O
+the	O
+structure	B-evidence
+suggests	O
+that	O
+the	O
+module	B-structure_element
+can	O
+adopt	O
+multiple	O
+positions	O
+with	O
+respect	O
+to	O
+the	O
+rest	O
+of	O
+the	O
+protein	O
+.	O
+The	O
+CtCBM62	B-structure_element
+,	O
+by	O
+binding	B-protein_state
+to	I-protein_state
+its	O
+ligands	O
+(	O
+d	B-chemical
+-	I-chemical
+Galp	I-chemical
+and	O
+l	B-chemical
+-	I-chemical
+Arap	I-chemical
+)	O
+in	O
+plant	B-taxonomy_domain
+cell	O
+walls	O
+,	O
+may	O
+be	O
+able	O
+to	O
+recruit	O
+the	O
+enzyme	O
+onto	O
+its	O
+target	O
+substrate	O
+.	O
+Xylans	B-chemical
+are	O
+not	O
+generally	O
+thought	O
+to	O
+contain	O
+such	O
+sugars	B-chemical
+.	O
+d	B-chemical
+-	I-chemical
+Galp	I-chemical
+,	O
+however	O
+,	O
+has	O
+been	O
+detected	O
+in	O
+xylans	B-chemical
+in	O
+the	O
+outer	O
+layer	O
+of	O
+cereal	B-taxonomy_domain
+grains	O
+and	O
+in	O
+eucalyptus	B-taxonomy_domain
+trees	I-taxonomy_domain
+,	O
+which	O
+are	O
+substrates	O
+used	O
+by	O
+CtXyl5A	B-protein
+.	O
+Thus	O
+,	O
+CtCBM62	B-structure_element
+may	O
+direct	O
+the	O
+enzyme	O
+to	O
+particularly	O
+complex	O
+xylans	B-chemical
+containing	O
+d	B-chemical
+-	I-chemical
+Galp	I-chemical
+at	O
+the	O
+non	O
+-	O
+reducing	O
+termini	O
+of	O
+the	O
+side	O
+chains	O
+,	O
+consistent	O
+with	O
+the	O
+open	B-protein_state
+substrate	B-site
+binding	I-site
+cleft	I-site
+of	O
+the	O
+arabinoxylanase	B-protein_type
+that	O
+is	O
+optimized	O
+to	O
+bind	O
+highly	O
+decorated	O
+forms	O
+of	O
+the	O
+hemicellulose	B-chemical
+.	O
+In	O
+general	O
+CBMs	B-structure_element
+have	O
+little	O
+influence	O
+on	O
+enzyme	O
+activity	O
+against	O
+soluble	O
+substrates	O
+but	O
+have	O
+a	O
+significant	O
+impact	O
+on	O
+glycans	B-chemical
+within	O
+plant	B-taxonomy_domain
+cell	O
+walls	O
+.	O
+Thus	O
+,	O
+the	O
+role	O
+of	O
+CBM62	B-structure_element
+will	O
+likely	O
+only	O
+be	O
+evident	O
+against	O
+insoluble	O
+composite	O
+substrates	O
+.	O
+Exploring	O
+GH5	B-protein_type
+Subfamily	I-protein_type
+34	I-protein_type
+CtXyl5A	B-protein
+is	O
+a	O
+member	O
+of	O
+a	O
+seven	O
+-	O
+protein	O
+subfamily	O
+of	O
+GH5	B-protein_type
+,	O
+GH5_34	B-protein_type
+.	O
+Four	O
+of	O
+these	O
+proteins	O
+are	O
+distinct	O
+,	O
+whereas	O
+the	O
+other	O
+three	O
+members	O
+are	O
+essentially	O
+identical	O
+(	O
+derived	O
+from	O
+different	O
+strains	O
+of	O
+C	B-species
+.	I-species
+thermocellum	I-species
+).	O
+To	O
+investigate	O
+further	O
+the	O
+substrate	O
+specificity	O
+within	O
+this	O
+subfamily	O
+,	O
+recombinant	O
+forms	O
+of	O
+three	O
+members	O
+of	O
+GH5_34	B-protein_type
+that	O
+were	O
+distinct	O
+from	O
+CtXyl5A	B-protein
+were	O
+generated	O
+.	O
+AcGH5	B-protein
+has	O
+a	O
+similar	O
+molecular	O
+architecture	O
+to	O
+CtXyl5A	B-protein
+with	O
+the	O
+exception	O
+of	O
+an	O
+additional	O
+carbohydrate	B-structure_element
+esterase	I-structure_element
+family	I-structure_element
+6	I-structure_element
+module	I-structure_element
+at	O
+the	O
+C	O
+terminus	O
+(	O
+Fig	O
+.	O
+1	O
+).	O
+The	O
+GH5_34	B-protein_type
+from	O
+Verrucomicrobiae	B-taxonomy_domain
+bacterium	B-taxonomy_domain
+,	O
+VbGH5	B-protein
+,	O
+contains	O
+the	O
+GH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+-	I-structure_element
+CBM13	I-structure_element
+core	O
+structure	O
+,	O
+but	O
+the	O
+C	O
+-	O
+terminal	O
+Fn3	B-structure_element
+-	I-structure_element
+CBM62	I-structure_element
+-	I-structure_element
+dockerin	I-structure_element
+modules	O
+,	O
+present	O
+in	O
+CtXyl5A	B-protein
+,	O
+are	O
+replaced	O
+with	O
+a	O
+Laminin_3_G	B-structure_element
+domain	I-structure_element
+,	O
+which	O
+,	O
+by	O
+analogy	O
+to	O
+homologous	O
+domains	O
+in	O
+other	O
+proteins	O
+that	O
+have	O
+affinity	O
+for	O
+carbohydrates	B-chemical
+,	O
+may	O
+display	O
+a	O
+glycan	B-chemical
+binding	O
+function	O
+.	O
+The	O
+Verrucomicobiae	B-taxonomy_domain
+enzyme	O
+also	O
+has	O
+an	O
+N	O
+-	O
+terminal	O
+GH43	B-protein_type
+subfamily	I-protein_type
+10	I-protein_type
+(	O
+GH43_10	B-protein_type
+)	O
+catalytic	B-structure_element
+module	I-structure_element
+.	O
+The	O
+fungal	B-taxonomy_domain
+GH5_34	B-protein_type
+,	O
+GpGH5	B-protein
+,	O
+unlike	O
+the	O
+two	O
+bacterial	B-taxonomy_domain
+homologs	O
+,	O
+comprises	O
+a	O
+single	O
+GH5	B-protein_type
+catalytic	B-structure_element
+module	I-structure_element
+lacking	O
+all	O
+of	O
+the	O
+other	O
+accessory	O
+modules	O
+(	O
+Fig	O
+.	O
+1	O
+).	O
+GpGh5	B-protein
+is	O
+particularly	O
+interesting	O
+as	O
+Gonapodya	B-species
+prolifera	I-species
+is	O
+the	O
+only	O
+fungus	B-taxonomy_domain
+of	O
+the	O
+several	O
+hundred	O
+fungal	B-taxonomy_domain
+genomes	O
+that	O
+encodes	O
+a	O
+GH5_34	B-protein_type
+enzyme	O
+.	O
+In	O
+fact	O
+there	O
+are	O
+four	O
+potential	O
+GH5_34	B-protein_type
+sequences	O
+in	O
+the	O
+G	B-species
+.	I-species
+prolifera	I-species
+genome	O
+,	O
+all	O
+of	O
+which	O
+show	O
+high	O
+sequence	O
+homology	O
+to	O
+Clostridium	B-taxonomy_domain
+GH5_34	B-protein_type
+sequences	O
+.	O
+G	B-species
+.	I-species
+prolifera	I-species
+and	O
+Clostridium	B-taxonomy_domain
+occupy	O
+similar	O
+environments	O
+,	O
+suggesting	O
+that	O
+the	O
+GpGH5_34	B-protein
+gene	O
+was	O
+acquired	O
+from	O
+a	O
+Clostridium	B-taxonomy_domain
+species	O
+,	O
+which	O
+was	O
+followed	O
+by	O
+duplication	O
+of	O
+the	O
+gene	O
+in	O
+the	O
+fungal	B-taxonomy_domain
+genome	O
+.	O
+The	O
+sequence	O
+identity	O
+of	O
+the	O
+GH5_34	B-protein_type
+catalytic	B-structure_element
+modules	I-structure_element
+with	O
+CtXyl5A	B-protein
+ranged	O
+from	O
+55	O
+to	O
+80	O
+%	O
+(	O
+supplemental	O
+Fig	O
+.	O
+S1	O
+).	O
+All	O
+the	O
+GH5_34	B-protein_type
+enzymes	O
+were	O
+active	O
+on	O
+the	O
+arabinoxylans	B-chemical
+RAX	B-chemical
+,	O
+WAX	B-chemical
+,	O
+and	O
+CX	B-chemical
+but	O
+displayed	O
+no	O
+activity	O
+on	O
+BX	B-chemical
+(	O
+Table	O
+1	O
+and	O
+Fig	O
+.	O
+6	O
+)	O
+and	O
+are	O
+thus	O
+defined	O
+as	O
+arabinoxylanases	B-protein_type
+.	O
+The	O
+limit	O
+products	O
+generated	O
+by	O
+CtXyl5A	B-protein
+,	O
+AcGH5	B-protein
+,	O
+and	O
+GpGH5	B-protein
+comprised	O
+a	O
+range	O
+of	O
+oligosaccharides	B-chemical
+with	O
+some	O
+high	O
+molecular	O
+weight	O
+material	O
+.	O
+The	O
+oligosaccharides	B-chemical
+with	O
+low	O
+degrees	O
+of	O
+polymerization	O
+were	O
+absent	O
+in	O
+the	O
+VbGH5	B-protein
+reaction	O
+products	O
+.	O
+However	O
+,	O
+the	O
+enzyme	O
+generated	O
+a	O
+large	O
+amount	O
+of	O
+arabinose	B-chemical
+,	O
+which	O
+was	O
+not	O
+produced	O
+by	O
+the	O
+other	O
+arabinoxylanases	B-protein_type
+.	O
+Given	O
+that	O
+GH43_10	B-protein_type
+is	O
+predominantly	O
+an	O
+arabinofuranosidase	B-protein_type
+subfamily	O
+of	O
+GH43	B-protein_type
+,	O
+the	O
+arabinose	B-chemical
+generated	O
+by	O
+VbGH5	B-protein
+is	O
+likely	O
+mediated	O
+by	O
+the	O
+N	O
+-	O
+terminal	O
+catalytic	B-structure_element
+module	I-structure_element
+(	O
+see	O
+below	O
+).	O
+Kinetic	O
+analysis	O
+showed	O
+that	O
+AcGH5	B-protein
+displayed	O
+similar	O
+activity	O
+to	O
+CtXyl5A	B-protein
+against	O
+both	O
+WAX	B-chemical
+and	O
+RAX	B-chemical
+and	O
+was	O
+2	O
+-	O
+fold	O
+less	O
+active	O
+against	O
+CX	B-chemical
+.	O
+When	O
+initially	O
+measuring	O
+the	O
+activity	O
+of	O
+wild	B-protein_state
+type	I-protein_state
+VbGH5	B-protein
+against	O
+the	O
+different	O
+substrates	O
+,	O
+no	O
+clear	O
+data	O
+could	O
+be	O
+obtained	O
+,	O
+regardless	O
+of	O
+the	O
+concentration	O
+of	O
+enzyme	O
+used	O
+the	O
+reaction	O
+appeared	O
+to	O
+cease	O
+after	O
+a	O
+few	O
+minutes	O
+.	O
+We	O
+hypothesized	O
+that	O
+the	O
+N	O
+-	O
+terminal	O
+GH43_10	B-protein_type
+rapidly	O
+removed	O
+single	O
+arabinose	B-chemical
+decorations	O
+from	O
+the	O
+arabinoxylans	B-chemical
+depleting	O
+the	O
+substrate	O
+available	O
+to	O
+the	O
+arabinoxylanase	B-protein_type
+,	O
+explaining	O
+why	O
+this	O
+activity	O
+was	O
+short	O
+lived	O
+.	O
+To	O
+test	O
+this	O
+hypothesis	O
+,	O
+the	O
+conserved	B-protein_state
+catalytic	O
+base	O
+(	O
+Asp45	B-residue_name_number
+)	O
+of	O
+the	O
+GH43_10	B-structure_element
+module	O
+of	O
+VbGH5	B-protein
+was	O
+substituted	B-experimental_method
+with	I-experimental_method
+alanine	B-residue_name
+,	O
+which	O
+is	O
+predicted	O
+to	O
+inactivate	O
+this	O
+catalytic	B-structure_element
+module	I-structure_element
+.	O
+The	O
+D45A	B-mutant
+mutant	B-protein_state
+did	O
+not	O
+produce	O
+arabinose	B-chemical
+consistent	O
+with	O
+the	O
+arabinofuranosidase	B-protein_type
+activity	O
+displayed	O
+by	O
+the	O
+GH43_10	B-structure_element
+module	O
+in	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+enzyme	O
+(	O
+Fig	O
+.	O
+6	O
+).	O
+The	O
+kinetics	B-evidence
+of	O
+the	O
+GH5_34	B-protein_type
+arabinoxylanase	B-protein_type
+catalytic	B-structure_element
+module	I-structure_element
+was	O
+now	O
+measurable	O
+,	O
+and	O
+activities	O
+were	O
+determined	O
+to	O
+be	O
+between	O
+∼	O
+6	O
+-	O
+and	O
+10	O
+-	O
+fold	O
+lower	O
+than	O
+that	O
+of	O
+CtXyl5A	B-protein
+.	O
+Interestingly	O
+,	O
+the	O
+fungal	B-taxonomy_domain
+arabinoxylanase	B-protein_type
+displays	O
+the	O
+highest	O
+activities	O
+against	O
+WAX	B-chemical
+and	O
+RAX	B-chemical
+,	O
+∼	O
+4	O
+-	O
+and	O
+6	O
+-	O
+fold	O
+higher	O
+,	O
+respectively	O
+,	O
+than	O
+CtXyl5A	B-protein
+;	O
+however	O
+,	O
+there	O
+is	O
+very	O
+little	O
+difference	O
+in	O
+the	O
+activity	O
+between	O
+the	O
+eukaryotic	B-taxonomy_domain
+and	O
+prokaryotic	B-taxonomy_domain
+enzymes	O
+against	O
+CX	B-chemical
+.	O
+Attempts	O
+to	O
+express	O
+individual	O
+modules	O
+of	O
+a	O
+variety	O
+of	O
+truncations	O
+of	O
+AcGH5	B-protein
+and	O
+VbGH5	B-protein
+were	O
+unsuccessful	O
+.	O
+This	O
+may	O
+indicate	O
+that	O
+the	O
+individual	O
+modules	O
+can	O
+only	O
+fold	O
+correctly	O
+when	O
+incorporated	O
+into	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+enzyme	O
+,	O
+demonstrating	O
+the	O
+importance	O
+of	O
+intermodule	O
+interactions	O
+to	O
+maintain	O
+the	O
+structural	O
+integrity	O
+of	O
+these	O
+enzymes	O
+.	O
+Products	O
+profile	O
+generated	O
+of	O
+GH5_34	B-protein_type
+enzymes	O
+.	O
+The	O
+enzymes	O
+at	O
+1	O
+μm	O
+were	O
+incubated	B-experimental_method
+with	O
+the	O
+four	O
+different	O
+xylans	B-chemical
+at	O
+1	O
+%	O
+in	O
+50	O
+mm	O
+sodium	O
+phosphate	O
+buffer	O
+for	O
+16	O
+h	O
+at	O
+37	O
+°	O
+C	O
+(	O
+GpGH5	B-protein
+,	O
+VbGH5	B-protein
+,	O
+and	O
+AcGH5	B-protein
+)	O
+or	O
+60	O
+°	O
+C	O
+.	O
+The	O
+limit	O
+products	O
+were	O
+separated	O
+by	O
+TLC	B-experimental_method
+.	O
+The	O
+xylooligosaccharide	B-chemical
+standards	O
+(	O
+X	O
+)	O
+are	O
+indicated	O
+by	O
+their	O
+degrees	O
+of	O
+polymerization	O
+.	O
+A	O
+characteristic	O
+feature	O
+of	O
+enzymes	O
+that	O
+attack	O
+the	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+is	O
+their	O
+complex	O
+molecular	O
+architecture	O
+.	O
+The	O
+CBMs	B-structure_element
+in	O
+these	O
+enzymes	O
+generally	O
+play	O
+a	O
+role	O
+in	O
+substrate	O
+targeting	O
+and	O
+are	O
+appended	O
+to	O
+the	O
+catalytic	B-structure_element
+modules	I-structure_element
+through	O
+flexible	B-structure_element
+linker	I-structure_element
+sequences	I-structure_element
+.	O
+CtXyl5A	B-protein
+provides	O
+a	O
+rare	O
+visualization	O
+of	O
+the	O
+structure	B-evidence
+of	O
+multiple	O
+modules	O
+within	O
+a	O
+single	O
+enzyme	O
+.	O
+The	O
+central	O
+feature	O
+of	O
+these	O
+data	O
+is	O
+the	O
+structural	O
+role	O
+played	O
+by	O
+two	O
+of	O
+the	O
+CBMs	B-structure_element
+,	O
+CtCBM6	B-structure_element
+and	O
+CtCBM13	B-structure_element
+,	O
+in	O
+maintaining	O
+the	O
+active	B-protein_state
+conformation	O
+of	O
+the	O
+catalytic	B-structure_element
+module	I-structure_element
+,	O
+CtGH5	B-structure_element
+.	O
+The	O
+crystallographic	B-evidence
+data	I-evidence
+described	O
+here	O
+are	O
+supported	O
+by	O
+biochemical	O
+data	O
+showing	O
+either	O
+that	O
+these	O
+two	O
+modules	O
+do	O
+not	O
+bind	O
+to	O
+glycans	B-chemical
+(	O
+CtCBM13	B-structure_element
+)	O
+or	O
+that	O
+the	O
+recognition	O
+of	O
+the	O
+non	O
+-	O
+reducing	O
+end	O
+of	O
+xylan	B-chemical
+or	O
+cellulose	B-chemical
+chains	O
+(	O
+CtCBM6	B-structure_element
+)	O
+is	O
+unlikely	O
+to	O
+be	O
+biologically	O
+significant	O
+.	O
+It	O
+should	O
+be	O
+emphasized	O
+,	O
+however	O
+,	O
+that	O
+glycan	B-chemical
+binding	O
+and	O
+substrate	O
+targeting	O
+may	O
+only	O
+be	O
+evident	O
+in	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+enzyme	O
+acting	O
+on	O
+highly	O
+complex	O
+structures	O
+such	O
+as	O
+the	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+,	O
+as	O
+observed	O
+recently	O
+by	O
+a	O
+CBM46	B-structure_element
+module	O
+in	O
+the	O
+Bacillus	B-taxonomy_domain
+xyloglucanase	B-protein_type
+/	O
+mixed	B-protein_type
+linked	I-protein_type
+glucanase	I-protein_type
+BhCel5B	B-protein
+.	O
+CtXyl5A	B-protein
+is	O
+a	O
+member	O
+of	O
+GH5	B-protein_type
+that	O
+contains	O
+6644	O
+members	O
+.	O
+CtXyl5A	B-protein
+is	O
+a	O
+member	O
+of	O
+subfamily	O
+GH5_34	B-protein_type
+.	O
+Despite	O
+differences	O
+in	O
+sequence	O
+identity	O
+all	O
+of	O
+the	O
+homologs	O
+were	O
+shown	O
+to	O
+be	O
+arabinoxylanases	B-protein_type
+.	O
+Consistent	O
+with	O
+the	O
+conserved	O
+substrate	O
+specificity	O
+,	O
+all	O
+members	O
+of	O
+GH5_34	B-protein_type
+contained	O
+the	O
+specificity	B-site
+determinants	I-site
+Glu68	B-residue_name_number
+,	O
+Tyr92	B-residue_name_number
+,	O
+and	O
+Asn139	B-residue_name_number
+,	O
+which	O
+make	O
+critical	O
+interactions	O
+with	O
+the	O
+xylose	B-chemical
+or	O
+arabinose	B-chemical
+in	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+,	O
+which	O
+are	O
+1	O
+,	O
+3	O
+-	O
+linked	O
+to	O
+the	O
+xylose	B-chemical
+positioned	O
+in	O
+the	O
+active	B-site
+site	I-site
+.	O
+The	O
+presence	O
+of	O
+a	O
+CBM62	B-structure_element
+in	O
+CtXyl5A	B-protein
+and	O
+AcGH5	B-protein
+suggests	O
+that	O
+these	O
+enzymes	O
+target	O
+highly	O
+complex	O
+xylans	B-chemical
+that	O
+contain	O
+d	B-chemical
+-	I-chemical
+galactose	I-chemical
+in	O
+their	O
+side	O
+chains	O
+.	O
+The	O
+absence	B-protein_state
+of	I-protein_state
+a	O
+“	O
+non	O
+-	O
+structural	O
+”	O
+CBM	B-structure_element
+in	O
+GpGH5	B-protein
+may	O
+indicate	O
+that	O
+this	O
+arabinoxylanase	B-protein_type
+is	O
+designed	O
+to	O
+target	O
+simpler	O
+arabinoxylans	B-chemical
+present	O
+in	O
+the	O
+endosperm	O
+of	O
+cereals	B-taxonomy_domain
+.	O
+Although	O
+the	O
+characterization	O
+of	O
+all	O
+members	O
+of	O
+GH5_34	B-protein_type
+suggests	O
+that	O
+this	O
+subfamily	O
+is	O
+monospecific	O
+,	O
+differences	O
+in	O
+specificity	O
+are	O
+observed	O
+in	O
+other	O
+subfamilies	O
+of	O
+GHs	B-protein_type
+including	O
+GH43	B-protein_type
+and	O
+GH5	B-protein_type
+.	O
+Thus	O
+,	O
+as	O
+new	O
+members	O
+of	O
+GH5_34	B-protein_type
+are	O
+identified	O
+from	O
+genomic	O
+sequence	O
+data	O
+and	O
+subsequently	O
+characterized	O
+,	O
+the	O
+specificity	O
+of	O
+this	O
+family	O
+may	O
+require	O
+reinterpretation	O
+.	O
+An	O
+intriguing	O
+feature	O
+of	O
+VbGH5	B-protein
+is	O
+that	O
+the	O
+limited	O
+products	O
+generated	O
+by	O
+this	O
+enzymes	O
+are	O
+much	O
+larger	O
+than	O
+those	O
+produced	O
+by	O
+the	O
+other	O
+arabinoxylanases	B-protein_type
+.	O
+This	O
+suggests	O
+that	O
+although	O
+arabinose	B-chemical
+decorations	O
+contribute	O
+to	O
+enzyme	O
+specificity	O
+(	O
+VbGH5	B-protein
+is	O
+not	O
+active	O
+on	O
+xylans	B-chemical
+lacking	O
+arabinose	B-chemical
+side	O
+chains	O
+),	O
+the	O
+enzyme	O
+requires	O
+other	O
+specificity	O
+determinants	O
+that	O
+occur	O
+less	O
+frequently	O
+in	O
+arabinoxylans	B-chemical
+.	O
+This	O
+has	O
+some	O
+resonance	O
+with	O
+a	O
+recently	O
+described	O
+GH98	B-protein_type
+xylanase	B-protein_type
+that	O
+also	O
+exploits	O
+specificity	O
+determinants	O
+that	O
+occur	O
+infrequently	O
+and	O
+are	O
+only	O
+evident	O
+in	O
+highly	O
+complex	O
+xylans	B-chemical
+(	O
+e	O
+.	O
+g	O
+.	O
+CX	B-chemical
+).	O
+To	O
+conclude	O
+,	O
+this	O
+study	O
+provides	O
+the	O
+molecular	O
+basis	O
+for	O
+the	O
+specificity	O
+displayed	O
+by	O
+arabinoxylanases	B-protein_type
+.	O
+Substrate	O
+specificity	O
+is	O
+dominated	O
+by	O
+the	O
+pocket	B-site
+that	O
+binds	O
+single	O
+arabinose	B-chemical
+or	O
+xylose	B-chemical
+side	O
+chains	O
+.	O
+The	O
+open	B-protein_state
+xylan	B-site
+binding	I-site
+cleft	I-site
+explains	O
+why	O
+the	O
+enzyme	O
+is	O
+able	O
+to	O
+attack	O
+highly	O
+decorated	O
+forms	O
+of	O
+the	O
+hemicellulose	B-chemical
+.	O
+It	O
+is	O
+also	O
+evident	O
+that	O
+appending	O
+additional	O
+catalytic	B-structure_element
+modules	I-structure_element
+and	O
+CBMs	B-structure_element
+onto	O
+the	O
+core	O
+components	O
+of	O
+these	O
+enzymes	O
+generates	O
+bespoke	O
+arabinoxylanases	B-protein_type
+with	O
+activities	O
+optimized	O
+for	O
+specific	O
+functions	O
+.	O
+The	O
+specificities	O
+of	O
+the	O
+arabinoxylanases	B-protein_type
+described	O
+here	O
+are	O
+distinct	O
+from	O
+the	O
+classical	O
+endo	B-protein_type
+-	I-protein_type
+xylanases	I-protein_type
+and	O
+thus	O
+have	O
+the	O
+potential	O
+to	O
+contribute	O
+to	O
+the	O
+toolbox	O
+of	O
+biocatalysts	O
+required	O
+by	O
+industries	O
+that	O
+exploit	O
+the	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+as	O
+a	O
+sustainable	O
+substrate	O
+.	O
+Data	B-evidence
+collection	I-evidence
+and	I-evidence
+refinement	I-evidence
+statistics	I-evidence
+CtXyl5A	B-mutant
+-	I-mutant
+D	I-mutant
+GH5	B-complex_assembly
+-	I-complex_assembly
+CBM6	I-complex_assembly
+-	I-complex_assembly
+Arap	I-complex_assembly
+GH5	B-complex_assembly
+-	I-complex_assembly
+CBM6	I-complex_assembly
+-	I-complex_assembly
+Xylp	I-complex_assembly
+GH5	B-complex_assembly
+-	I-complex_assembly
+CBM6	I-complex_assembly
+-	I-complex_assembly
+(	I-complex_assembly
+Araf	I-complex_assembly
+-	I-complex_assembly
+Xylp4	I-complex_assembly
+)	I-complex_assembly
+Data	O
+collection	O
+Source	O
+ESRF	O
+-	O
+ID14	O
+-	O
+1	O
+Diamond	O
+I04	O
+–	O
+1	O
+Diamond	O
+I24	O
+Diamond	O
+I02	O
+Wavelength	O
+(	O
+Å	O
+)	O
+0	O
+.	O
+9334	O
+0	O
+.	O
+9173	O
+0	O
+.	O
+9772	O
+0	O
+.	O
+9791	O
+Space	O
+group	O
+P21212	O
+P212121	O
+P212121	O
+P212121	O
+Cell	O
+dimensions	O
+a	O
+,	O
+b	O
+,	O
+c	O
+(	O
+Å	O
+)	O
+147	O
+.	O
+4	O
+,	O
+191	O
+.	O
+7	O
+,	O
+50	O
+.	O
+7	O
+67	O
+.	O
+1	O
+,	O
+72	O
+.	O
+4	O
+,	O
+109	O
+.	O
+1	O
+67	O
+.	O
+9	O
+,	O
+72	O
+.	O
+5	O
+,	O
+109	O
+.	O
+5	O
+76	O
+.	O
+3	O
+,	O
+123	O
+.	O
+2	O
+,	O
+125	O
+.	O
+4	O
+α	O
+,	O
+β	O
+,	O
+γ	O
+(°)	O
+90	O
+,	O
+90	O
+,	O
+90	O
+90	O
+,	O
+90	O
+,	O
+90	O
+90	O
+,	O
+90	O
+,	O
+90	O
+90	O
+,	O
+90	O
+,	O
+90	O
+No	O
+.	O
+of	O
+measured	O
+reflections	O
+244	O
+,	O
+475	O
+(	O
+29	O
+,	O
+324	O
+)	O
+224	O
+,	O
+842	O
+(	O
+11	O
+,	O
+281	O
+)	O
+152	O
+,	O
+004	O
+(	O
+4	O
+,	O
+996	O
+)	O
+463	O
+,	O
+237	O
+(	O
+23	O
+,	O
+068	O
+)	O
+No	O
+.	O
+of	O
+independent	O
+reflections	O
+42246	O
+(	O
+5	O
+,	O
+920	O
+)	O
+63	O
+,	O
+523	O
+(	O
+3	O
+,	O
+175	O
+)	O
+42	O
+,	O
+716	O
+(	O
+2	O
+,	O
+334	O
+)	O
+140	O
+,	O
+288	O
+(	O
+6	O
+,	O
+879	O
+)	O
+Resolution	O
+(	O
+Å	O
+)	O
+50	O
+.	O
+70	O
+–	O
+2	O
+.	O
+64	O
+(	O
+2	O
+.	O
+78	O
+–	O
+2	O
+.	O
+64	O
+)	O
+44	O
+.	O
+85	O
+–	O
+1	O
+.	O
+65	O
+(	O
+1	O
+.	O
+68	O
+–	O
+1	O
+.	O
+65	O
+)	O
+45	O
+.	O
+16	O
+–	O
+1	O
+.	O
+90	O
+(	O
+1	O
+.	O
+94	O
+–	O
+1	O
+.	O
+90	O
+)	O
+48	O
+.	O
+43	O
+–	O
+1	O
+.	O
+65	O
+(	O
+1	O
+.	O
+68	O
+–	O
+1	O
+.	O
+65	O
+)	O
+Rmerge	O
+(%)	O
+16	O
+.	O
+5	O
+(	O
+69	O
+.	O
+5	O
+)	O
+6	O
+.	O
+7	O
+(	O
+65	O
+.	O
+1	O
+)	O
+2	O
+.	O
+8	O
+(	O
+8	O
+.	O
+4	O
+)	O
+5	O
+.	O
+7	O
+(	O
+74	O
+.	O
+9	O
+)	O
+CC1	O
+/	O
+2	O
+0	O
+.	O
+985	O
+(	O
+0	O
+.	O
+478	O
+)	O
+0	O
+.	O
+998	O
+(	O
+0	O
+.	O
+594	O
+)	O
+0	O
+.	O
+999	O
+(	O
+0	O
+.	O
+982	O
+)	O
+0	O
+.	O
+998	O
+(	O
+0	O
+.	O
+484	O
+)	O
+I	O
+/	O
+σI	O
+8	O
+.	O
+0	O
+(	O
+2	O
+.	O
+0	O
+)	O
+13	O
+(	O
+1	O
+.	O
+6	O
+)	O
+26	O
+.	O
+6	O
+(	O
+8	O
+.	O
+0	O
+)	O
+11	O
+.	O
+2	O
+(	O
+1	O
+.	O
+6	O
+)	O
+Completeness	O
+(%)	O
+98	O
+.	O
+5	O
+(	O
+96	O
+.	O
+4	O
+)	O
+98	O
+.	O
+5	O
+(	O
+99	O
+.	O
+4	O
+)	O
+98	O
+.	O
+6	O
+(	O
+85	O
+.	O
+0	O
+)	O
+98	O
+.	O
+8	O
+(	O
+99	O
+.	O
+4	O
+)	O
+Redundancy	O
+5	O
+.	O
+8	O
+(	O
+5	O
+.	O
+0	O
+)	O
+3	O
+.	O
+5	O
+(	O
+3	O
+.	O
+6	O
+)	O
+3	O
+.	O
+6	O
+(	O
+2	O
+.	O
+1	O
+)	O
+3	O
+.	O
+3	O
+(	O
+3	O
+.	O
+4	O
+)	O
+Refinement	O
+Rwork	B-evidence
+/	O
+Rfree	B-evidence
+23	O
+.	O
+7	O
+/	O
+27	O
+.	O
+8	O
+12	O
+.	O
+2	O
+/	O
+17	O
+.	O
+0	O
+12	O
+.	O
+9	O
+/	O
+16	O
+.	O
+1	O
+14	O
+.	O
+5	O
+/	O
+19	O
+.	O
+9	O
+No	O
+.	O
+atoms	O
+Protein	O
+5446	O
+3790	O
+3729	O
+7333	O
+Ligand	O
+19	O
+20	O
+20	O
+92	O
+Water	O
+227	O
+579	O
+601	O
+923	O
+B	O
+-	O
+factors	O
+Protein	O
+41	O
+.	O
+6	O
+17	O
+.	O
+8	O
+15	O
+.	O
+8	O
+21	O
+.	O
+0	O
+Ligand	O
+65	O
+.	O
+0	O
+19	O
+.	O
+4	O
+24	O
+.	O
+2	O
+39	O
+.	O
+5	O
+Water	O
+35	O
+.	O
+4	O
+38	O
+.	O
+5	O
+32	O
+.	O
+2	O
+37	O
+.	O
+6	O
+R	O
+.	O
+m	O
+.	O
+s	O
+deviations	O
+Bond	O
+lengths	O
+(	O
+Å	O
+)	O
+0	O
+.	O
+008	O
+0	O
+.	O
+015	O
+0	O
+.	O
+012	O
+0	O
+.	O
+012	O
+Bond	O
+angles	O
+(°)	O
+1	O
+.	O
+233	O
+1	O
+.	O
+502	O
+1	O
+.	O
+624	O
+1	O
+.	O
+554	O
+Protein	O
+Data	O
+Bank	O
+code	O
+5G56	O
+5LA0	O
+5LA1	O
+2LA2	O

annotation_IOB/PMC5173035.tsv ADDED Viewed

	@@ -0,0 +1,6195 @@

+Biochemical	B-experimental_method
+and	I-experimental_method
+structural	I-experimental_method
+characterization	I-experimental_method
+of	O
+a	O
+DNA	B-protein_type
+N6	I-protein_type
+-	I-protein_type
+adenine	I-protein_type
+methyltransferase	I-protein_type
+from	O
+Helicobacter	B-species
+pylori	I-species
+DNA	B-ptm
+N6	I-ptm
+-	I-ptm
+methyladenine	I-ptm
+modification	O
+plays	O
+an	O
+important	O
+role	O
+in	O
+regulating	O
+a	O
+variety	O
+of	O
+biological	O
+functions	O
+in	O
+bacteria	B-taxonomy_domain
+.	O
+However	O
+,	O
+the	O
+mechanism	O
+of	O
+sequence	O
+-	O
+specific	O
+recognition	O
+in	O
+N6	B-ptm
+-	I-ptm
+methyladenine	I-ptm
+modification	O
+remains	O
+elusive	O
+.	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+,	O
+a	O
+DNA	B-protein_type
+N6	I-protein_type
+-	I-protein_type
+adenine	I-protein_type
+methyltransferase	I-protein_type
+from	O
+Helicobacter	B-species
+pylori	I-species
+,	O
+shows	O
+more	O
+promiscuous	O
+substrate	O
+specificity	O
+than	O
+other	O
+enzymes	O
+.	O
+Here	O
+,	O
+we	O
+present	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+cofactor	B-protein_state
+-	I-protein_state
+free	I-protein_state
+and	O
+AdoMet	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+structures	B-evidence
+of	O
+this	O
+enzyme	O
+,	O
+which	O
+were	O
+determined	O
+at	O
+resolutions	O
+of	O
+3	O
+.	O
+0	O
+Å	O
+and	O
+3	O
+.	O
+1	O
+Å	O
+,	O
+respectively	O
+.	O
+The	O
+core	O
+structure	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+resembles	O
+the	O
+canonical	O
+AdoMet	B-protein_type
+-	I-protein_type
+dependent	I-protein_type
+MTase	I-protein_type
+fold	O
+,	O
+while	O
+the	O
+putative	O
+DNA	B-site
+binding	I-site
+regions	I-site
+considerably	O
+differ	O
+from	O
+those	O
+of	O
+the	O
+other	O
+MTases	B-protein_type
+,	O
+which	O
+may	O
+account	O
+for	O
+the	O
+substrate	O
+promiscuity	O
+of	O
+this	O
+enzyme	O
+.	O
+Site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutagenesis	I-experimental_method
+experiments	O
+identified	O
+residues	O
+D29	B-residue_name_number
+and	O
+E216	B-residue_name_number
+as	O
+crucial	O
+amino	O
+acids	O
+for	O
+cofactor	O
+binding	O
+and	O
+the	O
+methyl	B-chemical
+transfer	O
+activity	O
+of	O
+the	O
+enzyme	O
+,	O
+while	O
+P41	B-residue_name_number
+,	O
+located	O
+in	O
+a	O
+highly	B-protein_state
+flexible	I-protein_state
+loop	B-structure_element
+,	O
+playing	O
+a	O
+determinant	O
+role	O
+for	O
+substrate	O
+specificity	O
+.	O
+Taken	O
+together	O
+,	O
+our	O
+data	O
+revealed	O
+the	O
+structural	O
+basis	O
+underlying	O
+DNA	B-protein_type
+N6	I-protein_type
+-	I-protein_type
+adenine	I-protein_type
+methyltransferase	I-protein_type
+substrate	O
+promiscuity	O
+.	O
+DNA	B-ptm
+methylation	I-ptm
+is	O
+a	O
+common	O
+form	O
+of	O
+modification	O
+on	O
+nucleic	O
+acids	O
+occurring	O
+in	O
+both	O
+prokaryotes	B-taxonomy_domain
+and	O
+eukaryotes	B-taxonomy_domain
+.	O
+Such	O
+a	O
+modification	O
+creates	O
+a	O
+signature	O
+motif	O
+recognized	O
+by	O
+DNA	B-chemical
+-	O
+interacting	O
+proteins	O
+and	O
+functions	O
+as	O
+a	O
+mechanism	O
+to	O
+regulate	O
+gene	O
+expression	O
+.	O
+DNA	B-ptm
+methylation	I-ptm
+is	O
+mediated	O
+by	O
+DNA	B-protein_type
+methyltransferases	I-protein_type
+(	O
+MTases	B-protein_type
+),	O
+which	O
+catalyze	O
+the	O
+transfer	O
+of	O
+a	O
+methyl	B-chemical
+group	O
+from	O
+S	B-chemical
+-	I-chemical
+adenosyl	I-chemical
+-	I-chemical
+L	I-chemical
+-	I-chemical
+methionine	I-chemical
+(	O
+AdoMet	B-chemical
+)	O
+to	O
+a	O
+given	O
+position	O
+of	O
+a	O
+particular	O
+DNA	B-chemical
+base	O
+within	O
+a	O
+specific	O
+DNA	B-chemical
+sequence	O
+.	O
+Three	O
+classes	O
+of	O
+DNA	B-protein_type
+MTases	I-protein_type
+have	O
+been	O
+identified	O
+to	O
+transfer	O
+a	O
+methyl	B-chemical
+group	O
+to	O
+different	O
+positions	O
+of	O
+DNA	B-chemical
+bases	O
+.	O
+C5	B-protein_type
+-	I-protein_type
+cytosine	I-protein_type
+MTases	I-protein_type
+,	O
+for	O
+example	O
+,	O
+methylate	O
+C5	O
+of	O
+cytosine	B-residue_name
+(	O
+m5C	B-ptm
+).	O
+In	O
+eukaryotes	B-taxonomy_domain
+,	O
+m5C	B-ptm
+plays	O
+an	O
+important	O
+role	O
+in	O
+gene	O
+expression	O
+,	O
+chromatin	O
+organization	O
+,	O
+genome	O
+maintenance	O
+and	O
+parental	O
+imprinting	O
+,	O
+and	O
+is	O
+involved	O
+in	O
+a	O
+variety	O
+of	O
+human	B-species
+diseases	O
+including	O
+cancer	O
+.	O
+By	O
+contrast	O
+,	O
+the	O
+functions	O
+of	O
+the	O
+prokaryotic	B-taxonomy_domain
+DNA	B-protein_type
+cytosine	I-protein_type
+MTase	I-protein_type
+remain	O
+unknown	O
+.	O
+N4	B-protein_type
+-	I-protein_type
+cytosine	I-protein_type
+MTases	I-protein_type
+,	O
+which	O
+are	O
+frequently	O
+present	O
+in	O
+thermophilic	B-taxonomy_domain
+or	O
+mesophilic	B-taxonomy_domain
+bacteria	B-taxonomy_domain
+,	O
+transfer	O
+a	O
+methyl	B-chemical
+group	O
+to	O
+the	O
+exocyclic	O
+amino	O
+group	O
+of	O
+cytosine	B-residue_name
+(	O
+4mC	B-ptm
+).	O
+N4	B-ptm
+methylation	I-ptm
+seems	O
+to	O
+be	O
+primarily	O
+a	O
+component	O
+of	O
+bacterial	B-taxonomy_domain
+immune	O
+system	O
+against	O
+invasion	O
+by	O
+foreign	O
+DNA	B-chemical
+,	O
+such	O
+as	O
+conjugative	O
+plasmids	O
+and	O
+bacteriophages	B-taxonomy_domain
+.	O
+The	O
+third	O
+group	O
+,	O
+N6	B-protein_type
+-	I-protein_type
+adenine	I-protein_type
+MTases	I-protein_type
+methylate	O
+the	O
+exocyclic	O
+amino	O
+groups	O
+of	O
+adenine	B-residue_name
+(	O
+6mA	B-ptm
+),	O
+which	O
+exists	O
+in	O
+prokaryotes	B-taxonomy_domain
+as	O
+a	O
+signal	O
+for	O
+genome	O
+defense	O
+,	O
+DNA	B-chemical
+replication	O
+and	O
+repair	O
+,	O
+regulation	O
+of	O
+gene	O
+expression	O
+,	O
+control	O
+of	O
+transposition	O
+and	O
+host	O
+-	O
+pathogen	O
+interactions	O
+.	O
+Recent	O
+studies	O
+utilizing	O
+new	O
+sequencing	O
+approaches	O
+have	O
+showed	O
+the	O
+existence	O
+of	O
+6mA	B-ptm
+in	O
+several	O
+eukaryotic	B-taxonomy_domain
+species	O
+.	O
+DNA	B-chemical
+6mA	B-ptm
+modification	O
+is	O
+associated	O
+with	O
+important	O
+biological	O
+processes	O
+including	O
+nucleosome	O
+distribution	O
+close	O
+to	O
+the	O
+transcription	O
+start	O
+sites	O
+in	O
+Chlamydomonas	B-taxonomy_domain
+,	O
+carrying	O
+heritable	O
+epigenetic	O
+information	O
+in	O
+C	B-species
+.	I-species
+elegans	I-species
+or	O
+controlling	O
+development	O
+of	O
+Drosophila	B-taxonomy_domain
+.	O
+All	O
+the	O
+three	O
+types	O
+of	O
+methylation	B-ptm
+exist	O
+in	O
+prokaryotes	B-taxonomy_domain
+,	O
+and	O
+most	O
+DNA	B-protein_type
+MTases	I-protein_type
+are	O
+components	O
+of	O
+the	O
+restriction	O
+-	O
+modification	O
+(	O
+R	O
+-	O
+M	O
+)	O
+systems	O
+.	O
+“	O
+R	O
+”	O
+stands	O
+for	O
+a	O
+restriction	B-protein_type
+endonuclease	I-protein_type
+cleaving	O
+specific	O
+DNA	B-chemical
+sequences	O
+,	O
+while	O
+“	O
+M	O
+”	O
+symbolizes	O
+a	O
+modification	B-protein_type
+methyltransferase	I-protein_type
+rendering	O
+these	O
+sequences	O
+resistant	O
+to	O
+cleavage	O
+.	O
+The	O
+cooperation	O
+of	O
+these	O
+two	O
+enzymes	O
+provides	O
+a	O
+defensive	O
+mechanism	O
+to	O
+protect	O
+bacteria	B-taxonomy_domain
+from	O
+infection	O
+by	O
+bacteriophages	B-taxonomy_domain
+.	O
+The	O
+R	O
+-	O
+M	O
+systems	O
+are	O
+classified	O
+into	O
+three	O
+types	O
+based	O
+on	O
+specific	O
+structural	O
+features	O
+,	O
+position	O
+of	O
+DNA	B-chemical
+cleavage	O
+and	O
+cofactor	O
+requirements	O
+.	O
+In	O
+types	O
+I	O
+and	O
+III	O
+,	O
+the	O
+DNA	B-protein_type
+adenine	I-protein_type
+or	I-protein_type
+cytosine	I-protein_type
+methyltransferase	I-protein_type
+is	O
+part	O
+of	O
+a	O
+multi	O
+-	O
+subunit	O
+enzyme	O
+that	O
+catalyzes	O
+both	O
+restriction	O
+and	O
+modification	O
+.	O
+By	O
+contrast	O
+,	O
+two	O
+separate	O
+enzymes	O
+exist	O
+in	O
+type	O
+II	O
+systems	O
+,	O
+where	O
+a	O
+restriction	B-protein_type
+endonuclease	I-protein_type
+and	O
+a	O
+DNA	B-protein_type
+adenine	I-protein_type
+or	I-protein_type
+cytosine	I-protein_type
+methyltransferase	I-protein_type
+recognize	O
+the	O
+same	O
+targets	O
+.	O
+To	O
+date	O
+,	O
+a	O
+number	O
+of	O
+bacterial	B-taxonomy_domain
+DNA	B-protein_type
+MTases	I-protein_type
+have	O
+been	O
+structurally	B-experimental_method
+characterized	I-experimental_method
+,	O
+covering	O
+enzymes	O
+from	O
+all	O
+the	O
+three	O
+classes	O
+.	O
+All	O
+these	O
+MTases	B-protein_type
+exhibit	O
+high	O
+similarity	O
+in	O
+their	O
+overall	O
+architectures	O
+,	O
+which	O
+are	O
+generally	O
+folded	O
+into	O
+two	O
+domains	O
+:	O
+a	O
+conserved	B-protein_state
+larger	O
+catalytic	B-structure_element
+domain	I-structure_element
+comprising	O
+an	O
+active	B-site
+site	I-site
+for	O
+methyl	B-chemical
+transfer	O
+and	O
+a	O
+site	O
+for	O
+AdoMet	B-chemical
+-	O
+binding	O
+,	O
+and	O
+a	O
+smaller	O
+target	B-structure_element
+(	I-structure_element
+DNA	I-structure_element
+)-	I-structure_element
+recognition	I-structure_element
+domain	I-structure_element
+(	O
+TRD	B-structure_element
+)	O
+containing	O
+variable	O
+regions	O
+implicated	O
+in	O
+sequence	O
+-	O
+specific	O
+DNA	B-chemical
+recognition	O
+and	O
+the	O
+infiltration	O
+of	O
+the	O
+DNA	B-chemical
+to	O
+flip	O
+the	O
+target	O
+base	O
+.	O
+Conserved	B-protein_state
+amino	O
+acid	O
+motifs	O
+have	O
+been	O
+identified	O
+from	O
+reported	O
+structures	B-evidence
+,	O
+including	O
+ten	O
+motifs	O
+(	O
+I	B-structure_element
+-	I-structure_element
+X	I-structure_element
+)	O
+in	O
+cytosine	B-protein_type
+MTases	I-protein_type
+and	O
+nine	O
+motifs	O
+(	O
+I	B-structure_element
+-	I-structure_element
+VIII	I-structure_element
+and	O
+X	B-structure_element
+)	O
+in	O
+adenine	B-protein_type
+MTases	I-protein_type
+,	O
+all	O
+of	O
+which	O
+are	O
+arranged	O
+in	O
+an	O
+almost	O
+constant	O
+order	O
+.	O
+According	O
+to	O
+the	O
+linear	O
+arrangement	O
+of	O
+three	O
+conserved	B-protein_state
+domains	O
+,	O
+exocyclic	B-protein_type
+amino	I-protein_type
+MTases	I-protein_type
+are	O
+subdivided	O
+into	O
+six	O
+groups	O
+(	O
+namely	O
+α	B-protein_type
+,	O
+β	B-protein_type
+,	O
+γ	B-protein_type
+,	O
+ζ	B-protein_type
+,	O
+δ	B-protein_type
+and	O
+ε	B-protein_type
+).	O
+N6	B-protein_type
+-	I-protein_type
+adenine	I-protein_type
+and	I-protein_type
+N4	I-protein_type
+-	I-protein_type
+cytosine	I-protein_type
+MTases	I-protein_type
+,	O
+in	O
+particular	O
+,	O
+are	O
+closely	O
+related	O
+by	O
+sharing	O
+common	O
+structural	O
+features	O
+.	O
+Despite	O
+the	O
+considerable	O
+similarity	O
+among	O
+bacterial	B-taxonomy_domain
+MTases	B-protein_type
+,	O
+some	O
+differences	O
+were	O
+observed	O
+among	O
+the	O
+enzymes	O
+from	O
+various	O
+species	O
+.	O
+For	O
+example	O
+,	O
+the	O
+structural	O
+regions	O
+of	O
+MTases	B-protein_type
+beyond	O
+the	O
+catalytic	B-structure_element
+domain	I-structure_element
+are	O
+rather	O
+variable	O
+,	O
+such	O
+as	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+of	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+,	O
+the	O
+extended	O
+arm	O
+of	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+and	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+,	O
+the	O
+helix	B-structure_element
+bundle	I-structure_element
+of	O
+EcoDam	B-protein
+,	O
+and	O
+so	O
+on	O
+.	O
+DNA	B-ptm
+methylation	I-ptm
+is	O
+thought	O
+to	O
+influence	O
+bacterial	B-taxonomy_domain
+virulence	O
+.	O
+DNA	B-protein_type
+adenine	I-protein_type
+methyltransferase	I-protein_type
+has	O
+been	O
+shown	O
+to	O
+play	O
+a	O
+crucial	O
+role	O
+in	O
+colonization	O
+of	O
+deep	O
+tissue	O
+sites	O
+in	O
+Salmonella	B-species
+typhimurium	I-species
+and	O
+Aeromonas	B-species
+hydrophila	I-species
+.	O
+Importantly	O
+,	O
+DNA	B-ptm
+adenine	I-ptm
+methylation	I-ptm
+is	O
+a	O
+global	O
+regulator	O
+of	O
+genes	O
+expressed	O
+during	O
+infection	O
+and	O
+inhibitors	O
+of	O
+DNA	B-ptm
+adenine	I-ptm
+methylation	I-ptm
+are	O
+likely	O
+to	O
+have	O
+a	O
+broad	O
+antimicrobial	O
+action	O
+.	O
+Dam	B-protein_type
+was	O
+considered	O
+a	O
+promising	O
+target	O
+for	O
+antimicrobial	O
+drug	O
+development	O
+.	O
+Helicobacter	B-species
+pylori	I-species
+is	O
+a	O
+Gram	B-taxonomy_domain
+-	I-taxonomy_domain
+negative	I-taxonomy_domain
+bacterium	I-taxonomy_domain
+that	O
+persistently	O
+colonizes	O
+in	O
+human	B-species
+stomach	O
+worldwide	O
+.	O
+H	B-species
+.	I-species
+pylori	I-species
+is	O
+involved	O
+in	O
+90	O
+%	O
+of	O
+all	O
+gastric	O
+malignancies	O
+,	O
+infecting	O
+nearly	O
+50	O
+%	O
+of	O
+the	O
+world	O
+'	O
+s	O
+population	O
+and	O
+is	O
+the	O
+most	O
+crucial	O
+etiologic	O
+agent	O
+for	O
+gastric	O
+adenocarcinoma	O
+.	O
+H	B-species
+.	I-species
+pylori	I-species
+strains	O
+possess	O
+a	O
+few	O
+R	O
+-	O
+M	O
+systems	O
+like	O
+other	O
+bacteria	B-taxonomy_domain
+to	O
+function	O
+as	O
+defensive	O
+systems	O
+.	O
+H	B-species
+.	I-species
+pylori	I-species
+26695	I-species
+,	O
+for	O
+example	O
+,	O
+has	O
+23	O
+R	O
+-	O
+M	O
+systems	O
+.	O
+Methyltransferases	B-protein_type
+were	O
+suggested	O
+to	O
+be	O
+involved	O
+in	O
+H	B-species
+.	I-species
+pylori	I-species
+pathogenicity	O
+.	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+is	O
+a	O
+DNA	B-protein_type
+adenine	I-protein_type
+MTase	I-protein_type
+that	O
+belongs	O
+to	O
+the	O
+type	O
+II	O
+R	O
+-	O
+M	O
+system	O
+.	O
+This	O
+system	O
+contains	O
+two	O
+DNA	B-protein_type
+MTases	I-protein_type
+named	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+and	O
+M2	B-protein
+.	I-protein
+HpyAVI	I-protein
+,	O
+and	O
+a	O
+putative	O
+restriction	B-protein_type
+enzyme	I-protein_type
+.	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+encoded	O
+by	O
+ORF	O
+hp0050	B-gene
+is	O
+an	O
+N6	B-protein_type
+-	I-protein_type
+adenine	I-protein_type
+methyltransferase	I-protein_type
+belonging	O
+to	O
+the	O
+β	B-protein_type
+-	I-protein_type
+class	I-protein_type
+MTase	I-protein_type
+.	O
+It	O
+has	O
+been	O
+reported	O
+recently	O
+that	O
+this	O
+enzyme	O
+recognizes	O
+the	O
+sequence	O
+of	O
+5	B-chemical
+′-	I-chemical
+GAGG	I-chemical
+-	I-chemical
+3	I-chemical
+′,	I-chemical
+5	B-chemical
+′-	I-chemical
+GGAG	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+or	O
+5	B-chemical
+′-	I-chemical
+GAAG	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+and	O
+methylates	O
+adenines	B-residue_name
+in	O
+these	O
+sequences	O
+.	O
+Given	O
+that	O
+methylation	B-ptm
+of	O
+two	O
+adjacent	O
+adenines	B-residue_name
+on	O
+the	O
+same	O
+strand	O
+have	O
+never	O
+been	O
+observed	O
+for	O
+other	O
+N6	B-protein_type
+-	I-protein_type
+adenine	I-protein_type
+MTases	I-protein_type
+,	O
+the	O
+methylation	B-ptm
+activity	O
+on	O
+5	B-chemical
+′-	I-chemical
+GAAG	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+seems	O
+to	O
+be	O
+a	O
+unique	O
+feature	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+,	O
+compared	O
+with	O
+the	O
+homologs	O
+from	O
+other	O
+strains	O
+of	O
+H	B-species
+.	I-species
+pylori	I-species
+which	O
+is	O
+able	O
+to	O
+methylate	O
+only	O
+5	B-chemical
+′-	I-chemical
+GAGG	I-chemical
+-	I-chemical
+3	I-chemical
+′.	I-chemical
+The	O
+structural	O
+basis	O
+and	O
+the	O
+catalytic	O
+mechanism	O
+underlying	O
+such	O
+a	O
+distinct	O
+activity	O
+are	O
+not	O
+well	O
+understood	O
+due	O
+to	O
+the	O
+lack	O
+of	O
+an	O
+available	O
+3D	O
+structure	B-evidence
+of	O
+this	O
+enzyme	O
+.	O
+Here	O
+,	O
+we	O
+report	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+from	O
+H	B-species
+.	I-species
+pylori	I-species
+26695	I-species
+,	O
+which	O
+is	O
+the	O
+first	O
+determined	O
+N6	B-protein_type
+-	I-protein_type
+adenine	I-protein_type
+MTase	I-protein_type
+structure	B-evidence
+in	O
+H	B-species
+.	I-species
+pylori	I-species
+.	O
+The	O
+structure	B-evidence
+reveals	O
+a	O
+similar	O
+architecture	O
+as	O
+the	O
+canonical	O
+fold	O
+of	O
+homologous	O
+proteins	O
+,	O
+but	O
+displays	O
+several	O
+differences	O
+in	O
+the	O
+loop	B-structure_element
+regions	O
+and	O
+TRD	B-structure_element
+.	O
+Based	O
+on	O
+structural	B-experimental_method
+and	I-experimental_method
+biochemical	I-experimental_method
+analyses	I-experimental_method
+,	O
+we	O
+then	O
+identified	O
+two	O
+conserved	B-protein_state
+amino	O
+acids	O
+,	O
+D29	B-residue_name_number
+at	O
+the	O
+catalytic	B-site
+site	I-site
+and	O
+E216	B-residue_name_number
+close	O
+to	O
+the	O
+C	O
+-	O
+terminus	O
+,	O
+as	O
+crucial	O
+residues	O
+for	O
+cofactor	O
+binding	O
+and	O
+methyltransferase	B-protein_type
+activity	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+.	O
+In	O
+addition	O
+,	O
+a	O
+non	B-protein_state
+-	I-protein_state
+conserved	I-protein_state
+amino	O
+acid	O
+,	O
+P41	B-residue_name_number
+,	O
+seems	O
+to	O
+play	O
+a	O
+key	O
+role	O
+in	O
+substrate	O
+recognition	O
+.	O
+Overall	O
+structure	B-evidence
+Recombinant	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+was	O
+produced	O
+as	O
+a	O
+soluble	O
+protein	O
+in	O
+Escherichia	B-species
+coli	I-species
+,	O
+but	O
+was	O
+quite	O
+unstable	O
+and	O
+tended	O
+to	O
+aggregate	O
+in	O
+low	O
+salt	O
+environment	O
+.	O
+The	O
+protein	O
+,	O
+however	O
+,	O
+remained	O
+fully	O
+soluble	O
+in	O
+a	O
+buffer	O
+containing	O
+higher	O
+concentration	O
+of	O
+sodium	B-chemical
+chloride	I-chemical
+(>	O
+300	O
+mM	O
+),	O
+which	O
+prompted	O
+that	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+is	O
+likely	O
+a	O
+halophilic	B-protein_state
+protein	O
+.	O
+The	O
+cofactor	B-protein_state
+-	I-protein_state
+free	I-protein_state
+and	O
+AdoMet	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+proteins	O
+were	O
+crystallized	B-experimental_method
+at	O
+different	O
+conditions	O
+.	O
+Both	O
+structures	B-evidence
+were	O
+determined	O
+by	O
+means	O
+of	O
+molecular	B-experimental_method
+replacement	I-experimental_method
+,	O
+and	O
+refined	O
+to	O
+3	O
+.	O
+0	O
+Å	O
+and	O
+3	O
+.	O
+1	O
+Å	O
+,	O
+respectively	O
+.	O
+Statistics	O
+of	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+data	I-experimental_method
+collection	I-experimental_method
+and	O
+structure	B-experimental_method
+refinement	I-experimental_method
+were	O
+summarized	O
+in	O
+Table	O
+1	O
+.	O
+Data	O
+collection	O
+and	O
+structure	B-evidence
+refinement	I-evidence
+statistics	I-evidence
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+M1	B-complex_assembly
+.	I-complex_assembly
+HpyAVI	I-complex_assembly
+-	I-complex_assembly
+AdoMet	I-complex_assembly
+complex	O
+Data	O
+collection	O
+Wavelength	O
+(	O
+Å	O
+)	O
+1	O
+.	O
+0000	O
+0	O
+.	O
+97772	O
+Space	O
+group	O
+P43212	O
+P65	O
+Unit	O
+-	O
+cell	O
+parameters	O
+(	O
+Å	O
+,	O
+˚)	O
+a	O
+=	O
+b	O
+=	O
+69	O
+.	O
+73	O
+,	O
+c	O
+=	O
+532	O
+.	O
+75α	O
+=	O
+β	O
+=	O
+γ	O
+=	O
+90	O
+a	O
+=	O
+b	O
+=	O
+135	O
+.	O
+60	O
+,	O
+c	O
+=	O
+265	O
+.	O
+15α	O
+=	O
+β	O
+=	O
+90	O
+,	O
+γ	O
+=	O
+120	O
+Resolution	O
+range	O
+(	O
+Å	O
+)	O
+a	O
+49	O
+.	O
+09	O
+-	O
+3	O
+.	O
+00	O
+(	O
+3	O
+.	O
+09	O
+-	O
+3	O
+.	O
+00	O
+)	O
+48	O
+.	O
+91	O
+-	O
+3	O
+.	O
+10	O
+(	O
+3	O
+.	O
+18	O
+-	O
+3	O
+.	O
+10	O
+)	O
+Unique	O
+reflections	O
+a	O
+27243	O
+49833	O
+Multiplicity	O
+a	O
+3	O
+.	O
+7	O
+(	O
+3	O
+.	O
+8	O
+)	O
+5	O
+.	O
+6	O
+(	O
+4	O
+.	O
+0	O
+)	O
+Completeness	O
+(%)	O
+a	O
+98	O
+.	O
+7	O
+(	O
+98	O
+.	O
+9	O
+)	O
+99	O
+.	O
+7	O
+(	O
+97	O
+.	O
+8	O
+)	O
+Mean	O
+I	O
+/	O
+δ	O
+(	O
+I	O
+)	O
+a	O
+12	O
+.	O
+1	O
+(	O
+3	O
+.	O
+4	O
+)	O
+14	O
+.	O
+0	O
+(	O
+1	O
+.	O
+9	O
+)	O
+Solvent	O
+content	O
+(%)	O
+58	O
+.	O
+67	O
+61	O
+.	O
+96	O
+Rmergea	O
+0	O
+.	O
+073	O
+(	O
+0	O
+.	O
+378	O
+)	O
+0	O
+.	O
+106	O
+(	O
+0	O
+.	O
+769	O
+)	O
+Structure	O
+refinement	O
+Rwork	O
+0	O
+.	O
+251	O
+0	O
+.	O
+221	O
+Rfree	O
+0	O
+.	O
+308	O
+0	O
+.	O
+276	O
+R	B-evidence
+.	I-evidence
+m	I-evidence
+.	I-evidence
+s	I-evidence
+.	I-evidence
+d	I-evidence
+.,	O
+bond	O
+lengths	O
+(	O
+Å	O
+)	O
+0	O
+.	O
+007	O
+0	O
+.	O
+007	O
+R	B-evidence
+.	I-evidence
+m	I-evidence
+.	I-evidence
+s	I-evidence
+.	I-evidence
+d	I-evidence
+.,	O
+bond	O
+angles	O
+(˚)	O
+1	O
+.	O
+408	O
+1	O
+.	O
+651	O
+Ramachandran	O
+plot	O
+Favoured	O
+region	O
+(%)	O
+89	O
+.	O
+44	O
+91	O
+.	O
+44	O
+Allowed	O
+region	O
+(%)	O
+9	O
+.	O
+58	O
+7	O
+.	O
+11	O
+Outliers	O
+(%)	O
+0	O
+.	O
+99	O
+1	O
+.	O
+45	O
+Four	O
+and	O
+eight	O
+protein	O
+monomers	B-oligomeric_state
+resided	O
+in	O
+the	O
+asymmetric	O
+units	O
+of	O
+the	O
+two	O
+crystal	B-evidence
+structures	I-evidence
+.	O
+Some	O
+amino	O
+acids	O
+,	O
+particularly	O
+those	O
+within	O
+two	O
+loops	B-structure_element
+(	O
+residues	O
+32	B-residue_range
+-	I-residue_range
+61	I-residue_range
+and	O
+152	B-residue_range
+-	I-residue_range
+172	I-residue_range
+)	O
+in	O
+both	O
+structures	B-evidence
+,	O
+were	O
+poorly	O
+defined	O
+in	O
+electron	B-evidence
+density	I-evidence
+and	O
+had	O
+to	O
+be	O
+omitted	O
+from	O
+the	O
+refined	O
+models	O
+.	O
+The	O
+two	O
+structures	B-evidence
+are	O
+very	O
+similar	O
+to	O
+each	O
+other	O
+(	O
+Figure	O
+1	O
+)	O
+and	O
+could	O
+be	O
+well	O
+overlaid	O
+with	O
+an	O
+RMSD	B-evidence
+of	O
+0	O
+.	O
+76	O
+Å	O
+on	O
+191	O
+Cα	O
+atoms	O
+.	O
+The	O
+overall	O
+architecture	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+revealed	O
+in	O
+these	O
+structures	B-evidence
+resembles	O
+the	O
+AdoMet	B-protein_type
+-	I-protein_type
+dependent	I-protein_type
+MTase	I-protein_type
+fold	O
+in	O
+which	O
+a	O
+twisted	O
+seven	O
+-	O
+stranded	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+flanked	O
+by	O
+six	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+forms	O
+the	O
+structural	O
+core	O
+.	O
+Like	O
+the	O
+reported	O
+structures	B-evidence
+of	O
+the	O
+larger	O
+domain	O
+of	O
+MTases	B-protein_type
+,	O
+three	O
+helices	B-structure_element
+(	O
+αA	B-structure_element
+,	O
+αB	B-structure_element
+and	O
+αZ	B-structure_element
+)	O
+are	O
+located	O
+at	O
+one	O
+face	O
+of	O
+the	O
+central	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+,	O
+while	O
+the	O
+other	O
+three	O
+αD	B-structure_element
+,	O
+αE	B-structure_element
+and	O
+αC	B-structure_element
+sit	O
+at	O
+the	O
+other	O
+side	O
+.	O
+All	O
+these	O
+conserved	B-protein_state
+structural	O
+motifs	O
+form	O
+a	O
+typical	O
+α	B-structure_element
+/	I-structure_element
+β	I-structure_element
+Rossmann	I-structure_element
+fold	I-structure_element
+.	O
+The	O
+catalytic	B-structure_element
+motif	I-structure_element
+DPPY	B-structure_element
+lies	O
+in	O
+a	O
+loop	B-structure_element
+connecting	O
+αD	B-structure_element
+and	O
+β4	B-structure_element
+,	O
+and	O
+the	O
+cofactor	O
+AdoMet	B-chemical
+binds	O
+in	O
+a	O
+neighboring	O
+cavity	B-site
+.	O
+The	O
+loop	B-structure_element
+(	O
+residues	O
+136	B-residue_range
+-	I-residue_range
+166	I-residue_range
+)	O
+located	O
+between	O
+β7	B-structure_element
+and	O
+αZ	B-structure_element
+corresponds	O
+to	O
+a	O
+highly	B-protein_state
+diverse	I-protein_state
+region	O
+in	O
+other	O
+MTases	B-protein_type
+that	O
+is	O
+involved	O
+in	O
+target	O
+DNA	B-chemical
+recognition	O
+.	O
+The	O
+hairpin	B-structure_element
+loop	I-structure_element
+(	O
+residues	O
+101	B-residue_range
+-	I-residue_range
+133	I-residue_range
+)	O
+bridging	O
+β6	B-structure_element
+and	O
+β7	B-structure_element
+,	O
+which	O
+is	O
+proposed	O
+to	O
+bind	O
+DNA	B-chemical
+in	O
+the	O
+minor	B-structure_element
+groove	I-structure_element
+,	O
+displays	O
+a	O
+similar	O
+conformation	O
+as	O
+those	O
+observed	O
+in	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+,	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+and	O
+M	B-protein
+.	I-protein
+pvuII	I-protein
+.	O
+The	O
+missing	B-protein_state
+loop	B-structure_element
+(	O
+residues	O
+33	B-residue_range
+-	I-residue_range
+58	I-residue_range
+)	O
+in	O
+the	O
+structure	B-evidence
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+corresponds	O
+to	O
+loop	B-structure_element
+I	I-structure_element
+in	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+,	O
+which	O
+was	O
+also	O
+invisible	O
+in	O
+a	O
+structure	B-evidence
+without	B-protein_state
+DNA	I-protein_state
+.	O
+This	O
+loop	B-structure_element
+,	O
+however	O
+,	O
+was	O
+well	B-protein_state
+ordered	I-protein_state
+in	O
+an	O
+M	B-evidence
+.	I-evidence
+TaqI	I-evidence
+-	I-evidence
+DNA	I-evidence
+complex	I-evidence
+structure	I-evidence
+and	O
+was	O
+shown	O
+to	O
+play	O
+a	O
+crucial	O
+role	O
+in	O
+DNA	B-ptm
+methylation	I-ptm
+by	O
+contacting	O
+the	O
+flipping	O
+adenine	B-residue_name
+and	O
+recognizing	O
+specific	O
+DNA	B-chemical
+sequence	O
+.	O
+Overall	O
+structure	B-evidence
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+A	O
+.	O
+Free	B-protein_state
+form	O
+B	O
+.	O
+AdoMet	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+form	O
+.	O
+Ribbon	O
+diagram	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+resembles	O
+an	O
+“	O
+AdoMet	B-protein_type
+-	I-protein_type
+dependent	I-protein_type
+MTase	I-protein_type
+fold	O
+”,	O
+a	O
+mixed	O
+seven	O
+-	O
+stranded	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+flanked	O
+by	O
+six	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+,	O
+αA	B-structure_element
+,	O
+αB	B-structure_element
+,	O
+αZ	B-structure_element
+on	O
+one	O
+side	O
+and	O
+αD	B-structure_element
+,	O
+αE	B-structure_element
+,	O
+αC	B-structure_element
+on	O
+the	O
+other	O
+side	O
+,	O
+the	O
+cofactor	O
+AdoMet	B-chemical
+is	O
+bound	B-protein_state
+in	I-protein_state
+a	O
+cavity	B-site
+near	O
+the	O
+conserved	B-protein_state
+enzyme	O
+activity	O
+motif	O
+DPPY	B-structure_element
+.	O
+The	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+and	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+are	O
+labelled	O
+and	O
+numbered	O
+according	O
+to	O
+the	O
+commonly	O
+numbering	O
+rule	O
+for	O
+the	O
+known	O
+MTases	B-protein_type
+.	O
+The	O
+AdoMet	B-chemical
+molecule	O
+is	O
+shown	O
+in	O
+green	O
+.	O
+Dimeric	B-oligomeric_state
+state	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+in	O
+crystal	B-evidence
+and	O
+solution	B-experimental_method
+Previous	O
+studies	O
+showed	O
+that	O
+some	O
+DNA	B-protein_type
+MTases	I-protein_type
+,	O
+e	O
+.	O
+g	O
+.	O
+M	B-protein
+.	I-protein
+BamHI	I-protein
+and	O
+M	B-protein
+.	I-protein
+EcoRI	I-protein
+,	O
+exist	O
+as	O
+monomer	B-oligomeric_state
+in	O
+solution	O
+,	O
+in	O
+agreement	O
+with	O
+the	O
+fact	O
+that	O
+a	O
+DNA	B-chemical
+substrate	O
+for	O
+a	O
+typical	O
+MTase	B-protein_type
+is	O
+hemimethylated	B-protein_state
+and	O
+therefore	O
+needs	O
+only	O
+a	O
+single	O
+methylation	B-ptm
+event	O
+to	O
+convert	O
+it	O
+into	O
+a	O
+fully	B-protein_state
+methylated	I-protein_state
+state	O
+.	O
+Increasing	O
+number	O
+of	O
+dimeric	B-oligomeric_state
+DNA	B-protein_type
+MTases	I-protein_type
+,	O
+however	O
+,	O
+has	O
+been	O
+identified	O
+from	O
+later	O
+studies	O
+.	O
+For	O
+instance	O
+,	O
+M	B-protein
+.	I-protein
+DpnII	I-protein
+,	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+,	O
+M	B-protein
+.	I-protein
+KpnI	I-protein
+,	O
+and	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+have	O
+been	O
+found	O
+as	O
+dimers	B-oligomeric_state
+in	O
+solution	O
+.	O
+In	O
+addition	O
+,	O
+several	O
+MTases	B-protein_type
+including	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+,	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+and	O
+TTH0409	B-protein
+form	O
+tightly	O
+associated	O
+dimers	B-oligomeric_state
+in	O
+crystal	B-evidence
+structures	I-evidence
+.	O
+Nonetheless	O
+,	O
+some	O
+DNA	B-protein_type
+MTases	I-protein_type
+such	O
+as	O
+M	B-protein
+.	I-protein
+CcrMI	I-protein
+and	O
+the	O
+Bacillus	B-species
+amyloliquefaciens	I-species
+MTase	B-protein_type
+dissociate	O
+from	O
+dimer	B-oligomeric_state
+into	O
+monomer	B-oligomeric_state
+upon	O
+DNA	B-chemical
+-	O
+binding	O
+.	O
+According	O
+to	O
+the	O
+arrangement	O
+of	O
+the	O
+three	O
+conserved	B-protein_state
+domains	O
+,	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+belongs	O
+to	O
+the	O
+β	B-protein_type
+-	I-protein_type
+subgroup	I-protein_type
+,	O
+in	O
+which	O
+a	O
+conserved	B-protein_state
+motif	O
+NXXTX9	B-structure_element
+−	I-structure_element
+11AXRXFSXXHX4WX6	I-structure_element
+−	I-structure_element
+9	I-structure_element
+YXFXLX3RX9	I-structure_element
+−	I-structure_element
+26NPX1	I-structure_element
+−	I-structure_element
+6NVWX29	I-structure_element
+−	I-structure_element
+34A	I-structure_element
+has	O
+been	O
+identified	O
+at	O
+the	O
+dimerization	B-site
+interface	I-site
+in	O
+crystal	B-evidence
+structures	I-evidence
+.	O
+Most	O
+of	O
+conserved	B-protein_state
+amino	O
+acids	O
+within	O
+that	O
+motif	O
+are	O
+present	O
+in	O
+the	O
+sequence	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+(	O
+Figure	O
+2A	O
+),	O
+implying	O
+dimerization	B-oligomeric_state
+of	O
+this	O
+protein	O
+.	O
+In	O
+agreement	O
+,	O
+a	O
+dimer	B-oligomeric_state
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+was	O
+observed	O
+in	O
+our	O
+crystal	B-evidence
+structures	I-evidence
+with	O
+the	O
+two	O
+monomers	B-oligomeric_state
+related	O
+by	O
+a	O
+two	O
+-	O
+fold	O
+axis	O
+(	O
+Figure	O
+2B	O
+and	O
+2C	O
+).	O
+An	O
+area	O
+of	O
+~	O
+1900	O
+Å2	O
+was	O
+buried	O
+at	O
+the	O
+dimeric	B-site
+interface	I-site
+,	O
+taking	O
+up	O
+ca	O
+17	O
+%	O
+of	O
+the	O
+total	O
+area	O
+.	O
+The	O
+dimeric	B-oligomeric_state
+architecture	O
+was	O
+greatly	O
+stabilized	O
+by	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+and	O
+salt	B-bond_interaction
+bridges	I-bond_interaction
+formed	O
+among	O
+residues	O
+R86	B-residue_name_number
+,	O
+D93	B-residue_name_number
+and	O
+E96	B-residue_name_number
+.	O
+In	O
+addition	O
+,	O
+comparison	O
+of	O
+the	O
+dimer	B-oligomeric_state
+structure	B-evidence
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+with	O
+some	O
+other	O
+β	B-protein_type
+-	I-protein_type
+class	I-protein_type
+MTases	I-protein_type
+(	O
+M1	B-protein
+.	I-protein
+MboIIA	I-protein
+,	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+and	O
+TTHA0409	B-protein
+)	O
+suggested	O
+that	O
+the	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+dimer	B-oligomeric_state
+organized	O
+in	O
+a	O
+similar	O
+form	O
+as	O
+others	O
+(	O
+Figure	O
+S3	O
+).	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+exists	O
+as	O
+dimer	B-oligomeric_state
+in	O
+crystal	B-evidence
+and	O
+solution	O
+A	O
+.	O
+A	O
+conserved	B-protein_state
+interface	B-site
+area	I-site
+of	O
+β	B-protein_type
+-	I-protein_type
+class	I-protein_type
+MTases	I-protein_type
+is	O
+defined	O
+in	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+.	O
+Residues	O
+that	O
+involved	O
+are	O
+signed	O
+in	O
+red	O
+color	O
+;	O
+Dimerization	B-oligomeric_state
+of	O
+free	B-protein_state
+-	O
+form	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+B	O
+.	O
+and	O
+cofactor	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+C	O
+.	O
+The	O
+two	O
+monomers	B-oligomeric_state
+are	O
+marked	O
+in	O
+green	O
+and	O
+blue	O
+,	O
+AdoMet	B-chemical
+molecules	O
+are	O
+marked	O
+in	O
+magenta	O
+.	O
+D	O
+.	O
+Gel	B-experimental_method
+-	I-experimental_method
+filtration	I-experimental_method
+analysis	I-experimental_method
+revealed	O
+that	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+exist	O
+as	O
+a	O
+dimer	B-oligomeric_state
+in	O
+solution	O
+.	O
+FPLC	B-experimental_method
+system	O
+coupled	O
+to	O
+a	O
+Superdex	O
+75	O
+10	O
+/	O
+300	O
+column	O
+.	O
+Elution	B-evidence
+profiles	I-evidence
+at	O
+280	O
+nm	O
+(	O
+blue	O
+)	O
+and	O
+260	O
+nm	O
+(	O
+red	O
+)	O
+are	O
+:	O
+different	O
+concentration	O
+(	O
+0	O
+.	O
+05	O
+,	O
+0	O
+.	O
+1	O
+,	O
+0	O
+.	O
+2	O
+,	O
+0	O
+.	O
+5	O
+mg	O
+/	O
+ml	O
+)	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+protein	O
+.	O
+To	O
+probe	O
+the	O
+oligomeric	O
+form	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+in	O
+solution	O
+,	O
+different	O
+concentrations	O
+of	O
+purified	O
+enzyme	O
+was	O
+loaded	O
+onto	O
+a	O
+Superdex	O
+75	O
+10	O
+/	O
+300	O
+column	O
+.	O
+The	O
+protein	O
+was	O
+eluted	O
+at	O
+~	O
+10	O
+ml	O
+regardless	O
+of	O
+the	O
+protein	O
+concentrations	O
+,	O
+corresponding	O
+to	O
+a	O
+dimeric	B-oligomeric_state
+molecular	B-evidence
+mass	I-evidence
+of	O
+54	O
+kDa	O
+(	O
+Figure	O
+2D	O
+).	O
+Our	O
+results	O
+clearly	O
+showed	O
+that	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+forms	O
+a	O
+dimer	B-oligomeric_state
+in	O
+both	O
+crystal	B-evidence
+and	O
+solution	O
+as	O
+other	O
+β	B-protein_type
+-	I-protein_type
+class	I-protein_type
+MTases	I-protein_type
+,	O
+which	O
+however	O
+disagrees	O
+with	O
+a	O
+previous	O
+investigation	O
+using	O
+dynamic	B-experimental_method
+light	I-experimental_method
+scattering	I-experimental_method
+(	O
+DLS	B-experimental_method
+)	O
+measurement	O
+and	O
+gel	B-experimental_method
+-	I-experimental_method
+filtration	I-experimental_method
+chromatography	I-experimental_method
+,	O
+suggesting	O
+that	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+is	O
+taking	O
+a	O
+monomeric	B-oligomeric_state
+state	O
+in	O
+solution	O
+.	O
+This	O
+variance	O
+might	O
+be	O
+caused	O
+by	O
+an	O
+addition	O
+of	O
+100	O
+mM	O
+arginine	B-chemical
+before	O
+cell	O
+lysis	O
+to	O
+keep	O
+protein	O
+solubility	O
+and	O
+also	O
+by	O
+later	O
+replacement	O
+of	O
+arginine	B-chemical
+with	O
+30	O
+%	O
+glycerol	B-chemical
+by	O
+dialysis	O
+.	O
+Structure	B-experimental_method
+comparisons	I-experimental_method
+As	O
+a	O
+β	B-protein_type
+-	I-protein_type
+class	I-protein_type
+N6	I-protein_type
+adenine	I-protein_type
+MTase	I-protein_type
+,	O
+the	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+structure	B-evidence
+displayed	O
+a	O
+good	O
+similarity	O
+with	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+(	O
+PDB	O
+ID	O
+1G60	O
+)	O
+and	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+(	O
+PDB	O
+ID	O
+1NW7	O
+),	O
+which	O
+are	O
+falling	O
+into	O
+the	O
+same	O
+subgroup	O
+.	O
+Superimposition	B-experimental_method
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+onto	O
+them	O
+gave	O
+RMSDs	B-evidence
+of	O
+1	O
+.	O
+63	O
+Å	O
+and	O
+1	O
+.	O
+9	O
+Å	O
+on	O
+168	O
+and	O
+190	O
+Cα	O
+atoms	O
+,	O
+respectively	O
+.	O
+The	O
+most	O
+striking	O
+structural	O
+difference	O
+was	O
+found	O
+to	O
+locate	O
+on	O
+the	O
+TRD	B-structure_element
+region	O
+(	O
+residues	O
+133	B-residue_range
+-	I-residue_range
+163	I-residue_range
+in	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+)	O
+(	O
+Figure	O
+3A	O
+–	O
+3C	O
+),	O
+where	O
+the	O
+secondary	O
+structures	O
+vary	O
+among	O
+these	O
+structures	O
+.	O
+By	O
+comparison	O
+with	O
+the	O
+other	O
+two	O
+enzymes	O
+that	O
+possess	O
+protruding	O
+arms	O
+containing	O
+several	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+and	O
+/	O
+or	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+,	O
+the	O
+TRD	B-structure_element
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+is	O
+much	O
+shorter	O
+in	O
+length	O
+(	O
+Figure	O
+S1	O
+),	O
+wrapping	O
+more	O
+closely	O
+around	O
+the	O
+structural	O
+core	O
+and	O
+lacking	B-protein_state
+apparent	O
+secondary	O
+structures	O
+.	O
+Given	O
+the	O
+proposed	O
+role	O
+of	O
+the	O
+TRD	B-structure_element
+for	O
+DNA	B-chemical
+interaction	O
+at	O
+the	O
+major	B-structure_element
+groove	I-structure_element
+,	O
+some	O
+differences	O
+of	O
+DNA	B-chemical
+recognition	O
+mode	O
+can	O
+be	O
+expected	O
+.	O
+Another	O
+difference	O
+locates	O
+at	O
+the	O
+highly	B-protein_state
+flexible	I-protein_state
+loop	B-structure_element
+between	O
+β4	B-structure_element
+and	O
+αD	B-structure_element
+(	O
+residues	O
+33	B-residue_range
+-	I-residue_range
+58	I-residue_range
+)	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+,	O
+which	O
+was	O
+invisible	O
+in	O
+our	O
+structures	B-evidence
+but	O
+present	O
+in	O
+the	O
+structures	B-evidence
+of	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+and	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+.	O
+Sequence	B-experimental_method
+alignment	I-experimental_method
+revealed	O
+that	O
+this	O
+region	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+was	O
+longer	O
+than	O
+its	O
+counterparts	O
+by	O
+13	O
+and	O
+16	O
+amino	O
+acids	O
+respectively	O
+,	O
+which	O
+likely	O
+renders	O
+the	O
+H	B-species
+.	I-species
+pylori	I-species
+enzyme	O
+more	O
+flexible	B-protein_state
+.	O
+Structural	B-experimental_method
+comparisons	I-experimental_method
+between	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+and	O
+other	O
+DNA	B-protein_type
+MTases	I-protein_type
+A	O
+.	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+;	O
+B	O
+.	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+;	O
+C	O
+.	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+;	O
+D	O
+.	O
+TTHA0409	B-protein
+;	O
+E	O
+.	O
+DpnM	B-protein
+;	O
+F	O
+.	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+.	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+possesses	O
+only	O
+a	O
+long	B-protein_state
+disorder	I-protein_state
+TRD	B-structure_element
+region	O
+,	O
+compared	O
+with	O
+the	O
+structure	B-protein_state
+-	I-protein_state
+rich	I-protein_state
+TRD	B-structure_element
+of	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+,	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+and	O
+TTHA0409	B-protein
+,	O
+or	O
+the	O
+extra	O
+DNA	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+of	O
+DpnM	B-protein
+and	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+.	O
+The	O
+core	O
+structure	O
+is	O
+in	O
+cyan	O
+;	O
+TRD	B-structure_element
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+,	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+,	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+and	O
+TTHA0409	B-protein
+is	O
+in	O
+red	O
+;	O
+The	O
+region	O
+between	O
+β4	B-structure_element
+and	O
+αD	B-structure_element
+of	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+and	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+is	O
+in	O
+green	O
+;	O
+DNA	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+of	O
+DpnM	B-protein
+is	O
+in	O
+magenta	O
+;	O
+The	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+of	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+is	O
+in	O
+orange	O
+.	O
+Structural	B-experimental_method
+comparison	I-experimental_method
+between	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+and	O
+a	O
+putative	O
+β	B-protein_type
+-	I-protein_type
+class	I-protein_type
+N4	I-protein_type
+cytosine	I-protein_type
+MTase	I-protein_type
+named	O
+TTHA0409	B-protein
+(	O
+PDB	O
+ID	O
+2ZIF	O
+)	O
+showed	O
+a	O
+good	O
+similarity	O
+as	O
+well	O
+,	O
+giving	O
+an	O
+RMSD	B-evidence
+of	O
+1	O
+.	O
+73	O
+Å	O
+on	O
+164	O
+Cα	O
+atoms	O
+(	O
+Figure	O
+3D	O
+).	O
+Exactly	O
+like	O
+the	O
+above	O
+comparison	O
+,	O
+the	O
+most	O
+significant	O
+difference	O
+exists	O
+in	O
+the	O
+TRD	B-structure_element
+,	O
+where	O
+the	O
+structures	B-evidence
+vary	O
+in	O
+terms	O
+of	O
+length	O
+and	O
+presence	O
+of	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+(	O
+Figure	O
+S1	O
+).	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+displayed	O
+a	O
+considerable	O
+structural	O
+dissimilarity	O
+in	O
+comparison	O
+with	O
+N6	B-protein_type
+-	I-protein_type
+adenine	I-protein_type
+MTases	I-protein_type
+from	O
+other	O
+subgroups	O
+including	O
+the	O
+α	B-protein_type
+-	I-protein_type
+class	I-protein_type
+DpnM	B-protein
+(	O
+PDB	O
+ID	O
+2DPM	O
+)	O
+and	O
+the	O
+γ	B-protein_type
+-	I-protein_type
+class	I-protein_type
+M	B-protein
+.	I-protein
+TaqI	I-protein
+(	O
+PDB	O
+ID	O
+2ADM	O
+).	O
+Both	O
+comparisons	O
+gave	O
+RMSDs	B-evidence
+above	O
+3	O
+.	O
+0	O
+Å	O
+(	O
+Figure	O
+3E	O
+and	O
+3F	O
+).	O
+These	O
+two	O
+enzymes	O
+lack	B-protein_state
+a	O
+counterpart	B-structure_element
+loop	I-structure_element
+present	O
+in	O
+the	O
+TRD	B-structure_element
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+,	O
+but	O
+instead	O
+rely	O
+on	O
+an	O
+extra	O
+domain	O
+for	O
+DNA	B-chemical
+binding	O
+and	O
+sequence	O
+recognition	O
+.	O
+Collectively	O
+,	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+possesses	O
+a	O
+long	B-protein_state
+disordered	I-protein_state
+TRD	B-structure_element
+,	O
+which	O
+is	O
+in	O
+sharp	O
+contrast	O
+to	O
+the	O
+secondary	B-protein_state
+structure	I-protein_state
+-	I-protein_state
+rich	I-protein_state
+TRD	B-structure_element
+in	O
+other	O
+β	B-protein_type
+-	I-protein_type
+class	I-protein_type
+N6	I-protein_type
+adenine	I-protein_type
+or	I-protein_type
+N4	I-protein_type
+cytosine	I-protein_type
+MTases	I-protein_type
+or	O
+the	O
+extra	O
+DNA	O
+binding	O
+domain	O
+present	O
+in	O
+DNA	B-protein_type
+MTases	I-protein_type
+from	O
+other	O
+subgroups	O
+.	O
+This	O
+striking	O
+difference	O
+may	O
+be	O
+a	O
+significant	O
+determinant	O
+of	O
+the	O
+wider	O
+substrate	O
+spectrum	O
+of	O
+this	O
+H	B-species
+.	I-species
+pylori	I-species
+enzyme	O
+.	O
+AdoMet	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+The	O
+cofactor	B-site
+binding	I-site
+pocket	I-site
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+is	O
+surrounded	O
+by	O
+residues	O
+7	B-residue_range
+-	I-residue_range
+9	I-residue_range
+,	O
+29	B-residue_range
+-	I-residue_range
+31	I-residue_range
+,	O
+165	B-residue_range
+-	I-residue_range
+167	I-residue_range
+,	O
+216	B-residue_range
+-	I-residue_range
+218	I-residue_range
+and	O
+221	B-residue_number
+(	O
+Figure	O
+4A	O
+),	O
+which	O
+are	O
+conserved	B-protein_state
+among	O
+most	O
+of	O
+DNA	B-protein_type
+MTases	I-protein_type
+.	O
+A	O
+hydrogen	B-bond_interaction
+bond	I-bond_interaction
+between	O
+D29	B-residue_name_number
+in	O
+the	O
+catalytic	B-structure_element
+motif	I-structure_element
+DPPY	B-structure_element
+and	O
+the	O
+amino	O
+group	O
+of	O
+bound	B-protein_state
+AdoMet	B-chemical
+is	O
+preserved	O
+as	O
+other	O
+MTase	B-protein_type
+structures	B-evidence
+.	O
+Residues	O
+D8	B-residue_name_number
+and	O
+A9	B-residue_name_number
+from	O
+hydrogen	B-bond_interaction
+-	I-bond_interaction
+bonds	I-bond_interaction
+with	O
+N6	O
+and	O
+N1	O
+of	O
+the	O
+purine	B-chemical
+ring	O
+,	O
+respectively	O
+,	O
+and	O
+E216	B-residue_name_number
+also	O
+locates	O
+at	O
+hydrogen	B-bond_interaction
+bonding	I-bond_interaction
+distance	O
+with	O
+O2	O
+′	O
+and	O
+O3	O
+′	O
+of	O
+the	O
+ribose	B-chemical
+.	O
+In	O
+addition	O
+,	O
+H168	B-residue_name_number
+,	O
+T200	B-residue_name_number
+and	O
+S198	B-residue_name_number
+contact	O
+the	O
+terminal	O
+carboxyl	O
+of	O
+AdoMet	B-chemical
+.	O
+Superposition	B-experimental_method
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+with	O
+the	O
+five	O
+structures	B-evidence
+shown	O
+in	O
+Figure	O
+3	O
+reveals	O
+that	O
+the	O
+orientation	O
+of	O
+cofactor	O
+is	O
+rather	B-protein_state
+conserved	I-protein_state
+except	O
+for	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+(	O
+Figure	O
+4B	O
+).	O
+The	O
+different	O
+conformation	O
+of	O
+the	O
+bound	B-protein_state
+cofactor	O
+observed	O
+in	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+might	O
+be	O
+attributable	O
+to	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+corresponding	O
+residues	O
+of	O
+the	O
+conserved	B-protein_state
+AdoMet	B-chemical
+-	O
+binding	O
+motif	O
+FXGXG	B-structure_element
+in	O
+that	O
+structure	B-evidence
+.	O
+Structural	B-experimental_method
+and	I-experimental_method
+biochemical	I-experimental_method
+analyses	I-experimental_method
+define	O
+two	O
+conserved	B-protein_state
+residues	O
+D29	B-residue_name_number
+and	O
+E216	B-residue_name_number
+to	O
+be	O
+the	O
+key	O
+sites	O
+for	O
+AdoMet	B-chemical
+binding	O
+A	O
+.	O
+The	O
+cofactor	B-site
+-	I-site
+binding	I-site
+cavity	I-site
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+.	O
+Residues	O
+(	O
+yellow	O
+)	O
+that	O
+form	O
+direct	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+with	O
+AdoMet	B-chemical
+(	O
+green	O
+)	O
+are	O
+indicated	O
+,	O
+distance	O
+of	O
+the	O
+hydrogen	B-bond_interaction
+bond	I-bond_interaction
+is	O
+marked	O
+.	O
+B	O
+.	O
+Superposition	B-experimental_method
+of	O
+AdoMet	B-chemical
+in	O
+the	O
+structures	B-evidence
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+(	O
+green	O
+),	O
+DpnM	B-protein
+(	O
+yellow	O
+)	O
+and	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+(	O
+orange	O
+).	O
+The	O
+AdoMet	B-chemical
+terminal	O
+carboxyl	O
+of	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+reveals	O
+different	O
+orientations	O
+.	O
+C	O
+.	O
+Cofactor	B-evidence
+binding	I-evidence
+affinity	I-evidence
+of	O
+wt	B-protein_state
+-/	O
+mutants	B-protein_state
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+proteins	O
+analyzed	O
+by	O
+microscale	B-experimental_method
+thermophoresis	I-experimental_method
+(	O
+MST	B-experimental_method
+).	O
+The	O
+binding	B-evidence
+affinity	I-evidence
+was	O
+determined	O
+between	O
+fluorescently	O
+labelled	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+protein	O
+and	O
+unlabeled	B-protein_state
+AdoMet	B-chemical
+.	O
+AdoMet	B-chemical
+(	O
+15	O
+nM	O
+to	O
+1	O
+mM	O
+)	O
+was	O
+titrated	B-experimental_method
+into	O
+a	O
+fixed	O
+concentration	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+wt	B-protein_state
+/	O
+mutant	B-protein_state
+proteins	O
+(	O
+800	O
+nM	O
+).	O
+The	O
+dissociation	B-evidence
+constant	I-evidence
+(	O
+KD	B-evidence
+)	O
+is	O
+yielded	O
+according	O
+to	O
+the	O
+law	O
+of	O
+mass	O
+action	O
+from	O
+the	O
+isotherm	B-evidence
+derived	O
+of	O
+the	O
+raw	O
+data	O
+:	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+-	O
+wt	B-protein_state
+:	O
+41	O
+±	O
+6	O
+μM	O
+;	O
+M1	B-mutant
+.	I-mutant
+HpyAVI	I-mutant
+-	I-mutant
+D8A	I-mutant
+:	O
+212	O
+±	O
+11	O
+μM	O
+;	O
+M1	B-mutant
+.	I-mutant
+HpyAVI	I-mutant
+-	I-mutant
+D29A	I-mutant
+:	O
+0	O
+μM	O
+;	O
+M1	B-mutant
+.	I-mutant
+HpyAVI	I-mutant
+-	I-mutant
+H168A	I-mutant
+:	O
+471	O
+±	O
+51	O
+μM	O
+;	O
+M1	B-mutant
+.	I-mutant
+HpyAVI	I-mutant
+-	I-mutant
+S198A	I-mutant
+:	O
+242	O
+±	O
+32	O
+μM	O
+;	O
+M1	B-mutant
+.	I-mutant
+HpyAVI	I-mutant
+-	I-mutant
+T200A	I-mutant
+:	O
+252	O
+±	O
+28	O
+μM	O
+;	O
+M1	B-mutant
+.	I-mutant
+HpyAVI	I-mutant
+-	I-mutant
+E216A	I-mutant
+:	O
+0	O
+μM	O
+.	O
+Standard	O
+for	O
+three	O
+replicates	O
+is	O
+indicated	O
+.	O
+D	O
+.	O
+DNA	B-protein_type
+methyltransferase	I-protein_type
+activity	O
+of	O
+wide	B-protein_state
+type	I-protein_state
+protein	O
+and	O
+the	O
+mutants	B-protein_state
+is	O
+quantified	O
+using	O
+radioactive	B-experimental_method
+assay	I-experimental_method
+.	O
+[	B-chemical
+3H	I-chemical
+]-	I-chemical
+methyl	I-chemical
+transferred	O
+to	O
+duplex	O
+DNA	B-chemical
+containing	O
+5	B-chemical
+′-	I-chemical
+GAGG	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+was	O
+quantified	O
+by	O
+Beckman	O
+LS6500	O
+for	O
+10	O
+min	O
+,	O
+experiments	O
+were	O
+repeated	O
+for	O
+three	O
+times	O
+and	O
+data	O
+were	O
+corrected	O
+by	O
+subtraction	O
+of	O
+the	O
+background	O
+.	O
+E	O
+.	O
+Superposition	B-experimental_method
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+(	O
+green	O
+)	O
+with	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+(	O
+cyan	O
+)	O
+and	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+(	O
+magenta	O
+).	O
+Residues	O
+D29	B-residue_name_number
+and	O
+E216	B-residue_name_number
+are	O
+conserved	B-protein_state
+through	O
+all	O
+the	O
+DNA	B-protein_type
+MTases	I-protein_type
+mentioned	O
+in	O
+Figure	O
+3	O
+(	O
+not	O
+shown	O
+in	O
+Figure	O
+4	O
+).	O
+To	O
+confirm	O
+the	O
+key	O
+residues	O
+for	O
+ligand	O
+binding	O
+,	O
+we	O
+prepared	O
+a	O
+series	O
+of	O
+single	B-experimental_method
+mutants	I-experimental_method
+by	O
+replacing	B-experimental_method
+D8	B-residue_name_number
+,	O
+D29	B-residue_name_number
+,	O
+H168	B-residue_name_number
+,	O
+S198	B-residue_name_number
+,	O
+T200	B-residue_name_number
+,	O
+E216	B-residue_name_number
+with	O
+alanine	B-residue_name
+and	O
+investigated	O
+their	O
+ligand	B-evidence
+binding	I-evidence
+affinity	I-evidence
+using	O
+microscale	B-experimental_method
+thermophoresis	I-experimental_method
+(	O
+MST	B-experimental_method
+)	O
+assay	O
+.	O
+As	O
+shown	O
+in	O
+Figure	O
+4C	O
+,	O
+by	O
+contrast	O
+to	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+enzyme	O
+,	O
+most	O
+mutants	B-protein_state
+displayed	O
+variable	O
+reduction	O
+of	O
+KD	B-evidence
+value	O
+,	O
+among	O
+them	O
+the	O
+D29A	B-mutant
+and	O
+E216A	B-mutant
+mutants	B-protein_state
+displayed	O
+no	O
+protein	B-evidence
+-	I-evidence
+AdoMet	I-evidence
+affinity	I-evidence
+at	O
+all	O
+.	O
+The	O
+results	O
+suggested	O
+that	O
+the	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+formed	O
+by	O
+D29	B-residue_name_number
+and	O
+E216	B-residue_name_number
+with	O
+AdoMet	B-chemical
+were	O
+most	O
+crucial	O
+interactions	O
+for	O
+cofactor	O
+binding	O
+.	O
+Mutation	B-experimental_method
+of	O
+the	O
+two	O
+residues	O
+may	O
+directly	O
+prevent	O
+the	O
+methyl	B-chemical
+transfer	O
+reaction	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+.	O
+The	O
+importance	O
+of	O
+D29	B-residue_name_number
+is	O
+preserved	O
+because	O
+it	O
+belongs	O
+to	O
+the	O
+catalytic	B-site
+active	I-site
+site	I-site
+DPPY	B-structure_element
+,	O
+but	O
+the	O
+residue	O
+E216	B-residue_name_number
+has	O
+not	O
+been	O
+fully	O
+investigated	O
+even	O
+being	O
+a	O
+conserved	B-protein_state
+amino	B-chemical
+acid	I-chemical
+throughout	O
+MTases	B-protein_type
+(	O
+Figure	O
+4E	O
+).	O
+E216	B-residue_name_number
+is	O
+the	O
+last	O
+residue	O
+of	O
+β2	B-structure_element
+,	O
+which	O
+contacts	O
+the	O
+two	O
+hydroxyls	O
+of	O
+the	O
+ribose	B-chemical
+of	O
+AdoMet	B-chemical
+.	O
+Replacement	B-experimental_method
+of	O
+this	O
+residue	O
+by	O
+alanine	B-residue_name
+completely	O
+abolishes	O
+the	O
+key	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+for	O
+AdoMet	B-chemical
+-	O
+binding	O
+,	O
+and	O
+very	O
+likely	O
+blocks	O
+the	O
+methyl	B-chemical
+transfer	O
+reaction	O
+.	O
+To	O
+confirm	O
+this	O
+notion	O
+,	O
+[	B-experimental_method
+3H	I-experimental_method
+]	I-experimental_method
+AdoMet	I-experimental_method
+radiological	I-experimental_method
+assay	I-experimental_method
+was	O
+applied	O
+to	O
+quantify	O
+the	O
+methyl	B-chemical
+transfer	O
+activity	O
+of	O
+the	O
+mutants	B-protein_state
+.	O
+As	O
+shown	O
+in	O
+Figure	O
+4D	O
+,	O
+the	O
+result	O
+of	O
+radiological	B-experimental_method
+assay	I-experimental_method
+agreed	O
+well	O
+with	O
+the	O
+MST	B-experimental_method
+measurement	O
+.	O
+The	O
+D29A	B-mutant
+and	O
+E216A	B-mutant
+mutants	B-protein_state
+showed	O
+little	O
+or	O
+no	O
+methyl	B-chemical
+transfer	O
+activity	O
+,	O
+while	O
+other	O
+mutants	B-protein_state
+exhibited	O
+reduced	O
+methyltransferase	B-protein_type
+activity	O
+.	O
+As	O
+mentioned	O
+previously	O
+,	O
+FXGXG	B-structure_element
+is	O
+a	O
+conserved	B-protein_state
+AdoMet	B-chemical
+-	O
+binding	O
+motif	O
+of	O
+DNA	B-protein_type
+MTases	I-protein_type
+.	O
+We	O
+also	O
+made	O
+mutants	B-protein_state
+of	O
+“	O
+FMGSG	B-structure_element
+”	O
+to	O
+alanine	B-residue_name
+for	O
+every	O
+amino	B-chemical
+acid	I-chemical
+,	O
+and	O
+found	O
+that	O
+the	O
+F195A	B-mutant
+mutant	B-protein_state
+was	O
+insoluble	O
+probably	O
+due	O
+to	O
+decreasing	O
+the	O
+local	O
+hydrophobicity	O
+upon	O
+this	O
+mutation	O
+.	O
+We	O
+subsequently	O
+investigated	O
+the	O
+ligand	B-evidence
+binding	I-evidence
+affinity	I-evidence
+and	O
+methyl	B-chemical
+transfer	O
+reaction	O
+of	O
+the	O
+other	O
+mutants	B-protein_state
+using	O
+MST	B-experimental_method
+and	O
+a	O
+radiological	B-experimental_method
+assay	I-experimental_method
+.	O
+We	O
+found	O
+that	O
+G197	B-residue_name_number
+played	O
+a	O
+crucial	O
+role	O
+in	O
+AdoMet	B-chemical
+-	O
+binding	O
+,	O
+while	O
+mutagenesis	B-experimental_method
+of	O
+M196	B-residue_name_number
+and	O
+G199	B-residue_name_number
+did	O
+not	O
+influence	O
+cofactor	O
+binding	O
+and	O
+catalytic	O
+activity	O
+(	O
+Figure	O
+S2A	O
+and	O
+B	O
+).	O
+G197	B-residue_name_number
+is	O
+a	O
+conserved	B-protein_state
+residue	O
+throughout	O
+the	O
+DNA	B-protein_type
+MTases	I-protein_type
+,	O
+and	O
+replacing	B-experimental_method
+by	O
+alanine	B-residue_name
+at	O
+this	O
+site	O
+likely	O
+change	O
+the	O
+local	O
+conformation	O
+of	O
+cofactor	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+.	O
+Mutagenesis	B-experimental_method
+on	O
+this	O
+glycine	B-residue_name
+residue	O
+in	O
+M	B-protein
+.	I-protein
+EcoKI	I-protein
+or	O
+M	B-protein
+.	I-protein
+EcoP15I	I-protein
+also	O
+abolished	O
+the	O
+AdoMet	B-chemical
+-	O
+binding	O
+activity	O
+.	O
+Although	O
+mutational	B-experimental_method
+study	I-experimental_method
+could	O
+not	O
+tell	O
+the	O
+role	O
+of	O
+F195	B-residue_name_number
+in	O
+ligand	O
+binding	O
+due	O
+to	O
+the	O
+insolubility	O
+of	O
+the	O
+F195A	B-mutant
+mutant	B-protein_state
+,	O
+structural	B-experimental_method
+analysis	I-experimental_method
+suggested	O
+the	O
+importance	O
+of	O
+this	O
+residue	O
+in	O
+AdoMet	B-chemical
+-	O
+binding	O
+.	O
+The	O
+phenyl	O
+ring	O
+of	O
+F195	B-residue_name_number
+forms	O
+a	O
+perpendicular	O
+π	B-bond_interaction
+-	I-bond_interaction
+stacking	I-bond_interaction
+interaction	I-bond_interaction
+with	O
+the	O
+purine	O
+ring	O
+of	O
+AdoMet	B-chemical
+,	O
+which	O
+stabilizes	O
+the	O
+orientation	O
+of	O
+AdoMet	B-chemical
+bound	B-protein_state
+in	I-protein_state
+the	O
+pocket	B-site
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+(	O
+Figure	O
+S2C	O
+).	O
+In	O
+a	O
+separate	O
+scenario	O
+,	O
+mutagenesis	B-experimental_method
+of	O
+this	O
+residue	O
+in	O
+M	B-protein
+.	I-protein
+EcoRV	I-protein
+has	O
+been	O
+proven	O
+to	O
+play	O
+an	O
+important	O
+role	O
+in	O
+AdoMet	B-chemical
+binding	O
+.	O
+Potential	O
+DNA	B-site
+-	I-site
+binding	I-site
+sites	I-site
+The	O
+putative	O
+DNA	B-site
+binding	I-site
+region	I-site
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+involves	O
+the	O
+hairpin	B-structure_element
+loop	I-structure_element
+(	O
+residue	O
+101	B-residue_range
+-	I-residue_range
+133	I-residue_range
+),	O
+the	O
+TRD	B-structure_element
+(	O
+residues	O
+136	B-residue_range
+-	I-residue_range
+166	I-residue_range
+),	O
+and	O
+a	O
+highly	B-protein_state
+flexible	I-protein_state
+loop	B-structure_element
+(	O
+residues	O
+33	B-residue_range
+-	I-residue_range
+58	I-residue_range
+).	O
+The	O
+hairpin	B-structure_element
+loop	I-structure_element
+between	O
+β6	B-structure_element
+and	O
+β7	B-structure_element
+strands	O
+that	O
+carries	O
+a	O
+conserved	B-protein_state
+HRRY	B-structure_element
+sequence	O
+signature	O
+in	O
+the	O
+middle	O
+is	O
+proposed	O
+to	O
+insert	O
+into	O
+the	O
+minor	B-structure_element
+groove	I-structure_element
+of	O
+the	O
+bound	B-protein_state
+DNA	B-chemical
+.	O
+As	O
+aforementioned	O
+,	O
+the	O
+TRD	B-structure_element
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+shows	O
+striking	O
+difference	O
+from	O
+the	O
+other	O
+DNA	B-protein_type
+MTases	I-protein_type
+,	O
+and	O
+the	O
+relaxed	O
+specificity	O
+of	O
+substrate	O
+recognition	O
+may	O
+be	O
+at	O
+least	O
+partially	O
+attributable	O
+to	O
+the	O
+disordered	B-protein_state
+TRD	B-structure_element
+.	O
+In	O
+addition	O
+,	O
+the	O
+highly	B-protein_state
+flexible	I-protein_state
+loop	B-structure_element
+immediately	O
+following	O
+the	O
+DPPY	B-structure_element
+motif	O
+in	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+was	O
+poorly	O
+defined	O
+in	O
+electron	B-evidence
+density	I-evidence
+,	O
+exactly	O
+like	O
+the	O
+corresponding	O
+loops	B-structure_element
+in	O
+the	O
+AdoMet	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+structures	B-evidence
+of	O
+M	B-protein
+.	I-protein
+PvuII	I-protein
+,	O
+DpnM	B-protein
+or	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+that	O
+were	O
+invisible	O
+either	O
+.	O
+This	O
+loop	B-structure_element
+,	O
+however	O
+,	O
+was	O
+largely	O
+stabilized	O
+upon	O
+DNA	B-chemical
+binding	O
+,	O
+as	O
+observed	O
+in	O
+the	O
+protein	B-evidence
+-	I-evidence
+DNA	I-evidence
+complex	I-evidence
+structures	I-evidence
+of	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+(	O
+PDB	O
+ID	O
+2IBS	O
+),	O
+M	B-protein
+.	I-protein
+HhaI	I-protein
+(	O
+PDB	O
+ID	O
+1MHT	O
+)	O
+and	O
+M	B-protein
+.	I-protein
+HaeIII	I-protein
+(	O
+PDB	O
+ID	O
+1DCT	O
+).	O
+The	O
+well	B-protein_state
+-	I-protein_state
+ordered	I-protein_state
+loop	B-structure_element
+in	O
+those	O
+structures	B-evidence
+directly	O
+contacts	O
+the	O
+flipping	O
+adenine	B-residue_name
+and	O
+forms	O
+hydrogen	B-bond_interaction
+bond	I-bond_interaction
+with	O
+neighboring	O
+bases	O
+.	O
+These	O
+observations	O
+implied	O
+that	O
+the	O
+corresponding	O
+loop	B-structure_element
+in	O
+other	O
+MTases	B-protein_type
+,	O
+e	O
+.	O
+g	O
+.	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+,	O
+is	O
+likely	O
+responsible	O
+for	O
+reducing	O
+sequence	O
+recognition	O
+specificity	O
+and	O
+thus	O
+plays	O
+crucial	O
+roles	O
+in	O
+catalysis	O
+.	O
+Previous	O
+research	O
+suggested	O
+that	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+from	O
+strain	O
+26695	O
+was	O
+the	O
+first	O
+N6	B-protein_type
+adenine	I-protein_type
+MTase	I-protein_type
+that	O
+can	O
+methylate	O
+the	O
+adenine	B-residue_name
+of	O
+5	B-chemical
+′-	I-chemical
+GAGG	I-chemical
+-	I-chemical
+3	I-chemical
+′/	I-chemical
+5	B-chemical
+′-	I-chemical
+GGAG	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+or	O
+both	O
+two	O
+adenines	B-residue_name
+of	O
+5	B-chemical
+′-	I-chemical
+GAAG	I-chemical
+-	I-chemical
+3	I-chemical
+′,	I-chemical
+compared	O
+with	O
+the	O
+homologs	O
+from	O
+other	O
+strains	O
+that	O
+can	O
+methylate	O
+only	O
+one	O
+adenine	B-residue_name
+of	O
+5	B-chemical
+′-	I-chemical
+GAGG	I-chemical
+-	I-chemical
+3	I-chemical
+′.	I-chemical
+To	O
+answer	O
+why	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+displayed	O
+a	O
+wider	O
+specificity	O
+for	O
+DNA	B-chemical
+recognition	O
+,	O
+we	O
+randomly	O
+choose	O
+fifty	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+sequences	O
+from	O
+hundreds	O
+of	O
+H	B-species
+.	I-species
+pylori	I-species
+strains	O
+for	O
+multiple	B-experimental_method
+sequence	I-experimental_method
+alignment	I-experimental_method
+.	O
+Based	O
+on	O
+sequence	B-experimental_method
+comparison	I-experimental_method
+and	O
+structural	B-experimental_method
+analysis	I-experimental_method
+,	O
+four	O
+residues	O
+including	O
+P41	B-residue_name_number
+,	O
+N111	B-residue_name_number
+,	O
+K165	B-residue_name_number
+and	O
+T166	B-residue_name_number
+were	O
+selected	O
+and	O
+replaced	B-experimental_method
+by	O
+serine	B-residue_name
+,	O
+threonine	B-residue_name
+,	O
+threonine	B-residue_name
+and	O
+valine	B-residue_name
+,	O
+respectively	O
+(	O
+Figure	O
+5A	O
+).	O
+Then	O
+,	O
+a	O
+[	B-experimental_method
+3H	I-experimental_method
+]	I-experimental_method
+AdoMet	I-experimental_method
+radiological	I-experimental_method
+assay	I-experimental_method
+was	O
+applied	O
+to	O
+quantify	O
+the	O
+methyl	B-chemical
+transfer	O
+activity	O
+of	O
+the	O
+wide	B-protein_state
+type	I-protein_state
+protein	O
+and	O
+the	O
+mutants	B-protein_state
+.	O
+As	O
+shown	O
+in	O
+Figure	O
+5	O
+,	O
+when	O
+the	O
+substrate	O
+DNA	B-chemical
+contains	O
+5	B-chemical
+′-	I-chemical
+GAGG	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+or	O
+5	B-chemical
+′-	I-chemical
+GAAG	I-chemical
+-	I-chemical
+3	I-chemical
+′,	I-chemical
+all	O
+the	O
+mutants	B-protein_state
+showed	O
+no	O
+apparent	O
+difference	O
+of	O
+methyl	B-chemical
+transfer	O
+activity	O
+compared	O
+to	O
+the	O
+wt	B-protein_state
+-	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+;	O
+but	O
+when	O
+the	O
+recognition	O
+sequence	O
+was	O
+5	B-chemical
+′-	I-chemical
+GGAG	I-chemical
+-	I-chemical
+3	I-chemical
+′,	I-chemical
+the	O
+methyl	B-chemical
+transfer	O
+activity	O
+of	O
+the	O
+P41S	B-mutant
+mutant	B-protein_state
+was	O
+significantly	O
+reduced	O
+compared	O
+to	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+.	O
+Sequence	B-experimental_method
+alignment	I-experimental_method
+,	O
+structural	B-experimental_method
+analysis	I-experimental_method
+and	O
+radioactive	B-experimental_method
+methyl	I-experimental_method
+transfer	I-experimental_method
+activity	I-experimental_method
+define	O
+the	O
+key	O
+residue	O
+for	O
+wider	O
+substrate	O
+specificity	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+A	O
+.	O
+Sequence	B-experimental_method
+alignment	I-experimental_method
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+from	O
+50	O
+H	B-species
+.	I-species
+pylori	I-species
+strains	O
+including	O
+26695	O
+revealed	O
+several	O
+variant	O
+residues	O
+.	O
+Residues	O
+P41	B-residue_name_number
+,	O
+N111	B-residue_name_number
+,	O
+K165	B-residue_name_number
+and	O
+T166	B-residue_name_number
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+from	O
+strain	O
+26695	B-species
+were	O
+chosen	O
+based	O
+on	O
+structural	B-experimental_method
+analysis	I-experimental_method
+and	O
+sequence	B-experimental_method
+alignment	I-experimental_method
+(	O
+shown	O
+in	O
+red	O
+arrow	O
+).	O
+Amino	O
+-	O
+acid	O
+conservation	O
+is	O
+depicted	O
+using	O
+WebLogo	B-experimental_method
+(	O
+Crooks	O
+et	O
+al	O
+,	O
+2004	O
+).	O
+B	O
+.,	O
+C	O
+.,	O
+D	O
+.	O
+Methyl	B-chemical
+transfer	O
+reactions	O
+were	O
+performed	O
+using	O
+wt	B-protein_state
+-	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+,	O
+M1	B-mutant
+.	I-mutant
+HpyAVI	I-mutant
+-	I-mutant
+P41S	I-mutant
+,	O
+M1	B-mutant
+.	I-mutant
+HpyAVI	I-mutant
+-	I-mutant
+N111T	I-mutant
+,	O
+and	O
+M1	B-mutant
+.	I-mutant
+HpyAVI	I-mutant
+-	I-mutant
+K165R	I-mutant
+T166V	I-mutant
+,	O
+respectively	O
+.	O
+Radioactivity	O
+incorporated	O
+into	O
+the	O
+duplex	O
+DNA	B-chemical
+containing	O
+5	B-chemical
+′-	I-chemical
+GAGG	I-chemical
+-	I-chemical
+3	I-chemical
+′,	I-chemical
+5	B-chemical
+′-	I-chemical
+GAAG	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+or	O
+5	B-chemical
+′-	I-chemical
+GGAG	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+was	O
+quantified	O
+by	O
+Beckman	O
+LS6500	O
+for	O
+10	O
+min	O
+.	O
+Our	O
+experimental	O
+data	O
+identified	O
+P41	B-residue_name_number
+as	O
+a	O
+key	O
+residue	O
+determining	O
+the	O
+recognition	O
+of	O
+GGAG	B-structure_element
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+.	O
+This	O
+amino	O
+acid	O
+locates	O
+in	O
+the	O
+highly	B-protein_state
+flexible	I-protein_state
+loop	B-structure_element
+between	O
+residues	O
+33	B-residue_range
+and	I-residue_range
+58	I-residue_range
+,	O
+which	O
+is	O
+involved	O
+in	O
+DNA	B-chemical
+binding	O
+and	O
+substrate	O
+recognition	O
+as	O
+shown	O
+above	O
+.	O
+Replacement	B-experimental_method
+by	O
+serine	B-residue_name
+at	O
+this	O
+position	O
+definitely	O
+changes	O
+the	O
+local	O
+conformation	O
+and	O
+hydrophobicity	O
+,	O
+and	O
+probably	O
+some	O
+structural	O
+properties	O
+of	O
+the	O
+whole	O
+loop	B-structure_element
+,	O
+which	O
+may	O
+in	O
+turn	O
+result	O
+in	O
+reduced	O
+specificity	O
+for	O
+sequence	O
+recognition	O
+of	O
+the	O
+enzyme	O
+from	O
+strain	O
+26695	B-species
+.	O
+Although	O
+the	O
+DNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+structure	B-evidence
+of	O
+previous	O
+investigation	O
+on	O
+a	O
+γ	B-protein_type
+-	I-protein_type
+class	I-protein_type
+N6	I-protein_type
+-	I-protein_type
+adenine	I-protein_type
+MTase	I-protein_type
+revealed	O
+that	O
+the	O
+target	O
+adenine	B-residue_name
+was	O
+rotated	O
+out	O
+of	O
+DNA	B-chemical
+helix	O
+,	O
+details	O
+of	O
+the	O
+methyl	B-chemical
+transfer	O
+process	O
+were	O
+still	O
+unclear	O
+.	O
+Additionally	O
+,	O
+recent	O
+studies	O
+reported	O
+the	O
+importance	O
+of	O
+N6	B-ptm
+-	I-ptm
+methyladenine	I-ptm
+in	O
+some	O
+eukaryotic	B-taxonomy_domain
+species	O
+,	O
+but	O
+until	O
+now	O
+there	O
+has	O
+not	O
+been	O
+any	O
+N6	B-protein_type
+-	I-protein_type
+adenine	I-protein_type
+MTases	I-protein_type
+being	O
+identified	O
+in	O
+eukaryotes	B-taxonomy_domain
+.	O
+Biochemical	B-experimental_method
+and	I-experimental_method
+structural	I-experimental_method
+characterization	I-experimental_method
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+provides	O
+a	O
+new	O
+model	O
+for	O
+uncovering	O
+the	O
+methyl	B-chemical
+transfer	O
+mechanism	O
+and	O
+for	O
+investigating	O
+the	O
+N6	B-ptm
+-	I-ptm
+methyladenine	I-ptm
+in	O
+eukaryotes	B-taxonomy_domain
+.	O
+Oligomeric	O
+state	O
+of	O
+DNA	B-protein_type
+MTases	I-protein_type
+was	O
+long	O
+accepted	O
+as	O
+monomer	B-oligomeric_state
+,	O
+but	O
+our	O
+study	O
+indicated	O
+here	O
+that	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+exists	O
+as	O
+a	O
+dimer	B-oligomeric_state
+both	O
+in	O
+crystal	B-evidence
+and	O
+solution	O
+.	O
+Interestingly	O
+,	O
+some	O
+other	O
+β	B-protein_type
+-	I-protein_type
+class	I-protein_type
+DNA	I-protein_type
+exocyclic	I-protein_type
+MTases	I-protein_type
+showed	O
+similar	O
+oligomeric	O
+state	O
+in	O
+crystal	B-evidence
+and	O
+in	O
+solution	O
+,	O
+indicating	O
+that	O
+dimer	B-oligomeric_state
+may	O
+be	O
+the	O
+functional	O
+state	O
+shared	O
+by	O
+a	O
+subgroup	O
+of	O
+DNA	B-protein_type
+MTases	I-protein_type
+.	O
+The	O
+highly	B-protein_state
+flexible	I-protein_state
+region	O
+(	O
+residues	O
+33	B-residue_range
+-	I-residue_range
+58	I-residue_range
+)	O
+and	O
+TRD	B-structure_element
+(	O
+residues	O
+133	B-residue_range
+-	I-residue_range
+163	I-residue_range
+)	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+are	O
+supposed	O
+to	O
+interact	O
+with	O
+DNA	B-chemical
+at	O
+minor	B-structure_element
+and	I-structure_element
+major	I-structure_element
+grooves	I-structure_element
+,	O
+respectively	O
+.	O
+And	O
+residue	O
+P41	B-residue_name_number
+might	O
+be	O
+a	O
+key	O
+residue	O
+partially	O
+determining	O
+the	O
+substrate	O
+spectrum	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+.	O
+The	O
+missing	B-protein_state
+loop	B-structure_element
+between	O
+residues	O
+33	B-residue_range
+and	I-residue_range
+58	I-residue_range
+may	O
+need	O
+DNA	B-chemical
+binding	O
+so	O
+as	O
+to	O
+form	O
+a	O
+stable	B-protein_state
+conformation	O
+,	O
+which	O
+is	O
+similar	O
+to	O
+the	O
+condition	O
+of	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+.	O
+Crystallization	B-experimental_method
+of	O
+M1	B-complex_assembly
+.	I-complex_assembly
+HpyAVI	I-complex_assembly
+-	I-complex_assembly
+DNA	I-complex_assembly
+complex	O
+warrants	O
+future	O
+investigations	O
+,	O
+with	O
+the	O
+purpose	O
+of	O
+revealing	O
+the	O
+mechanism	O
+behind	O
+the	O
+wider	O
+substrate	O
+specificity	O
+of	O
+this	O
+enzyme	O
+.	O
+DNA	B-ptm
+methylation	I-ptm
+plays	O
+an	O
+important	O
+role	O
+in	O
+bacterial	B-taxonomy_domain
+pathogenicity	O
+.	O
+DNA	B-ptm
+adenine	I-ptm
+methylation	I-ptm
+was	O
+known	O
+to	O
+regulate	O
+the	O
+expression	O
+of	O
+some	O
+virulence	O
+genes	O
+in	O
+bacteria	B-taxonomy_domain
+including	O
+H	B-species
+.	I-species
+pylori	I-species
+.	O
+Inhibitors	O
+of	O
+DNA	B-ptm
+adenine	I-ptm
+methylation	I-ptm
+may	O
+have	O
+a	O
+broad	O
+antimicrobial	O
+action	O
+by	O
+targeting	O
+DNA	B-protein_type
+adenine	I-protein_type
+methyltransferase	I-protein_type
+.	O
+As	O
+an	O
+important	O
+biological	O
+modification	O
+,	O
+DNA	B-ptm
+methylation	I-ptm
+directly	O
+influences	O
+bacterial	B-taxonomy_domain
+survival	O
+.	O
+Knockout	B-experimental_method
+of	I-experimental_method
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+largely	O
+prevents	O
+the	O
+growth	O
+of	O
+H	B-species
+.	I-species
+pylori	I-species
+.	O
+Importantly	O
+,	O
+H	B-species
+.	I-species
+pylori	I-species
+is	O
+involved	O
+in	O
+90	O
+%	O
+of	O
+all	O
+gastric	O
+malignancies	O
+.	O
+Appropriate	O
+antibiotic	O
+regimens	O
+could	O
+successfully	O
+cure	O
+gastric	O
+diseases	O
+caused	O
+by	O
+H	B-species
+.	I-species
+pylori	I-species
+infection	O
+.	O
+However	O
+,	O
+eradication	O
+of	O
+H	B-species
+.	I-species
+pylori	I-species
+infection	O
+remains	O
+a	O
+big	O
+challenge	O
+for	O
+the	O
+significantly	O
+increasing	O
+prevalence	O
+of	O
+its	O
+resistance	O
+to	O
+antibiotics	O
+.	O
+The	O
+development	O
+of	O
+new	O
+drugs	O
+targeting	O
+adenine	B-protein_type
+MTases	I-protein_type
+such	O
+as	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+offers	O
+a	O
+new	O
+opportunity	O
+for	O
+inhibition	O
+of	O
+H	B-species
+.	I-species
+pylori	I-species
+infection	O
+.	O
+Residues	O
+that	O
+play	O
+crucial	O
+roles	O
+for	O
+catalytic	O
+activity	O
+like	O
+D29	B-residue_name_number
+or	O
+E216	B-residue_name_number
+may	O
+influence	O
+the	O
+H	B-species
+.	I-species
+pylori	I-species
+survival	O
+.	O
+Small	O
+molecules	O
+targeting	O
+these	O
+highly	B-protein_state
+conserved	I-protein_state
+residues	O
+are	O
+likely	O
+to	O
+emerge	O
+less	O
+drug	O
+resistance	O
+.	O
+In	O
+summary	O
+,	O
+the	O
+structure	B-evidence
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+is	O
+featured	O
+with	O
+a	O
+disordered	B-protein_state
+TRD	B-structure_element
+and	O
+a	O
+key	O
+residue	O
+P41that	B-residue_name_number
+located	O
+in	O
+the	O
+putative	O
+DNA	B-site
+binding	I-site
+region	I-site
+that	O
+may	O
+associate	O
+with	O
+the	O
+wider	O
+substrate	O
+specificity	O
+.	O
+Residues	O
+D29	B-residue_name_number
+and	O
+E216	B-residue_name_number
+were	O
+identified	O
+to	O
+play	O
+a	O
+crucial	O
+role	O
+in	O
+cofactor	O
+binding	O
+.	O
+As	O
+the	O
+first	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+N6	B-protein_type
+-	I-protein_type
+adenine	I-protein_type
+MTase	I-protein_type
+in	O
+H	B-species
+.	I-species
+pylori	I-species
+,	O
+this	O
+model	O
+may	O
+shed	O
+light	O
+on	O
+design	O
+of	O
+new	O
+antibiotics	O
+to	O
+interfere	O
+the	O
+growth	O
+and	O
+pathogenesis	O
+of	O
+H	B-species
+.	I-species
+pylori	I-species
+in	O
+human	B-species
+.	O

annotation_IOB/PMC5603727.tsv ADDED Viewed

	@@ -0,0 +1,8414 @@

+Roquin	B-protein
+recognizes	O
+a	O
+non	O
+-	O
+canonical	O
+hexaloop	B-structure_element
+structure	O
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+Ox40	B-protein
+The	O
+RNA	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+protein	I-protein_type
+Roquin	B-protein
+is	O
+required	O
+to	O
+prevent	O
+autoimmunity	O
+.	O
+Roquin	B-protein
+controls	O
+T	O
+-	O
+helper	O
+cell	O
+activation	O
+and	O
+differentiation	O
+by	O
+limiting	O
+the	O
+induced	O
+expression	O
+of	O
+costimulatory	B-protein_type
+receptors	I-protein_type
+such	O
+as	O
+tumor	B-protein
+necrosis	I-protein
+factor	I-protein
+receptor	I-protein
+superfamily	I-protein
+4	I-protein
+(	O
+Tnfrs4	B-protein
+or	O
+Ox40	B-protein
+).	O
+A	O
+constitutive	B-structure_element
+decay	I-structure_element
+element	I-structure_element
+(	O
+CDE	B-structure_element
+)	O
+with	O
+a	O
+characteristic	O
+triloop	B-structure_element
+hairpin	I-structure_element
+was	O
+previously	O
+shown	O
+to	O
+be	O
+recognized	O
+by	O
+Roquin	B-protein
+.	O
+Here	O
+we	O
+use	O
+SELEX	B-experimental_method
+assays	I-experimental_method
+to	O
+identify	O
+a	O
+novel	O
+U	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+hexaloop	I-structure_element
+motif	I-structure_element
+,	O
+representing	O
+an	O
+alternative	B-structure_element
+decay	I-structure_element
+element	I-structure_element
+(	O
+ADE	B-structure_element
+).	O
+Crystal	B-evidence
+structures	I-evidence
+and	O
+NMR	B-experimental_method
+data	O
+show	O
+that	O
+the	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+ROQ	B-structure_element
+domain	O
+recognizes	O
+hexaloops	B-structure_element
+in	O
+the	O
+SELEX	B-experimental_method
+-	O
+derived	O
+ADE	B-structure_element
+and	O
+in	O
+an	O
+ADE	B-structure_element
+-	O
+like	O
+variant	O
+present	O
+in	O
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+with	O
+identical	O
+binding	O
+modes	O
+.	O
+In	O
+cells	O
+,	O
+ADE	B-structure_element
+-	O
+like	O
+and	O
+CDE	B-structure_element
+-	O
+like	O
+motifs	O
+cooperate	O
+in	O
+the	O
+repression	O
+of	O
+Ox40	B-protein
+by	O
+Roquin	B-protein
+.	O
+Our	O
+data	O
+reveal	O
+an	O
+unexpected	O
+recognition	O
+of	O
+hexaloop	B-structure_element
+cis	I-structure_element
+elements	I-structure_element
+for	O
+the	O
+posttranscriptional	O
+regulation	O
+of	O
+target	O
+messenger	B-chemical
+RNAs	I-chemical
+by	O
+Roquin	B-protein
+.	O
+Roquin	B-protein
+is	O
+an	O
+RNA	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+protein	I-protein_type
+that	O
+prevents	O
+autoimmunity	O
+by	O
+limiting	O
+expression	O
+of	O
+receptors	O
+such	O
+as	O
+Ox40	B-protein
+.	O
+Here	O
+,	O
+the	O
+authors	O
+identify	O
+an	O
+RNA	B-chemical
+structure	B-evidence
+that	O
+they	O
+describe	O
+as	O
+an	O
+alternative	B-structure_element
+decay	I-structure_element
+element	I-structure_element
+,	O
+and	O
+they	O
+characterise	O
+its	O
+interaction	O
+with	O
+Roquin	B-protein
+using	O
+structural	B-experimental_method
+and	I-experimental_method
+biochemical	I-experimental_method
+techniques	I-experimental_method
+.	O
+The	O
+Roquin	B-protein
+protein	O
+is	O
+essential	O
+in	O
+T	O
+cells	O
+for	O
+the	O
+prevention	O
+of	O
+autoimmune	O
+disease	O
+.	O
+This	O
+is	O
+evident	O
+from	O
+the	O
+so	O
+-	O
+called	O
+sanroque	O
+mutation	O
+in	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+,	O
+a	O
+single	O
+amino	O
+acid	O
+exchange	O
+from	O
+Met199	B-residue_name_number
+to	O
+Arg	B-residue_name
+that	O
+causes	O
+the	O
+development	O
+of	O
+systemic	O
+lupus	O
+erythematosus	O
+-	O
+like	O
+symptoms	O
+in	O
+homozygous	O
+mice	B-taxonomy_domain
+.	O
+The	O
+Rc3h1	B-gene
+and	O
+Rc3h2	B-gene
+genes	O
+,	O
+encoding	O
+for	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+and	O
+Roquin	B-protein
+-	I-protein
+2	I-protein
+proteins	O
+in	O
+vertebrates	B-taxonomy_domain
+,	O
+respectively	O
+,	O
+have	O
+both	O
+been	O
+shown	O
+to	O
+be	O
+essential	O
+for	O
+the	O
+survival	O
+of	O
+mice	B-taxonomy_domain
+,	O
+but	O
+apparently	O
+serve	O
+redundant	O
+functions	O
+in	O
+T	O
+cells	O
+.	O
+Consistently	O
+,	O
+CD4	O
++	O
+and	O
+CD8	O
++	O
+T	O
+cells	O
+with	O
+the	O
+combined	O
+deletion	B-experimental_method
+of	I-experimental_method
+Roquin	B-protein
+-	O
+encoding	O
+genes	O
+are	O
+spontaneously	O
+activated	O
+and	O
+CD4	O
++	O
+T	O
+-	O
+helper	O
+cells	O
+preferentially	O
+differentiate	O
+into	O
+the	O
+Th1	O
+,	O
+Tfh	O
+or	O
+Th17	O
+subsets	O
+.	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+was	O
+shown	O
+to	O
+negatively	O
+regulate	O
+expression	O
+of	O
+transcripts	O
+encoding	O
+for	O
+co	B-protein_type
+-	I-protein_type
+stimulatory	I-protein_type
+receptors	I-protein_type
+such	O
+as	O
+Icos	B-protein
+,	O
+Ox40	B-protein
+and	O
+CTLA	B-protein
+-	I-protein
+4	I-protein
+,	O
+for	O
+cytokines	B-protein_type
+such	O
+as	O
+interleukin	B-protein
+(	I-protein
+IL	I-protein
+)-	I-protein
+6	I-protein
+and	O
+tumour	B-protein
+necrosis	I-protein
+factor	I-protein
+or	O
+for	O
+transcription	B-protein_type
+factors	I-protein_type
+such	O
+as	O
+IRF4	B-protein
+,	O
+IκBNS	B-protein
+and	O
+IκBζ	B-protein
+(	O
+refs	O
+).	O
+We	O
+have	O
+recently	O
+reported	O
+structural	B-evidence
+and	I-evidence
+functional	I-evidence
+data	I-evidence
+of	O
+the	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+ROQ	B-structure_element
+domain	O
+bound	B-protein_state
+to	I-protein_state
+a	O
+canonical	O
+constitutive	B-structure_element
+decay	I-structure_element
+element	I-structure_element
+(	O
+CDE	B-structure_element
+),	O
+a	O
+short	B-structure_element
+stem	I-structure_element
+loop	I-structure_element
+(	O
+SL	B-structure_element
+)	O
+that	O
+acts	O
+as	O
+a	O
+cis	O
+-	O
+regulatory	O
+RNA	B-chemical
+element	O
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+untranslated	I-structure_element
+regions	I-structure_element
+(	O
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+)	O
+of	O
+target	O
+genes	O
+such	O
+as	O
+Tnf	B-protein
+(	O
+ref	O
+).	O
+The	O
+ROQ	B-structure_element
+domain	O
+adopts	O
+an	O
+extended	B-structure_element
+winged	I-structure_element
+helix	I-structure_element
+fold	I-structure_element
+that	O
+engages	O
+predominantly	O
+non	O
+-	O
+sequence	O
+-	O
+specific	O
+protein	O
+–	O
+RNA	B-chemical
+contacts	O
+and	O
+mainly	O
+recognizes	O
+the	O
+shape	O
+of	O
+the	O
+canonical	O
+Tnf	B-protein
+CDE	B-structure_element
+RNA	B-chemical
+.	O
+The	O
+structural	B-evidence
+data	I-evidence
+and	O
+mutational	B-experimental_method
+analysis	I-experimental_method
+indicated	O
+that	O
+a	O
+broader	O
+,	O
+extended	O
+range	O
+of	O
+sequence	O
+variations	O
+in	O
+both	O
+the	O
+loop	B-structure_element
+and	O
+stem	B-structure_element
+of	O
+the	O
+CDE	B-structure_element
+element	O
+is	O
+recognized	O
+and	O
+regulated	O
+by	O
+Roquin	B-protein
+.	O
+At	O
+the	O
+same	O
+time	O
+,	O
+Tan	O
+et	O
+al	O
+.	O
+described	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+and	O
+supporting	O
+functional	O
+data	O
+of	O
+a	O
+similar	O
+interaction	O
+with	O
+a	O
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+,	O
+and	O
+reported	O
+a	O
+second	B-site
+binding	I-site
+site	I-site
+for	O
+a	O
+double	B-chemical
+-	I-chemical
+stranded	I-chemical
+RNA	I-chemical
+(	O
+dsRNA	B-chemical
+)	O
+within	O
+an	O
+extended	B-protein_state
+ROQ	B-structure_element
+domain	O
+.	O
+The	O
+structural	O
+basis	O
+for	O
+CDE	B-structure_element
+recognition	O
+by	O
+the	O
+Roquin	B-protein
+-	I-protein
+2	I-protein
+ROQ	B-structure_element
+domain	O
+has	O
+also	O
+been	O
+recently	O
+reported	O
+.	O
+We	O
+found	O
+that	O
+the	O
+posttranscriptional	O
+activity	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+and	O
+Roquin	B-protein
+-	I-protein
+2	I-protein
+is	O
+regulated	O
+through	O
+cleavage	O
+by	O
+the	O
+paracaspase	B-protein_type
+MALT1	B-protein
+(	O
+refs	O
+).	O
+Enhanced	O
+MALT1	B-protein
+-	O
+dependent	O
+cleavage	O
+and	O
+inactivation	O
+of	O
+Roquin	B-protein
+,	O
+and	O
+thus	O
+less	O
+effective	O
+repression	O
+of	O
+target	O
+genes	O
+,	O
+result	O
+from	O
+increased	O
+strength	O
+of	O
+antigen	O
+recognition	O
+in	O
+T	O
+cells	O
+.	O
+These	O
+findings	O
+suggest	O
+that	O
+dependent	O
+on	O
+the	O
+strength	O
+of	O
+cognate	O
+antigen	O
+recognition	O
+differential	O
+gene	O
+expression	O
+and	O
+cell	O
+fate	O
+decisions	O
+can	O
+be	O
+established	O
+in	O
+naive	O
+T	O
+cells	O
+by	O
+a	O
+graded	O
+cleavage	O
+and	O
+inactivation	O
+of	O
+Roquin	B-protein
+.	O
+In	O
+addition	O
+to	O
+this	O
+mechanism	O
+,	O
+the	O
+composition	O
+and	O
+binding	B-evidence
+affinity	I-evidence
+of	O
+cis	O
+-	O
+regulatory	O
+SL	B-structure_element
+elements	O
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+of	O
+target	O
+mRNAs	B-chemical
+may	O
+determine	O
+the	O
+sensitivity	O
+to	O
+repression	O
+by	O
+the	O
+trans	O
+-	O
+acting	O
+factor	O
+Roquin	B-protein
+.	O
+Defining	O
+the	O
+SL	B-structure_element
+RNA	B-chemical
+structures	O
+that	O
+are	O
+recognized	O
+by	O
+Roquin	B-protein
+is	O
+therefore	O
+essential	O
+for	O
+our	O
+understanding	O
+of	O
+posttranscriptional	O
+gene	O
+regulation	O
+by	O
+Roquin	B-protein
+and	O
+its	O
+involvement	O
+in	O
+T	O
+-	O
+cell	O
+biology	O
+and	O
+T	O
+-	O
+cell	O
+-	O
+driven	O
+pathology	O
+.	O
+Here	O
+we	O
+present	O
+structural	O
+and	O
+functional	O
+evidence	O
+for	O
+a	O
+greatly	O
+expanded	O
+repertoire	O
+of	O
+RNA	B-chemical
+elements	O
+that	O
+are	O
+regulated	O
+by	O
+Roquin	B-protein
+as	O
+demonstrated	O
+with	O
+a	O
+novel	O
+U	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+hexaloop	I-structure_element
+SL	B-structure_element
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+Ox40	B-protein
+bound	B-protein_state
+to	I-protein_state
+the	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+ROQ	B-structure_element
+domain	O
+.	O
+We	O
+find	O
+an	O
+additive	O
+regulation	O
+of	O
+Ox40	B-protein
+gene	O
+expression	O
+based	O
+on	O
+both	O
+its	O
+CDE	B-structure_element
+-	O
+like	O
+and	O
+hexaloop	B-structure_element
+SL	B-structure_element
+RNAs	B-chemical
+that	O
+we	O
+identified	O
+using	O
+Systematic	B-experimental_method
+Evolution	I-experimental_method
+of	I-experimental_method
+Ligands	I-experimental_method
+by	I-experimental_method
+Exponential	I-experimental_method
+Enrichment	I-experimental_method
+(	O
+SELEX	B-experimental_method
+)	O
+experiments	O
+.	O
+Our	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallographic	I-experimental_method
+,	O
+NMR	B-experimental_method
+,	O
+biochemical	B-evidence
+and	I-evidence
+functional	I-evidence
+data	I-evidence
+combined	O
+with	O
+mutational	B-experimental_method
+analysis	I-experimental_method
+demonstrate	O
+that	O
+both	O
+triloop	B-structure_element
+and	O
+hexaloop	B-structure_element
+SL	B-structure_element
+RNAs	B-chemical
+contribute	O
+to	O
+the	O
+functional	O
+activity	O
+of	O
+Roquin	B-protein
+in	O
+T	O
+cells	O
+.	O
+SELEX	B-experimental_method
+identifies	O
+novel	O
+RNA	B-chemical
+ligands	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+We	O
+set	O
+out	O
+to	O
+identify	O
+Roquin	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+RNA	B-chemical
+motifs	O
+in	O
+an	O
+unbiased	O
+manner	O
+by	O
+performing	O
+SELEX	B-experimental_method
+experiments	O
+.	O
+A	O
+biotinylated	B-protein_state
+amino	O
+-	O
+terminal	O
+protein	O
+fragment	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+(	O
+residues	O
+2	B-residue_range
+–	I-residue_range
+440	I-residue_range
+)	O
+was	O
+used	O
+to	O
+enrich	O
+RNAs	B-chemical
+from	O
+a	O
+library	O
+containing	O
+47	O
+random	O
+nucleotides	O
+over	O
+three	O
+sequential	O
+selection	O
+rounds	O
+.	O
+Next	B-experimental_method
+-	I-experimental_method
+generation	I-experimental_method
+sequencing	I-experimental_method
+(	O
+NGS	B-experimental_method
+)	O
+of	O
+the	O
+RNA	B-chemical
+before	O
+and	O
+after	O
+each	O
+selection	O
+round	O
+revealed	O
+that	O
+the	O
+starting	O
+pool	O
+represented	O
+about	O
+99	O
+.	O
+6	O
+%	O
+unique	O
+reads	O
+in	O
+∼	O
+4	O
+.	O
+2	O
+×	O
+106	O
+sequences	O
+.	O
+Bioinformatic	B-experimental_method
+analysis	I-experimental_method
+of	O
+NGS	B-experimental_method
+data	O
+sets	O
+derived	O
+from	O
+the	O
+starting	O
+pool	O
+and	O
+enriched	O
+selection	O
+rounds	O
+revealed	O
+that	O
+the	O
+complexity	O
+was	O
+reduced	O
+to	O
+78	O
+.	O
+6	O
+%	O
+unique	O
+reads	O
+in	O
+3	O
+.	O
+7	O
+×	O
+106	O
+sequences	O
+that	O
+were	O
+analysed	O
+after	O
+3	O
+rounds	O
+of	O
+selection	O
+and	O
+enrichment	O
+.	O
+For	O
+NGS	B-experimental_method
+data	O
+analysis	O
+,	O
+the	O
+COMPAS	O
+software	O
+(	O
+AptaIT	O
+,	O
+Munich	O
+,	O
+Germany	O
+)	O
+was	O
+applied	O
+.	O
+Enriched	O
+sequences	B-experimental_method
+were	I-experimental_method
+clustered	I-experimental_method
+into	O
+so	O
+-	O
+called	O
+patterns	O
+with	O
+highly	O
+homologous	O
+sequences	O
+.	O
+Based	O
+on	O
+this	O
+so	O
+-	O
+called	O
+co	B-experimental_method
+-	I-experimental_method
+occurrence	I-experimental_method
+approach	I-experimental_method
+,	O
+patterns	O
+on	O
+the	O
+basis	O
+of	O
+frequent	O
+motifs	O
+were	O
+generated	O
+and	O
+were	O
+searched	O
+for	O
+prominent	O
+hexamer	O
+sequences	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1a	O
+).	O
+We	O
+identified	O
+5	B-chemical
+′-	I-chemical
+CGTTTT	I-chemical
+-	I-chemical
+3	I-chemical
+′,	I-chemical
+5	B-chemical
+′-	I-chemical
+GCGTTT	I-chemical
+-	I-chemical
+3	I-chemical
+′,	I-chemical
+5	B-chemical
+′-	I-chemical
+TGCGTT	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+and	O
+5	B-chemical
+′-	I-chemical
+GTTTTA	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+motifs	O
+that	O
+were	O
+also	O
+reconfirmed	O
+in	O
+an	O
+independent	O
+experiment	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1a	O
+)	O
+and	O
+are	O
+located	O
+within	O
+highly	O
+similar	O
+sequences	O
+(	O
+Fig	O
+.	O
+1a	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1b	O
+).	O
+Consistent	O
+with	O
+previous	O
+findings	O
+showing	O
+that	O
+the	O
+sanroque	B-mutant
+mutation	I-mutant
+does	O
+not	O
+impair	O
+RNA	B-chemical
+binding	O
+of	O
+Roquin	B-protein
+,	O
+we	O
+found	O
+similarly	O
+enriched	O
+sequences	O
+in	O
+SELEX	B-experimental_method
+approaches	O
+using	O
+a	O
+corresponding	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+fragment	O
+harbouring	O
+the	O
+M199R	B-mutant
+mutation	O
+(	O
+Fig	O
+.	O
+1a	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1b	O
+).	O
+Notably	O
+,	O
+our	O
+SELEX	B-experimental_method
+approach	O
+did	O
+not	O
+reveal	O
+the	O
+previously	O
+identified	O
+CDE	B-structure_element
+sequence	O
+.	O
+We	O
+assume	O
+that	O
+the	O
+region	O
+of	O
+sequence	O
+identity	O
+in	O
+the	O
+CDE	B-structure_element
+is	O
+too	O
+short	O
+for	O
+our	O
+sequence	B-experimental_method
+clustering	I-experimental_method
+algorithm	I-experimental_method
+.	O
+Evaluation	O
+of	O
+the	O
+structural	O
+context	O
+for	O
+the	O
+SELEX	B-experimental_method
+-	O
+derived	O
+motif	O
+suggested	O
+a	O
+putative	O
+SL	B-structure_element
+formation	O
+with	O
+six	O
+unpaired	O
+nucleotides	O
+in	O
+a	O
+loop	B-structure_element
+followed	O
+by	O
+a	O
+5	O
+–	O
+8	O
+nt	O
+stem	B-structure_element
+,	O
+with	O
+one	O
+base	O
+in	O
+the	O
+stem	B-structure_element
+not	O
+being	O
+paired	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1c	O
+).	O
+Searching	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+of	O
+known	O
+Roquin	B-protein
+targets	O
+with	O
+the	O
+consensus	O
+5	B-chemical
+′-	I-chemical
+TGCGTTTTAGGA	I-chemical
+-	I-chemical
+3	I-chemical
+′,	I-chemical
+obtained	O
+by	O
+Motif	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+sequence	I-experimental_method
+analysis	I-experimental_method
+(	O
+MEME	B-experimental_method
+),	O
+revealed	O
+a	O
+homologous	O
+sequence	O
+with	O
+the	O
+potential	O
+to	O
+form	O
+a	O
+hexaloop	B-structure_element
+structure	O
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+Ox40	B-protein
+(	O
+Fig	O
+.	O
+1b	O
+).	O
+Importantly	O
+,	O
+this	O
+motif	O
+is	O
+present	O
+across	O
+species	O
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+of	O
+respective	O
+mRNAs	B-chemical
+and	O
+showed	O
+highest	O
+conservation	O
+in	O
+the	O
+loop	B-structure_element
+and	O
+the	O
+upper	O
+stem	B-structure_element
+sequences	O
+with	O
+a	O
+drop	O
+of	O
+conservation	O
+towards	O
+the	O
+boundaries	O
+of	O
+the	O
+motif	O
+(	O
+Fig	O
+.	O
+1c	O
+,	O
+d	O
+).	O
+The	O
+predicted	O
+SL	B-structure_element
+for	O
+the	O
+consensus	O
+SELEX	B-experimental_method
+-	O
+derived	O
+motif	O
+(	O
+from	O
+here	O
+on	O
+referred	O
+to	O
+as	O
+alternative	B-structure_element
+decay	I-structure_element
+element	I-structure_element
+SL	B-structure_element
+,	O
+ADE	B-structure_element
+SL	B-structure_element
+),	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+,	O
+is	O
+positioned	O
+5	O
+′	O
+to	O
+another	O
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+Ox40	B-protein
+mRNA	B-chemical
+.	O
+This	O
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+differs	O
+in	O
+the	O
+sequence	O
+of	O
+the	O
+upper	O
+stem	O
+from	O
+the	O
+canonical	O
+CDE	B-structure_element
+from	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+Tnf	B-protein
+mRNA	B-chemical
+(	O
+CDE	B-structure_element
+SL	B-structure_element
+)	O
+(	O
+Fig	O
+.	O
+1d	O
+).	O
+NMR	B-experimental_method
+analysis	O
+of	O
+Roquin	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+SL	B-structure_element
+RNAs	B-chemical
+We	O
+used	O
+NMR	B-experimental_method
+to	O
+analyse	O
+the	O
+secondary	O
+structure	O
+of	O
+Roquin	B-structure_element
+-	I-structure_element
+1	I-structure_element
+-	I-structure_element
+binding	I-structure_element
+motifs	I-structure_element
+derived	O
+from	O
+SELEX	B-experimental_method
+.	O
+Imino	B-experimental_method
+one	I-experimental_method
+-	I-experimental_method
+and	I-experimental_method
+two	I-experimental_method
+-	I-experimental_method
+dimensional	I-experimental_method
+nuclear	I-experimental_method
+Overhauser	I-experimental_method
+enhancement	I-experimental_method
+spectroscopy	I-experimental_method
+(	O
+NOESY	B-experimental_method
+)	O
+NMR	B-experimental_method
+spectra	B-evidence
+of	O
+the	O
+free	B-protein_state
+RNA	B-chemical
+and	O
+when	O
+bound	B-protein_state
+to	I-protein_state
+the	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+ROQ	B-structure_element
+domain	O
+were	O
+recorded	O
+for	O
+the	O
+ADE	B-structure_element
+SL	B-structure_element
+,	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+Ox40	B-protein
+and	O
+the	O
+previously	O
+identified	O
+Ox40	B-protein
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+(	O
+Fig	O
+.	O
+2	O
+).	O
+The	O
+NMR	B-experimental_method
+data	O
+of	O
+the	O
+free	B-protein_state
+RNAs	B-chemical
+show	O
+that	O
+almost	O
+all	O
+predicted	O
+base	O
+pairs	O
+in	O
+the	O
+stem	B-structure_element
+regions	I-structure_element
+of	O
+the	O
+hexa	B-structure_element
+-	I-structure_element
+and	I-structure_element
+triloop	I-structure_element
+SL	B-structure_element
+including	O
+the	O
+closing	O
+base	O
+pairs	O
+are	O
+formed	O
+in	O
+all	O
+three	O
+RNAs	B-chemical
+.	O
+Notably	O
+,	O
+we	O
+also	O
+found	O
+an	O
+unambiguous	O
+imino	O
+proton	O
+signal	O
+for	O
+G15	B-residue_name_number
+,	O
+but	O
+not	O
+G6	B-residue_name_number
+,	O
+in	O
+the	O
+ADE	B-structure_element
+SL	B-structure_element
+,	O
+indicating	O
+a	O
+non	B-bond_interaction
+-	I-bond_interaction
+Watson	I-bond_interaction
+–	I-bond_interaction
+Crick	I-bond_interaction
+G	I-bond_interaction
+–	I-bond_interaction
+G	I-bond_interaction
+base	I-bond_interaction
+pair	I-bond_interaction
+at	O
+this	O
+position	O
+(	O
+Fig	O
+.	O
+2a	O
+).	O
+Significant	O
+chemical	B-evidence
+shift	I-evidence
+perturbations	I-evidence
+(	O
+CSPs	B-evidence
+)	O
+are	O
+observed	O
+for	O
+imino	O
+proton	O
+signals	O
+on	O
+binding	O
+to	O
+the	O
+ROQ	B-structure_element
+domain	O
+,	O
+demonstrating	O
+that	O
+formation	O
+of	O
+protein	O
+–	O
+RNA	B-chemical
+complexes	O
+involves	O
+contacts	O
+of	O
+the	O
+ROQ	B-structure_element
+domain	O
+to	O
+the	O
+stem	B-structure_element
+region	I-structure_element
+of	O
+the	O
+RNA	B-chemical
+ligands	O
+(	O
+Fig	O
+.	O
+2	O
+,	O
+bases	O
+coloured	O
+red	O
+).	O
+No	O
+imino	O
+correlations	O
+are	O
+observed	O
+for	O
+the	O
+predicted	O
+Watson	B-bond_interaction
+–	I-bond_interaction
+Crick	I-bond_interaction
+base	I-bond_interaction
+pairs	I-bond_interaction
+at	O
+the	O
+bottom	O
+of	O
+the	O
+ADE	B-structure_element
+SL	B-structure_element
+and	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+,	O
+as	O
+well	O
+as	O
+for	O
+the	O
+A	B-residue_name
+–	O
+U	B-residue_name
+base	O
+pair	O
+flanking	O
+the	O
+bulge	B-structure_element
+in	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNA	B-chemical
+(	O
+Fig	O
+.	O
+2a	O
+,	O
+b	O
+),	O
+suggesting	O
+that	O
+these	O
+base	O
+pairs	O
+are	O
+dynamic	O
+.	O
+In	O
+contrast	O
+,	O
+all	O
+expected	O
+base	O
+pairs	O
+are	O
+observed	O
+for	O
+the	O
+Ox40	B-protein
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNA	B-chemical
+(	O
+Fig	O
+.	O
+2c	O
+;	O
+see	O
+also	O
+Supplementary	O
+Notes	O
+).	O
+Structures	B-evidence
+of	O
+ROQ	B-structure_element
+bound	B-protein_state
+to	I-protein_state
+ADE	B-structure_element
+SL	B-structure_element
+RNAs	B-chemical
+To	O
+elucidate	O
+how	O
+Roquin	B-protein
+can	O
+recognize	O
+the	O
+novel	O
+SL	B-structure_element
+elements	O
+identified	O
+in	O
+the	O
+SELEX	B-experimental_method
+approach	O
+,	O
+we	O
+solved	B-experimental_method
+crystal	B-evidence
+structures	I-evidence
+of	O
+the	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+ROQ	B-structure_element
+domain	O
+bound	B-protein_state
+to	I-protein_state
+these	O
+non	O
+-	O
+canonical	O
+RNA	B-chemical
+elements	O
+.	O
+The	O
+structures	B-evidence
+of	O
+ROQ	B-structure_element
+bound	B-protein_state
+to	I-protein_state
+the	O
+20	O
+-	O
+mer	O
+ADE	B-structure_element
+SL	B-structure_element
+(	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+)	O
+and	O
+to	O
+the	O
+22	O
+-	O
+mer	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+(	O
+Fig	O
+.	O
+3a	O
+)	O
+were	O
+refined	O
+to	O
+a	O
+resolution	O
+of	O
+3	O
+.	O
+0	O
+and	O
+2	O
+.	O
+2	O
+Å	O
+,	O
+respectively	O
+.	O
+In	O
+both	O
+structures	B-evidence
+the	O
+RNA	B-chemical
+adopts	O
+an	O
+SL	B-structure_element
+fold	O
+,	O
+where	O
+the	O
+hexaloop	B-structure_element
+is	O
+located	O
+in	O
+the	O
+vicinity	O
+of	O
+the	O
+carboxy	O
+-	O
+terminal	O
+end	O
+of	O
+ROQ	B-structure_element
+helix	B-structure_element
+α4	B-structure_element
+and	O
+the	O
+N	O
+-	O
+terminal	O
+part	O
+of	O
+β3	B-structure_element
+(	O
+Fig	O
+.	O
+3a	O
+,	O
+b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+,	O
+b	O
+).	O
+The	O
+dsRNA	B-chemical
+stem	B-structure_element
+is	O
+recognized	O
+in	O
+the	O
+same	O
+way	O
+as	O
+previously	O
+reported	O
+for	O
+the	O
+Tnf	B-protein
+CDE	B-structure_element
+SL	B-structure_element
+RNA	B-chemical
+(	O
+Supplementary	O
+Fig	O
+.	O
+2c	O
+–	O
+e	O
+).	O
+As	O
+may	O
+be	O
+expected	O
+,	O
+the	O
+recognition	O
+of	O
+the	O
+hexaloop	B-structure_element
+is	O
+significantly	O
+different	O
+from	O
+the	O
+triloop	B-structure_element
+in	O
+the	O
+CDE	B-structure_element
+RNA	B-chemical
+(	O
+Fig	O
+.	O
+3b	O
+,	O
+c	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2b	O
+).	O
+Interestingly	O
+,	O
+although	O
+the	O
+sequences	O
+of	O
+the	O
+ADE	B-structure_element
+SL	B-structure_element
+and	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+are	O
+different	O
+,	O
+the	O
+overall	O
+structures	B-evidence
+and	O
+protein	O
+–	O
+RNA	B-chemical
+contacts	O
+are	O
+virtually	O
+identical	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+,	O
+d	O
+,	O
+e	O
+).	O
+The	O
+only	O
+differences	O
+are	O
+a	O
+C19	B-residue_name_number
+bulge	B-structure_element
+,	O
+the	O
+non	B-bond_interaction
+-	I-bond_interaction
+Watson	I-bond_interaction
+–	I-bond_interaction
+Crick	I-bond_interaction
+G6	B-residue_name_number
+–	O
+G15	B-residue_name_number
+base	B-bond_interaction
+pair	I-bond_interaction
+and	O
+the	O
+interaction	O
+of	O
+U1	B-residue_name_number
+with	O
+Trp184	B-residue_name_number
+and	O
+Phe194	B-residue_name_number
+in	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNA	B-chemical
+(	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+,	O
+e	O
+–	O
+g	O
+).	O
+Given	O
+their	O
+highly	O
+similar	O
+binding	O
+modes	O
+we	O
+focus	O
+the	O
+following	O
+discussion	O
+on	O
+the	O
+structure	B-evidence
+of	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNA	B-chemical
+,	O
+as	O
+it	O
+naturally	O
+exists	O
+in	O
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+and	O
+was	O
+solved	O
+at	O
+higher	O
+resolution	O
+.	O
+The	O
+overall	O
+orientation	O
+and	O
+recognition	O
+of	O
+the	O
+double	B-structure_element
+-	I-structure_element
+stranded	I-structure_element
+stem	I-structure_element
+in	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+is	O
+similar	O
+to	O
+the	O
+CDE	B-structure_element
+triloop	B-structure_element
+.	O
+Notably	O
+,	O
+the	O
+U	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+hexaloop	I-structure_element
+in	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNA	B-chemical
+binds	O
+to	O
+an	O
+extended	O
+surface	B-site
+on	O
+the	O
+ROQ	B-structure_element
+domain	O
+that	O
+cannot	O
+be	O
+accessed	O
+by	O
+the	O
+CDE	B-structure_element
+triloop	B-structure_element
+(	O
+Fig	O
+.	O
+3b	O
+,	O
+c	O
+)	O
+and	O
+includes	O
+a	O
+few	O
+pyrimidine	O
+-	O
+specific	O
+contacts	O
+.	O
+For	O
+example	O
+,	O
+the	O
+main	O
+chain	O
+atoms	O
+of	O
+Phe255	B-residue_name_number
+form	O
+two	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+with	O
+the	O
+Watson	O
+–	O
+Crick	O
+face	O
+of	O
+the	O
+U11	B-residue_name_number
+base	O
+(	O
+Fig	O
+.	O
+3d	O
+).	O
+Although	O
+in	O
+the	O
+structure	B-evidence
+of	O
+the	O
+Tnf	B-protein
+CDE	B-structure_element
+triloop	B-structure_element
+the	O
+Tyr250	B-residue_name_number
+side	O
+chain	O
+engages	O
+only	O
+one	O
+hydrogen	B-bond_interaction
+bond	I-bond_interaction
+to	O
+the	O
+phosphate	O
+group	O
+of	O
+G12	B-residue_name_number
+(	O
+ref	O
+.),	O
+a	O
+number	O
+of	O
+contacts	O
+are	O
+observed	O
+with	O
+the	O
+hexaloop	B-structure_element
+(	O
+Fig	O
+.	O
+3d	O
+–	O
+f	O
+):	O
+the	O
+side	O
+chain	O
+hydroxyl	O
+of	O
+Tyr250	B-residue_name_number
+contacts	O
+the	O
+phosphate	O
+group	O
+of	O
+U11	B-residue_name_number
+,	O
+while	O
+the	O
+aromatic	O
+ring	O
+is	O
+positioned	O
+by	O
+parallel	O
+and	O
+orthogonal	O
+stacking	B-bond_interaction
+interactions	I-bond_interaction
+with	O
+the	O
+U10	B-residue_name_number
+and	O
+U11	B-residue_name_number
+bases	O
+,	O
+on	O
+either	O
+side	O
+,	O
+respectively	O
+(	O
+Fig	O
+.	O
+3e	O
+).	O
+In	O
+addition	O
+,	O
+the	O
+Tyr250	B-residue_name_number
+main	O
+-	O
+chain	O
+carbonyl	O
+interacts	O
+with	O
+U13	B-residue_name_number
+imino	O
+proton	O
+(	O
+Fig	O
+.	O
+3d	O
+,	O
+e	O
+).	O
+Val257	B-residue_name_number
+and	O
+Lys259	B-residue_name_number
+in	O
+strand	B-structure_element
+β3	B-structure_element
+are	O
+too	O
+far	O
+to	O
+contact	O
+the	O
+UGU	B-structure_element
+triloop	B-structure_element
+in	O
+the	O
+Tnf	B-protein
+CDE	B-structure_element
+RNA	B-chemical
+,	O
+but	O
+mediate	O
+a	O
+number	O
+of	O
+contacts	O
+with	O
+the	O
+longer	O
+hexaloop	B-structure_element
+.	O
+The	O
+side	O
+chain	O
+of	O
+Lys259	B-residue_name_number
+forms	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+with	O
+the	O
+phosphate	O
+groups	O
+of	O
+U10	B-residue_name_number
+and	O
+U11	B-residue_name_number
+(	O
+Fig	O
+.	O
+3e	O
+,	O
+f	O
+)	O
+and	O
+the	O
+hydrophobic	O
+side	O
+chain	O
+of	O
+Val257	B-residue_name_number
+stacks	B-bond_interaction
+with	O
+the	O
+U11	B-residue_name_number
+base	O
+(	O
+Fig	O
+.	O
+3d	O
+,	O
+f	O
+).	O
+The	O
+RNA	B-chemical
+stem	B-structure_element
+is	O
+closed	O
+by	O
+a	O
+Watson	B-bond_interaction
+–	I-bond_interaction
+Crick	I-bond_interaction
+base	I-bond_interaction
+pair	I-bond_interaction
+(	O
+C8	B-residue_name_number
+–	O
+G15	B-residue_name_number
+in	O
+the	O
+hexaloop	B-structure_element
+SL	B-structure_element
+RNA	B-chemical
+).	O
+Interestingly	O
+,	O
+the	O
+G9	B-residue_name_number
+base	O
+stacks	B-bond_interaction
+on	O
+top	O
+of	O
+this	O
+closing	O
+base	O
+pair	O
+and	O
+takes	O
+a	O
+position	O
+that	O
+is	O
+very	O
+similar	O
+to	O
+the	O
+purine	O
+base	O
+of	O
+G12	B-residue_name_number
+in	O
+the	O
+CDE	B-structure_element
+triloop	B-structure_element
+(	O
+Fig	O
+.	O
+3b	O
+,	O
+c	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2b	O
+).	O
+The	O
+G9	B-residue_name_number
+base	O
+does	O
+not	O
+form	O
+a	O
+base	O
+pair	O
+with	O
+A14	B-residue_name_number
+but	O
+rather	O
+the	O
+A14	B-residue_name_number
+base	O
+packs	O
+into	O
+the	O
+minor	B-site
+groove	I-site
+of	O
+the	O
+RNA	B-chemical
+duplex	O
+.	O
+This	O
+arrangement	O
+provides	O
+an	O
+extended	O
+stacking	B-bond_interaction
+interaction	I-bond_interaction
+of	O
+G9	B-residue_name_number
+,	O
+U10	B-residue_name_number
+and	O
+Tyr250	B-residue_name_number
+in	O
+the	O
+ROQ	B-structure_element
+domain	O
+at	O
+the	O
+5	O
+′-	O
+side	O
+of	O
+the	O
+RNA	B-chemical
+stem	B-structure_element
+(	O
+Fig	O
+.	O
+3e	O
+).	O
+The	O
+U11	B-residue_name_number
+and	O
+U13	B-residue_name_number
+bases	O
+stack	B-bond_interaction
+with	O
+each	O
+other	O
+in	O
+the	O
+vicinity	O
+of	O
+the	O
+ROQ	B-structure_element
+domain	O
+wing	B-structure_element
+(	O
+Fig	O
+.	O
+3b	O
+,	O
+d	O
+,	O
+f	O
+).	O
+This	O
+is	O
+possible	O
+by	O
+exposing	O
+the	O
+base	O
+C12	B-residue_name_number
+of	O
+the	O
+Ox	B-protein
+-	I-protein
+40	I-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+towards	O
+the	O
+solvent	O
+,	O
+which	O
+accordingly	O
+does	O
+not	O
+show	O
+any	O
+contacts	O
+to	O
+the	O
+protein	O
+.	O
+In	O
+summary	O
+,	O
+similar	O
+to	O
+the	O
+CDE	B-structure_element
+SL	B-structure_element
+,	O
+both	O
+the	O
+ADE	B-structure_element
+SL	B-structure_element
+and	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+are	O
+recognized	O
+mainly	O
+by	O
+non	O
+-	O
+sequence	O
+-	O
+specific	O
+contacts	O
+.	O
+However	O
+,	O
+these	O
+involve	O
+an	O
+extended	O
+binding	O
+surface	O
+on	O
+the	O
+ROQ	B-structure_element
+domain	O
+with	O
+a	O
+number	O
+of	O
+additional	O
+residues	O
+compared	O
+with	O
+the	O
+triloop	O
+RNA	B-chemical
+.	O
+NMR	B-experimental_method
+analysis	O
+of	O
+ROQ	B-structure_element
+interactions	O
+with	O
+ADE	B-structure_element
+SLs	B-structure_element
+We	O
+next	O
+used	O
+NMR	B-experimental_method
+spectroscopy	I-experimental_method
+to	O
+compare	O
+the	O
+ROQ	B-structure_element
+domain	O
+interaction	O
+of	O
+ADE	B-structure_element
+-	O
+like	O
+and	O
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+in	O
+solution	O
+.	O
+CSPs	B-evidence
+observed	O
+for	O
+amides	O
+in	O
+the	O
+ROQ	B-structure_element
+domain	O
+on	O
+binding	O
+to	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNA	B-chemical
+(	O
+Fig	O
+.	O
+4a	O
+,	O
+b	O
+)	O
+map	O
+to	O
+residues	O
+that	O
+also	O
+mediate	O
+key	O
+interactions	O
+with	O
+CDE	B-structure_element
+SLs	B-structure_element
+,	O
+such	O
+as	O
+Lys220	B-residue_name_number
+,	O
+Lys239	B-residue_name_number
+/	O
+Thr240	B-residue_name_number
+and	O
+Lys259	B-residue_name_number
+/	O
+Arg260	B-residue_name_number
+(	O
+Fig	O
+.	O
+4b	O
+).	O
+This	O
+is	O
+fully	O
+consistent	O
+with	O
+the	O
+interactions	O
+observed	O
+in	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+(	O
+Supplementary	O
+Fig	O
+.	O
+2c	O
+–	O
+e	O
+)	O
+and	O
+indicates	O
+a	O
+similar	O
+binding	B-site
+surface	I-site
+.	O
+However	O
+,	O
+there	O
+are	O
+also	O
+notable	O
+CSP	B-evidence
+differences	I-evidence
+when	O
+comparing	O
+binding	O
+of	O
+the	O
+ROQ	B-structure_element
+domain	O
+to	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+and	O
+to	O
+the	O
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNA	B-chemical
+in	O
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+(	O
+Fig	O
+.	O
+4c	O
+),	O
+or	O
+to	O
+the	O
+Tnf	B-protein
+CDE	B-structure_element
+SL	B-structure_element
+RNA	B-chemical
+(	O
+Supplementary	O
+Fig	O
+.	O
+3	O
+and	O
+Supplementary	O
+Notes	O
+).	O
+For	O
+example	O
+,	O
+Ser253	B-residue_name_number
+is	O
+strongly	O
+affected	O
+only	O
+on	O
+binding	O
+to	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+(	O
+Fig	O
+.	O
+4a	O
+,	O
+b	O
+)	O
+in	O
+line	O
+with	O
+tight	O
+interactions	O
+with	O
+the	O
+hexaloop	B-structure_element
+(	O
+Fig	O
+.	O
+3d	O
+).	O
+On	O
+the	O
+other	O
+hand	O
+,	O
+comparison	O
+of	O
+ROQ	B-structure_element
+domain	O
+binding	O
+with	O
+the	O
+ADE	B-structure_element
+and	O
+with	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+indicates	O
+almost	O
+identical	O
+NMR	B-experimental_method
+spectra	B-evidence
+and	O
+CSPs	B-evidence
+.	O
+This	O
+is	O
+consistent	O
+with	O
+the	O
+very	O
+similar	O
+structural	O
+features	O
+and	O
+mode	O
+of	O
+RNA	B-chemical
+recognition	O
+of	O
+the	O
+ROQ	B-structure_element
+domain	O
+with	O
+these	O
+RNAs	B-chemical
+(	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+,	O
+d	O
+,	O
+e	O
+).	O
+Mutational	B-experimental_method
+analysis	I-experimental_method
+of	O
+the	O
+ROQ	B-structure_element
+-	O
+ADE	B-structure_element
+interaction	O
+To	O
+examine	O
+the	O
+individual	O
+contributions	O
+of	O
+ROQ	B-structure_element
+–	O
+hexaloop	O
+interactions	O
+for	O
+complex	O
+formation	O
+,	O
+we	O
+performed	O
+electrophoretic	B-experimental_method
+mobility	I-experimental_method
+shift	I-experimental_method
+assays	I-experimental_method
+(	O
+EMSAs	B-experimental_method
+)	O
+with	O
+variants	O
+of	O
+the	O
+ROQ	B-structure_element
+domain	O
+and	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+RNA	B-chemical
+(	O
+Fig	O
+.	O
+5a	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+).	O
+Analysis	O
+of	O
+the	O
+interaction	O
+with	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+ROQ	B-structure_element
+revealed	O
+an	O
+apparent	O
+affinity	B-evidence
+in	O
+a	O
+similar	O
+range	O
+as	O
+for	O
+the	O
+Tnf	B-protein
+CDE	B-structure_element
+(	O
+Fig	O
+.	O
+5a	O
+and	O
+)	O
+Table	O
+2	O
+).	O
+We	O
+next	O
+tested	O
+a	O
+set	O
+of	O
+mutants	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+),	O
+which	O
+were	O
+designed	O
+based	O
+on	O
+contacts	O
+observed	O
+in	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+(	O
+Fig	O
+.	O
+3	O
+)	O
+and	O
+the	O
+NMR	B-experimental_method
+CSPs	B-evidence
+(	O
+Fig	O
+.	O
+4a	O
+,	O
+b	O
+).	O
+In	O
+line	O
+with	O
+expectations	O
+from	O
+ROQ	B-complex_assembly
+-	I-complex_assembly
+Tnf	I-complex_assembly
+CDE	I-complex_assembly
+binding	O
+(	O
+see	O
+comparison	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+)	O
+and	O
+based	O
+on	O
+our	O
+structural	B-experimental_method
+analysis	I-experimental_method
+,	O
+the	O
+key	O
+residues	O
+Lys220	B-residue_name_number
+,	O
+Lys239	B-residue_name_number
+,	O
+Lys259	B-residue_name_number
+and	O
+Arg260	B-residue_name_number
+strongly	O
+reduce	O
+or	O
+abolish	O
+binding	O
+after	O
+replacement	B-experimental_method
+by	O
+alanine	B-residue_name
+.	O
+We	O
+also	O
+observe	O
+an	O
+almost	O
+complete	O
+loss	O
+of	O
+binding	O
+in	O
+the	O
+Y250A	B-mutant
+mutant	B-protein_state
+to	O
+the	O
+hexaloop	B-structure_element
+SL	B-structure_element
+RNA	B-chemical
+,	O
+which	O
+had	O
+not	O
+been	O
+seen	O
+for	O
+the	O
+Tnf	B-protein
+CDE	B-structure_element
+previously	O
+(	O
+Fig	O
+.	O
+5a	O
+).	O
+This	O
+underlines	O
+the	O
+central	O
+role	O
+of	O
+Tyr250	B-residue_name_number
+for	O
+stabilization	O
+of	O
+the	O
+hexaloop	B-structure_element
+structure	O
+and	O
+recognition	O
+by	O
+stacking	B-bond_interaction
+interactions	I-bond_interaction
+(	O
+Fig	O
+.	O
+3b	O
+,	O
+e	O
+).	O
+Mutation	B-experimental_method
+of	O
+Ser253	B-residue_name_number
+,	O
+which	O
+shows	O
+large	O
+CSPs	B-evidence
+in	O
+the	O
+NMR	B-experimental_method
+titrations	I-experimental_method
+(	O
+Fig	O
+.	O
+4a	O
+,	O
+b	O
+),	O
+does	O
+not	O
+significantly	O
+impair	O
+complex	O
+formation	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+).	O
+The	O
+large	O
+chemical	B-evidence
+shift	I-evidence
+change	I-evidence
+is	O
+probably	O
+caused	O
+by	O
+ring	O
+current	O
+effects	O
+induced	O
+by	O
+the	O
+close	O
+proximity	O
+of	O
+the	O
+U11	B-residue_name_number
+and	O
+U13	B-residue_name_number
+bases	O
+.	O
+Finally	O
+,	O
+a	O
+mutant	B-protein_state
+in	O
+the	O
+wing	B-structure_element
+of	O
+the	O
+ROQ	B-structure_element
+domain	O
+(	O
+S265Y	B-mutant
+)	O
+does	O
+only	O
+slightly	O
+impair	O
+binding	O
+,	O
+as	O
+has	O
+been	O
+previously	O
+observed	O
+for	O
+the	O
+interaction	O
+with	O
+the	O
+Tnf	B-protein
+CDE	B-structure_element
+(	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+).	O
+This	O
+indicates	O
+that	O
+replacement	B-experimental_method
+by	O
+Tyr	B-residue_name
+does	O
+not	O
+strongly	O
+affect	O
+the	O
+RNA	B-chemical
+interaction	O
+,	O
+and	O
+that	O
+some	O
+conformational	O
+variations	O
+are	O
+tolerated	O
+.	O
+Thus	O
+,	O
+the	O
+mutational	B-experimental_method
+analysis	I-experimental_method
+is	O
+fully	O
+consistent	O
+with	O
+the	O
+recognition	O
+of	O
+the	O
+hexaloop	B-structure_element
+observed	O
+in	O
+our	O
+crystal	B-evidence
+structures	I-evidence
+.	O
+To	O
+prove	O
+the	O
+contribution	O
+of	O
+the	O
+key	O
+residue	O
+Tyr250	B-residue_name_number
+in	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+to	O
+Ox40	B-protein
+mRNA	B-chemical
+recognition	O
+and	O
+regulation	O
+,	O
+we	O
+set	O
+up	O
+a	O
+retroviral	B-experimental_method
+reconstitution	I-experimental_method
+system	I-experimental_method
+in	O
+Roquin	B-protein
+-	O
+deficient	O
+CD4	O
++	O
+T	O
+cells	O
+.	O
+Isolated	O
+CD4	O
++	O
+T	O
+cells	O
+from	O
+Rc3h1	B-gene
+/	O
+2fl	B-gene
+/	O
+fl	B-gene
+;	O
+Cd4	O
+-	O
+Cre	O
+-	O
+ERT2	O
+;	O
+rtTA	O
+mice	B-taxonomy_domain
+harbouring	O
+floxed	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+/	O
+2	B-protein
+encoding	O
+alleles	O
+,	O
+a	O
+tamoxifen	B-chemical
+-	O
+inducible	O
+Cre	O
+recombinase	O
+and	O
+the	O
+reverse	B-protein_type
+tetracycline	I-protein_type
+-	I-protein_type
+controlled	I-protein_type
+transactivator	I-protein_type
+rtTA	B-protein
+were	O
+treated	O
+in	O
+vitro	O
+with	O
+4	B-chemical
+-	I-chemical
+hydroxy	I-chemical
+tamoxifen	I-chemical
+,	O
+to	O
+induce	O
+deletion	O
+.	O
+The	O
+cells	O
+were	O
+then	O
+transduced	O
+with	O
+doxycycline	B-chemical
+-	O
+inducible	O
+retroviral	O
+vectors	O
+to	O
+reconstitute	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+expression	O
+(	O
+Fig	O
+.	O
+5b	O
+).	O
+Depletion	O
+of	O
+Roquin	B-protein
+proteins	O
+on	O
+tamoxifen	B-chemical
+treatment	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5a	O
+)	O
+strongly	O
+increased	O
+surface	O
+expression	O
+of	O
+Ox40	B-protein
+and	O
+Icos	B-protein
+(	O
+Fig	O
+.	O
+5c	O
+).	O
+This	O
+increase	O
+in	O
+surface	O
+expression	O
+of	O
+both	O
+costimulatory	B-protein_type
+receptors	I-protein_type
+was	O
+partially	O
+corrected	O
+by	O
+the	O
+doxycycline	B-chemical
+-	O
+induced	O
+reconstitution	O
+with	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+WT	B-protein_state
+protein	O
+(	O
+Fig	O
+.	O
+5c	O
+left	O
+panels	O
+).	O
+Importantly	O
+,	O
+no	O
+effect	O
+was	O
+observed	O
+on	O
+expression	O
+of	O
+the	O
+Y250A	B-mutant
+mutant	B-protein_state
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+or	O
+the	O
+K220A	B-mutant
+,	O
+K239A	B-mutant
+and	O
+R260	B-mutant
+mutant	B-protein_state
+,	O
+which	O
+is	O
+strongly	O
+impaired	O
+in	O
+CDE	B-structure_element
+SL	B-structure_element
+interactions	O
+(	O
+Fig	O
+.	O
+5c	O
+middle	O
+and	O
+right	O
+panels	O
+).	O
+However	O
+,	O
+it	O
+is	O
+also	O
+possible	O
+that	O
+continuous	O
+overexpression	B-experimental_method
+of	O
+targets	O
+following	O
+Roquin	B-protein
+deletion	O
+induces	O
+a	O
+hyperactivated	O
+state	O
+in	O
+the	O
+T	O
+cells	O
+.	O
+This	O
+hyperactivation	O
+,	O
+compared	O
+with	O
+the	O
+actual	O
+posttranscriptional	O
+derepression	O
+,	O
+may	O
+contribute	O
+even	O
+stronger	O
+to	O
+the	O
+increased	O
+Icos	B-protein
+and	O
+Ox40	B-protein
+expression	O
+levels	O
+.	O
+Hence	O
+,	O
+our	O
+structure	B-experimental_method
+–	I-experimental_method
+function	I-experimental_method
+analyses	I-experimental_method
+conclusively	O
+show	O
+that	O
+the	O
+Y250	B-residue_name_number
+residue	O
+is	O
+essential	O
+for	O
+Roquin	B-protein
+interaction	O
+and	O
+regulation	O
+of	O
+Ox40	B-protein
+,	O
+and	O
+potentially	O
+also	O
+for	O
+other	O
+Roquin	B-protein
+targets	O
+such	O
+as	O
+Icos	B-protein
+.	O
+We	O
+also	O
+investigated	O
+the	O
+role	O
+of	O
+individual	O
+nucleotides	O
+in	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+for	O
+complex	O
+formation	O
+with	O
+the	O
+ROQ	B-structure_element
+domain	O
+.	O
+We	O
+designed	O
+four	O
+mutants	O
+(	O
+Mut1	O
+–	O
+4	O
+,	O
+see	O
+Supplementary	O
+Fig	O
+.	O
+6	O
+)	O
+that	O
+were	O
+expected	O
+to	O
+disrupt	O
+key	O
+interactions	O
+with	O
+the	O
+protein	O
+according	O
+to	O
+our	O
+co	B-evidence
+-	I-evidence
+crystal	I-evidence
+structure	I-evidence
+(	O
+Fig	O
+.	O
+3d	O
+–	O
+f	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+).	O
+NMR	B-experimental_method
+analysis	O
+confirmed	O
+that	O
+all	O
+mutant	B-protein_state
+RNAs	B-chemical
+formed	O
+the	O
+same	O
+base	O
+pairs	O
+in	O
+the	O
+stem	B-structure_element
+region	I-structure_element
+,	O
+identical	O
+to	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+(	O
+Fig	O
+.	O
+2b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+6	O
+).	O
+We	O
+next	O
+used	O
+surface	B-experimental_method
+plasmon	I-experimental_method
+resonance	I-experimental_method
+experiments	O
+to	O
+determine	O
+dissociation	B-evidence
+constants	I-evidence
+for	O
+the	O
+ROQ	B-structure_element
+-	O
+RNA	B-chemical
+interaction	O
+(	O
+Table	O
+2	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+7	O
+).	O
+Although	O
+the	O
+replacement	B-experimental_method
+of	O
+a	O
+C8	B-residue_name_number
+–	O
+G15	B-residue_name_number
+closing	O
+base	O
+pair	O
+by	O
+A	B-residue_name
+-	O
+U	B-residue_name
+(	O
+Mut	B-mutant
+4	I-mutant
+)	O
+only	O
+reduces	O
+the	O
+affinity	B-evidence
+threefold	O
+,	O
+reduction	O
+of	O
+loop	B-structure_element
+size	O
+in	O
+the	O
+A14C	B-mutant
+mutant	B-protein_state
+(	O
+Mut	B-mutant
+1	I-mutant
+,	O
+see	O
+Table	O
+2	O
+)	O
+reduces	O
+the	O
+affinity	B-evidence
+and	O
+binding	O
+is	O
+not	O
+detected	O
+by	O
+surface	B-experimental_method
+plasmon	I-experimental_method
+resonance	I-experimental_method
+.	O
+As	O
+intended	O
+,	O
+the	O
+mutation	O
+Mut	B-mutant
+1	I-mutant
+allows	O
+the	O
+formation	O
+of	O
+an	O
+additional	O
+base	O
+pair	O
+and	O
+thus	O
+leads	O
+to	O
+the	O
+formation	O
+of	O
+a	O
+tetraloop	B-structure_element
+with	O
+a	O
+new	O
+G	B-residue_name
+-	O
+C	B-residue_name
+closing	O
+base	O
+pair	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+6a	O
+).	O
+Consistent	O
+with	O
+the	O
+structural	B-experimental_method
+analysis	I-experimental_method
+,	O
+we	O
+assume	O
+that	O
+this	O
+variant	O
+alters	O
+the	O
+hexaloop	B-structure_element
+conformation	O
+and	O
+thus	O
+reduces	O
+the	O
+interaction	O
+with	O
+ROQ	B-structure_element
+.	O
+Disruption	O
+of	O
+stacking	B-bond_interaction
+interactions	I-bond_interaction
+between	O
+G15	B-residue_name_number
+,	O
+G9	B-residue_name_number
+and	O
+Y250	B-residue_name_number
+in	O
+the	O
+G9C	B-mutant
+mutant	B-protein_state
+(	O
+Mut	B-mutant
+2	I-mutant
+)	O
+completely	O
+abolished	O
+binding	O
+of	O
+ROQ	B-structure_element
+to	O
+the	O
+SL	B-structure_element
+RNA	B-chemical
+(	O
+Table	O
+2	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+7	O
+).	O
+No	O
+binding	O
+is	O
+also	O
+observed	O
+for	O
+the	O
+U11AU13G	B-mutant
+double	B-protein_state
+mutant	I-protein_state
+(	O
+Mut	B-mutant
+3	I-mutant
+)	O
+(	O
+Table	O
+2	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+7	O
+),	O
+which	O
+abolishes	O
+specific	O
+interactions	O
+mediated	O
+by	O
+U11	B-residue_name_number
+and	O
+U13	B-residue_name_number
+in	O
+the	O
+hexaloop	B-structure_element
+with	O
+ROQ	B-structure_element
+(	O
+Fig	O
+.	O
+3d	O
+).	O
+Consistent	O
+with	O
+the	O
+SELEX	B-experimental_method
+consensus	O
+(	O
+Fig	O
+.	O
+1b	O
+),	O
+all	O
+of	O
+the	O
+tested	O
+mutations	B-experimental_method
+of	O
+conserved	B-protein_state
+nucleotides	B-chemical
+in	O
+the	O
+loop	B-structure_element
+reduce	O
+or	O
+abolish	O
+the	O
+interaction	O
+with	O
+ROQ	B-structure_element
+.	O
+Interestingly	O
+,	O
+the	O
+affinity	B-evidence
+of	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+Tnf	B-protein
+CDE	B-structure_element
+and	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SLs	B-structure_element
+to	O
+ROQ	B-structure_element
+are	O
+very	O
+similar	O
+(	O
+42	O
+and	O
+81	O
+nM	O
+,	O
+respectively	O
+,	O
+Table	O
+2	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+7	O
+).	O
+Roquin	B-protein
+binding	O
+to	O
+different	O
+SLs	B-structure_element
+in	O
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+We	O
+have	O
+recently	O
+shown	O
+that	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+binds	O
+to	O
+a	O
+CDE	B-structure_element
+-	O
+like	O
+motif	O
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+Ox40	B-protein
+mRNA	B-chemical
+(	O
+Figs	O
+1d	O
+and	O
+4c	O
+).	O
+We	O
+therefore	O
+investigated	O
+whether	O
+the	O
+interactions	O
+with	O
+the	O
+CDE	B-structure_element
+-	O
+like	O
+and	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+both	O
+contribute	O
+to	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+binding	O
+in	O
+the	O
+context	O
+of	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+.	O
+The	O
+binding	B-evidence
+affinities	I-evidence
+of	O
+either	O
+motif	O
+for	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+(	O
+residues	O
+2	B-residue_range
+–	I-residue_range
+440	I-residue_range
+)	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+8a	O
+,	O
+b	O
+)	O
+or	O
+the	O
+ROQ	B-structure_element
+domain	O
+alone	B-protein_state
+are	O
+in	O
+a	O
+similar	O
+range	O
+(	O
+Table	O
+2	O
+).	O
+The	O
+dissociation	B-evidence
+constants	I-evidence
+for	O
+the	O
+ROQ	B-structure_element
+interaction	O
+with	O
+the	O
+Ox40	B-protein
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+and	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+are	O
+1	O
+,	O
+460	O
+and	O
+81	O
+nM	O
+,	O
+respectively	O
+(	O
+Table	O
+2	O
+).	O
+This	O
+is	O
+consistent	O
+with	O
+the	O
+extended	O
+binding	B-site
+interface	I-site
+and	O
+additional	O
+interactions	O
+observed	O
+with	O
+the	O
+hexaloop	B-structure_element
+,	O
+and	O
+suggests	O
+a	O
+preferential	O
+binding	O
+to	O
+the	O
+hexaloop	B-structure_element
+SL	B-structure_element
+RNA	B-chemical
+in	O
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+.	O
+We	O
+designed	O
+different	O
+variants	O
+of	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+by	O
+point	B-experimental_method
+mutagenesis	I-experimental_method
+abrogating	O
+base	O
+pairing	O
+in	O
+the	O
+stem	B-structure_element
+region	I-structure_element
+,	O
+where	O
+none	O
+,	O
+individual	O
+,	O
+or	O
+both	O
+SL	B-structure_element
+RNA	B-chemical
+motifs	O
+were	O
+mutated	B-experimental_method
+to	O
+impair	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+binding	O
+(	O
+Fig	O
+.	O
+6a	O
+).	O
+These	O
+RNAs	B-chemical
+were	O
+then	O
+tested	O
+in	O
+EMSAs	B-experimental_method
+with	O
+the	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+N	O
+terminus	O
+(	O
+residues	O
+2	B-residue_range
+–	I-residue_range
+440	I-residue_range
+)	O
+(	O
+Fig	O
+.	O
+6b	O
+).	O
+Gel	B-experimental_method
+shift	I-experimental_method
+assays	I-experimental_method
+show	O
+that	O
+binding	O
+to	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+construct	O
+leads	O
+to	O
+two	O
+distinct	O
+bands	O
+during	O
+the	O
+titrations	B-experimental_method
+,	O
+which	O
+should	O
+reflect	O
+binding	O
+to	O
+one	O
+and	O
+both	O
+RNA	B-chemical
+motifs	O
+,	O
+respectively	O
+.	O
+Consistent	O
+with	O
+this	O
+,	O
+both	O
+bands	O
+are	O
+strongly	O
+reduced	O
+when	O
+mutations	O
+are	O
+introduced	O
+that	O
+interfere	O
+with	O
+the	O
+formation	O
+of	O
+both	O
+SLs	B-structure_element
+.	O
+Notably	O
+,	O
+among	O
+these	O
+,	O
+the	O
+slower	O
+migrating	O
+band	O
+disappears	O
+when	O
+either	O
+of	O
+the	O
+two	O
+SL	B-structure_element
+RNA	B-chemical
+motifs	O
+is	O
+altered	O
+to	O
+impair	O
+Roquin	B-protein
+binding	O
+,	O
+indicating	O
+an	O
+interaction	O
+with	O
+the	O
+remaining	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+SL	B-structure_element
+.	O
+We	O
+thus	O
+conclude	O
+that	O
+Roquin	B-protein
+is	O
+able	O
+to	O
+bind	O
+to	O
+both	O
+SL	B-structure_element
+RNA	B-chemical
+motifs	O
+in	O
+the	O
+context	O
+of	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+.	O
+Regulation	O
+of	O
+Ox40	B-protein
+expression	O
+via	O
+two	O
+motifs	O
+in	O
+its	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+To	O
+investigate	O
+the	O
+role	O
+of	O
+the	O
+new	O
+ADE	B-structure_element
+-	O
+like	O
+motif	O
+in	O
+target	O
+mRNA	B-chemical
+regulation	O
+,	O
+we	O
+introduced	B-experimental_method
+Ox40	B-protein
+mRNA	B-chemical
+variants	O
+harbouring	O
+altered	B-protein_state
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+in	O
+cells	O
+.	O
+Considering	O
+the	O
+close	O
+proximity	O
+of	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+and	O
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+(	O
+Fig	O
+.	O
+6a	O
+),	O
+which	O
+is	O
+essential	O
+for	O
+Roquin	B-protein
+-	O
+mediated	O
+posttranscriptional	O
+regulation	O
+of	O
+Ox40	B-protein
+(	O
+ref	O
+.)	O
+we	O
+tested	O
+individual	O
+contributions	O
+and	O
+the	O
+functional	O
+cooperation	O
+of	O
+the	O
+two	O
+RNA	B-chemical
+elements	O
+by	O
+deletion	B-experimental_method
+and	I-experimental_method
+point	I-experimental_method
+mutagenesis	I-experimental_method
+abrogating	B-protein_state
+base	B-bond_interaction
+pairing	I-bond_interaction
+in	O
+the	O
+stem	B-structure_element
+region	I-structure_element
+(	O
+Fig	O
+.	O
+6a	O
+,	O
+c	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+8c	O
+).	O
+Specifically	O
+,	O
+using	O
+retroviruses	B-taxonomy_domain
+we	O
+introduced	O
+Ox40	B-protein
+expression	O
+constructs	O
+placed	O
+under	O
+the	O
+control	O
+of	O
+different	O
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+into	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+/	O
+2	B-protein
+-	O
+deficient	O
+mouse	B-taxonomy_domain
+embryonic	O
+fibroblasts	O
+.	O
+Doxycycline	B-chemical
+treatment	O
+of	O
+cells	O
+from	O
+this	O
+cell	O
+line	O
+enabled	O
+ectopic	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+and	O
+co	O
+-	O
+translational	O
+mCherry	O
+expression	O
+due	O
+to	O
+the	O
+stable	O
+integration	O
+of	O
+an	O
+inducible	O
+lentiviral	B-taxonomy_domain
+vector	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+8c	O
+).	O
+The	O
+expression	O
+of	O
+Ox40	B-protein
+in	O
+cells	O
+with	O
+and	O
+without	O
+doxycycline	B-chemical
+treatment	O
+was	O
+then	O
+quantified	O
+by	O
+flow	B-experimental_method
+cytometry	I-experimental_method
+(	O
+Supplementary	O
+Fig	O
+.	O
+8c	O
+).	O
+Comparing	O
+the	O
+ratio	O
+of	O
+Ox40	B-protein
+mean	B-evidence
+fluorescence	I-evidence
+intensities	I-evidence
+in	O
+cells	O
+with	O
+and	O
+without	O
+doxycycline	B-chemical
+treatment	O
+normalized	O
+to	O
+the	O
+values	O
+from	O
+cells	O
+that	O
+expressed	O
+Ox40	B-protein
+constructs	O
+without	B-protein_state
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+revealed	O
+a	O
+comparable	O
+importance	O
+of	O
+both	O
+structural	O
+elements	O
+(	O
+Fig	O
+.	O
+6c	O
+).	O
+In	O
+fact	O
+,	O
+only	O
+deletion	B-experimental_method
+or	I-experimental_method
+point	I-experimental_method
+mutagenesis	I-experimental_method
+of	O
+the	O
+sequences	O
+encoding	O
+both	O
+structures	O
+at	O
+the	O
+same	O
+time	O
+(	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+1	B-residue_range
+–	I-residue_range
+80	I-residue_range
+and	O
+double	B-protein_state
+mut	I-protein_state
+)	O
+neutralized	O
+Roquin	B-protein
+-	O
+dependent	O
+repression	O
+of	O
+Ox40	B-protein
+.	O
+In	O
+contrast	O
+,	O
+individual	O
+mutations	B-experimental_method
+that	O
+left	O
+the	O
+hexaloop	B-structure_element
+(	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+1	B-residue_range
+–	I-residue_range
+120	I-residue_range
+or	O
+CDE	B-mutant
+mut	I-mutant
+)	O
+or	O
+the	O
+CDE	B-structure_element
+-	O
+like	O
+triloop	B-structure_element
+intact	B-protein_state
+still	O
+enabled	O
+Roquin	B-protein
+-	O
+dependent	O
+repression	O
+,	O
+which	O
+occurred	O
+in	O
+an	O
+attenuated	O
+manner	O
+compared	O
+with	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+(	O
+Fig	O
+.	O
+6c	O
+).	O
+To	O
+further	O
+analyse	O
+the	O
+functional	O
+consequences	O
+of	O
+Roquin	B-protein
+binding	O
+to	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+,	O
+we	O
+also	O
+measured	O
+mRNA	B-evidence
+decay	I-evidence
+rates	I-evidence
+after	O
+introducing	O
+the	O
+different	O
+Ox40	B-protein
+constructs	O
+into	O
+HeLa	O
+tet	O
+-	O
+off	O
+cells	O
+that	O
+allow	O
+to	O
+turn	O
+off	O
+transcription	O
+from	O
+the	O
+tetracycline	O
+-	O
+repressed	O
+vectors	O
+by	O
+addition	O
+of	O
+doxycycline	B-chemical
+(	O
+Fig	O
+.	O
+6d	O
+).	O
+Quantitative	B-experimental_method
+reverse	I-experimental_method
+transcriptase	I-experimental_method
+–	I-experimental_method
+PCR	I-experimental_method
+revealed	O
+a	O
+strong	O
+stabilization	O
+of	O
+the	O
+Ox40	B-protein
+mRNA	B-chemical
+by	O
+deletion	B-experimental_method
+of	I-experimental_method
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+(	O
+CDS	B-structure_element
+t1	B-evidence
+/	I-evidence
+2	I-evidence
+=	O
+311	O
+min	O
+vs	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+t1	B-evidence
+/	I-evidence
+2	I-evidence
+=	O
+96	O
+min	O
+).	O
+A	O
+comparable	O
+stabilization	O
+was	O
+achieved	O
+by	O
+combined	B-experimental_method
+mutation	I-experimental_method
+of	O
+the	O
+CDE	B-structure_element
+-	O
+like	O
+and	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+SLs	B-structure_element
+(	O
+ADE	B-structure_element
+/	O
+CDE	B-structure_element
+-	O
+like	O
+mut	B-protein_state
+t1	B-evidence
+/	I-evidence
+2	I-evidence
+=	O
+255	O
+min	O
+).	O
+Individual	O
+mutations	B-experimental_method
+of	O
+either	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+or	O
+the	O
+CDE	B-structure_element
+-	O
+like	O
+SLs	B-structure_element
+showed	O
+intermediate	O
+effects	O
+(	O
+ADE	B-structure_element
+-	O
+like	O
+mut	B-protein_state
+t1	B-evidence
+/	I-evidence
+2	I-evidence
+=	O
+170	O
+min	O
+,	O
+CDE	B-structure_element
+-	O
+like	O
+mut	B-protein_state
+t1	B-evidence
+/	I-evidence
+2	I-evidence
+=	O
+167	O
+min	O
+),	O
+respectively	O
+.	O
+These	O
+findings	O
+underscore	O
+the	O
+importance	O
+of	O
+both	O
+structural	O
+motifs	O
+and	O
+reveal	O
+that	O
+they	O
+have	O
+an	O
+additive	O
+effect	O
+on	O
+the	O
+regulation	O
+of	O
+Ox40	B-protein
+mRNA	B-chemical
+expression	O
+in	O
+cells	O
+.	O
+Recent	O
+structural	B-experimental_method
+and	I-experimental_method
+functional	I-experimental_method
+studies	I-experimental_method
+have	O
+provided	O
+first	O
+insight	O
+into	O
+the	O
+RNA	B-chemical
+binding	O
+of	O
+Roquin	B-protein
+.	O
+Structures	B-evidence
+of	O
+Roquin	B-protein
+bound	B-protein_state
+to	I-protein_state
+CDE	B-structure_element
+SL	B-structure_element
+RNAs	B-chemical
+indicated	O
+mainly	O
+shape	O
+recognition	O
+of	O
+the	O
+SL	B-structure_element
+RNA	B-chemical
+in	O
+the	O
+so	O
+-	O
+called	O
+A	B-site
+-	I-site
+site	I-site
+of	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+the	O
+Roquin	B-protein
+protein	O
+with	O
+no	O
+sequence	O
+specificity	O
+,	O
+except	O
+the	O
+requirement	O
+for	O
+a	O
+pyrimidine	B-structure_element
+–	I-structure_element
+purine	I-structure_element
+–	I-structure_element
+pyrimidine	I-structure_element
+triloop	I-structure_element
+.	O
+Considering	O
+that	O
+the	O
+CDE	B-structure_element
+RNA	B-chemical
+recognition	O
+is	O
+mostly	O
+structure	O
+specific	O
+and	O
+not	O
+sequence	O
+dependent	O
+,	O
+a	O
+wide	O
+spectrum	O
+of	O
+target	O
+mRNA	B-chemical
+might	O
+be	O
+recognized	O
+by	O
+Roquin	B-protein
+.	O
+Here	O
+we	O
+have	O
+used	O
+SELEX	B-experimental_method
+assays	I-experimental_method
+to	O
+identify	O
+a	O
+novel	O
+RNA	B-structure_element
+recognition	I-structure_element
+motif	I-structure_element
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+,	O
+which	O
+is	O
+present	O
+in	O
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+and	O
+variations	O
+of	O
+which	O
+may	O
+be	O
+found	O
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+of	O
+many	O
+other	O
+genes	O
+.	O
+Our	O
+experiments	O
+show	O
+that	O
+this	O
+SELEX	B-experimental_method
+-	O
+derived	O
+ADE	B-structure_element
+shows	O
+functional	O
+activity	O
+comparable	O
+to	O
+the	O
+previously	O
+established	O
+CDE	B-structure_element
+motif	O
+.	O
+The	O
+ADE	B-structure_element
+and	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+adopt	O
+SL	B-structure_element
+folds	O
+with	O
+a	O
+hexaloop	B-structure_element
+instead	O
+of	O
+a	O
+triloop	B-structure_element
+.	O
+Notably	O
+,	O
+the	O
+recognition	O
+of	O
+the	O
+respective	O
+RNA	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+stem	I-structure_element
+regions	I-structure_element
+by	O
+the	O
+ROQ	B-structure_element
+domain	O
+is	O
+identical	O
+for	O
+the	O
+triloop	B-structure_element
+and	O
+hexaloop	B-structure_element
+motifs	O
+.	O
+However	O
+,	O
+the	O
+U	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+hexaloops	I-structure_element
+in	O
+the	O
+ADE	B-structure_element
+and	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+mediate	O
+a	O
+number	O
+of	O
+additional	O
+contacts	O
+with	O
+the	O
+helix	B-structure_element
+α4	B-structure_element
+and	O
+strand	B-structure_element
+β3	B-structure_element
+in	O
+the	O
+ROQ	B-structure_element
+domain	O
+that	O
+are	O
+absent	O
+in	O
+the	O
+triloop	B-structure_element
+CDE	B-structure_element
+(	O
+Fig	O
+.	O
+3b	O
+–	O
+f	O
+).	O
+Of	O
+particular	O
+importance	O
+for	O
+the	O
+hexaloop	B-structure_element
+recognition	O
+is	O
+Tyr250	B-residue_name_number
+,	O
+which	O
+acts	O
+as	O
+a	O
+stabilizing	O
+element	O
+for	O
+the	O
+integrity	O
+of	O
+a	O
+defined	O
+loop	B-structure_element
+conformation	O
+.	O
+It	O
+stacks	B-bond_interaction
+with	O
+nucleotides	O
+in	O
+the	O
+hexaloop	B-structure_element
+but	O
+not	O
+the	O
+CDE	B-structure_element
+triloop	B-structure_element
+(	O
+Fig	O
+.	O
+3b	O
+,	O
+c	O
+).	O
+The	O
+functional	O
+role	O
+of	O
+Tyr250	B-residue_name_number
+for	O
+ADE	B-structure_element
+-	O
+mediated	O
+mRNA	B-chemical
+regulation	O
+by	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+is	O
+thus	O
+explained	O
+by	O
+our	O
+experiments	O
+(	O
+Fig	O
+.	O
+5b	O
+,	O
+c	O
+).	O
+The	O
+preference	O
+for	O
+U	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+hexaloops	I-structure_element
+depends	O
+on	O
+nucleotide	O
+-	O
+specific	O
+interactions	O
+of	O
+ROQ	B-structure_element
+with	O
+U10	B-residue_name_number
+,	O
+U11	B-residue_name_number
+and	O
+U13	B-residue_name_number
+in	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+.	O
+Consistent	O
+with	O
+this	O
+,	O
+loss	O
+of	O
+ROQ	B-structure_element
+binding	O
+is	O
+observed	O
+on	O
+replacement	B-experimental_method
+of	O
+U11	B-residue_name_number
+and	O
+U13	B-residue_name_number
+by	O
+other	O
+bases	O
+(	O
+Table	O
+2	O
+).	O
+In	O
+spite	O
+of	O
+these	O
+differences	O
+in	O
+some	O
+aspects	O
+of	O
+the	O
+RNA	B-chemical
+recognition	O
+,	O
+overall	O
+features	O
+of	O
+Roquin	B-protein
+targets	O
+are	O
+conserved	O
+in	O
+ADE	B-structure_element
+and	O
+CDE	B-structure_element
+-	O
+like	O
+RNAs	B-chemical
+,	O
+namely	O
+,	O
+a	O
+crucial	O
+role	O
+of	O
+non	O
+-	O
+sequence	O
+-	O
+specific	O
+contacts	O
+to	O
+the	O
+RNA	B-chemical
+stem	B-structure_element
+and	O
+mainly	O
+shape	O
+recognition	O
+of	O
+the	O
+hexa	B-structure_element
+-	I-structure_element
+and	I-structure_element
+triloops	I-structure_element
+,	O
+respectively	O
+.	O
+A	O
+unique	O
+feature	O
+of	O
+the	O
+bound	B-protein_state
+RNA	B-chemical
+structure	B-evidence
+,	O
+common	O
+to	O
+both	O
+tri	B-structure_element
+-	I-structure_element
+and	I-structure_element
+hexaloops	I-structure_element
+,	O
+is	O
+the	O
+stacking	B-bond_interaction
+of	O
+a	O
+purine	O
+base	O
+onto	O
+the	O
+closing	O
+base	O
+pair	O
+(	O
+Fig	O
+.	O
+3b	O
+,	O
+c	O
+).	O
+Previous	O
+structural	B-evidence
+data	I-evidence
+and	O
+the	O
+results	O
+presented	O
+here	O
+therefore	O
+suggest	O
+that	O
+Roquin	B-protein
+may	O
+recognize	O
+additional	O
+SL	B-structure_element
+RNA	B-chemical
+motifs	O
+,	O
+potentially	O
+with	O
+larger	O
+loops	B-structure_element
+.	O
+Interestingly	O
+,	O
+the	O
+SELEX	B-experimental_method
+-	O
+derived	O
+motif	O
+resembles	O
+the	O
+U	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+motifs	I-structure_element
+that	O
+were	O
+identified	O
+recently	O
+by	O
+Murakawa	O
+et	O
+al	O
+..	O
+In	O
+their	O
+study	O
+,	O
+several	O
+U	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+loops	I-structure_element
+of	O
+various	O
+sizes	O
+were	O
+identified	O
+by	O
+crosslinking	B-experimental_method
+and	I-experimental_method
+immunoprecipitation	I-experimental_method
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+using	O
+PAR	B-experimental_method
+-	I-experimental_method
+CLIP	I-experimental_method
+and	O
+the	O
+data	O
+also	O
+included	O
+sequences	O
+comprising	O
+the	O
+U	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+hexaloop	I-structure_element
+identified	O
+in	O
+our	O
+present	O
+work	O
+.	O
+Most	O
+probably	O
+,	O
+the	O
+experimental	O
+setup	O
+of	O
+Murakawa	O
+et	O
+al	O
+.	O
+revealed	O
+both	O
+high	O
+-	O
+and	O
+low	O
+-	O
+affinity	O
+target	O
+motifs	O
+for	O
+Roquin	B-protein
+,	O
+whereas	O
+our	O
+structural	B-experimental_method
+study	I-experimental_method
+reports	O
+on	O
+a	O
+high	O
+-	O
+affinity	O
+binding	O
+motif	O
+.	O
+Notably	O
+,	O
+Murakawa	O
+et	O
+al	O
+.	O
+neither	O
+found	O
+the	O
+Roquin	B-protein
+-	O
+regulated	O
+Ox40	B-protein
+nor	O
+the	O
+Tnf	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+,	O
+as	O
+both	O
+genes	O
+are	O
+not	O
+expressed	O
+in	O
+HEK	O
+293	O
+cells	O
+.	O
+However	O
+,	O
+their	O
+newly	O
+identified	O
+U	O
+-	O
+rich	O
+target	O
+SL	B-structure_element
+within	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+A20	B-protein
+mRNA	B-chemical
+supports	O
+our	O
+conclusion	O
+that	O
+Roquin	B-protein
+can	O
+accept	O
+alternative	O
+target	O
+motifs	O
+apart	O
+from	O
+the	O
+classical	O
+CDE	B-structure_element
+triloop	B-structure_element
+arrangement	O
+.	O
+It	O
+remains	O
+to	O
+be	O
+seen	O
+which	O
+exact	O
+features	O
+govern	O
+the	O
+recognition	O
+of	O
+the	O
+A20	B-protein
+SL	B-structure_element
+by	O
+Roquin	B-protein
+.	O
+The	O
+regulatory	O
+cis	B-structure_element
+RNA	I-structure_element
+elements	I-structure_element
+in	O
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+may	O
+also	O
+be	O
+targeted	O
+by	O
+additional	O
+trans	O
+-	O
+acting	O
+factors	O
+.	O
+We	O
+have	O
+recently	O
+identified	O
+the	O
+endonuclease	B-protein_type
+Regnase	B-protein
+-	I-protein
+1	I-protein
+as	O
+a	O
+cofactor	O
+of	O
+Roquin	B-protein
+function	O
+that	O
+shares	O
+an	O
+overlapping	O
+set	O
+of	O
+target	O
+mRNAs	B-chemical
+.	O
+In	O
+another	O
+study	O
+,	O
+the	O
+overlap	O
+in	O
+targets	O
+was	O
+confirmed	O
+,	O
+but	O
+a	O
+mutually	O
+exclusive	O
+regulation	O
+was	O
+proposed	O
+based	O
+on	O
+studies	O
+in	O
+lipopolysaccharide	B-chemical
+(	O
+LPS	B-chemical
+)-	O
+stimulated	O
+myeloid	O
+cells	O
+.	O
+In	O
+these	O
+cells	O
+,	O
+Roquin	B-protein
+induced	O
+mRNA	B-chemical
+decay	O
+only	O
+for	O
+translationally	O
+inactive	B-protein_state
+mRNAs	B-chemical
+,	O
+while	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+-	O
+induced	O
+mRNA	B-chemical
+decay	O
+depended	O
+on	O
+active	O
+translation	O
+of	O
+the	O
+target	O
+.	O
+In	O
+CD4	O
++	O
+T	O
+cells	O
+,	O
+Ox40	B-protein
+does	O
+not	O
+show	O
+derepression	O
+in	O
+individual	O
+knockouts	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+or	O
+Roquin	B-protein
+-	I-protein
+2	I-protein
+encoding	O
+genes	O
+,	O
+but	O
+is	O
+strongly	O
+induced	O
+upon	O
+combined	O
+deficiency	B-experimental_method
+of	O
+both	O
+genes	O
+.	O
+In	O
+addition	O
+,	O
+conditional	O
+deletion	B-experimental_method
+of	I-experimental_method
+the	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+-	O
+encoding	O
+gene	O
+induced	O
+Ox40	B-protein
+expression	O
+in	O
+these	O
+cells	O
+.	O
+Whether	O
+induced	O
+decay	O
+of	O
+Ox40	B-protein
+mRNA	B-chemical
+by	O
+Roquin	B-protein
+or	O
+Regnase	B-protein_type
+proteins	O
+occurs	O
+in	O
+a	O
+mutually	O
+exclusive	O
+manner	O
+at	O
+different	O
+points	O
+during	O
+T	O
+-	O
+cell	O
+activation	O
+or	O
+shows	O
+cooperative	O
+regulation	O
+will	O
+have	O
+to	O
+await	O
+a	O
+direct	O
+comparison	O
+of	O
+T	O
+cells	O
+with	O
+single	O
+,	O
+double	B-experimental_method
+and	I-experimental_method
+triple	I-experimental_method
+knockouts	I-experimental_method
+of	O
+these	O
+genes	O
+.	O
+However	O
+,	O
+in	O
+cultures	O
+of	O
+CD4	O
++	O
+T	O
+cells	O
+,	O
+Ox40	B-protein
+is	O
+translated	O
+on	O
+day	O
+4	O
+–	O
+5	O
+and	O
+is	O
+expressed	O
+much	O
+higher	O
+in	O
+T	O
+cells	O
+with	O
+combined	O
+deficiency	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+and	O
+Roquin	B-protein
+-	I-protein
+2	I-protein
+.	O
+At	O
+this	O
+time	O
+point	O
+,	O
+the	O
+short	O
+-	O
+term	O
+inducible	O
+reconstitution	B-experimental_method
+with	O
+WT	B-protein_state
+Roquin	B-protein
+-	I-protein
+1	I-protein
+was	O
+effective	O
+to	O
+reduced	O
+Ox40	B-protein
+expression	O
+,	O
+demonstrating	O
+the	O
+regulation	O
+of	O
+a	O
+translationally	O
+active	B-protein_state
+mRNA	B-chemical
+by	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+in	O
+T	O
+cells	O
+(	O
+Fig	O
+.	O
+5c	O
+).	O
+Recombinant	O
+N	O
+-	O
+terminal	O
+protein	O
+fragments	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+or	O
+Roquin	B-protein
+-	I-protein
+2	I-protein
+bind	O
+with	O
+comparable	O
+affinity	O
+to	O
+Ox40	B-protein
+mRNA	B-chemical
+in	O
+EMSAs	B-experimental_method
+and	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+Ox40	B-protein
+is	O
+similarly	O
+retained	O
+by	O
+the	O
+two	O
+recombinant	O
+proteins	O
+in	O
+filter	B-experimental_method
+binding	I-experimental_method
+assays	I-experimental_method
+.	O
+Given	O
+the	O
+almost	O
+identical	O
+RNA	B-chemical
+contacts	O
+in	O
+both	O
+paralogues	O
+,	O
+we	O
+assume	O
+a	O
+similar	O
+recognition	O
+of	O
+ADE	B-structure_element
+and	O
+CDE	B-structure_element
+motifs	O
+in	O
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+by	O
+both	O
+proteins	O
+.	O
+In	O
+contrast	O
+,	O
+structural	O
+details	O
+on	O
+how	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+can	O
+interact	O
+with	O
+these	O
+SL	B-structure_element
+RNAs	B-chemical
+are	O
+currently	O
+missing	O
+.	O
+Surprisingly	O
+,	O
+transcriptome	O
+-	O
+wide	O
+mapping	O
+of	O
+Regnase	B-site
+-	I-site
+1	I-site
+-	I-site
+binding	I-site
+sites	I-site
+in	O
+crosslinking	B-experimental_method
+and	I-experimental_method
+immunoprecipitation	I-experimental_method
+experiments	I-experimental_method
+identified	O
+specific	O
+triloop	B-structure_element
+structures	O
+with	O
+pyrimidine	B-structure_element
+–	I-structure_element
+purine	I-structure_element
+–	I-structure_element
+pyrimidine	I-structure_element
+loops	I-structure_element
+in	O
+3	O
+-	O
+to	O
+7	O
+-	O
+nt	O
+-	O
+long	O
+stems	B-structure_element
+,	O
+as	O
+well	O
+as	O
+a	O
+novel	O
+hexaloop	B-structure_element
+structure	O
+in	O
+the	O
+Ptgs2	B-gene
+gene	O
+.	O
+Both	O
+were	O
+required	O
+for	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+-	O
+mediated	O
+repression	O
+.	O
+These	O
+findings	O
+therefore	O
+raise	O
+the	O
+possibility	O
+that	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+interacts	O
+with	O
+ADE	B-structure_element
+-	O
+like	O
+hexaloop	B-structure_element
+structures	O
+either	O
+in	O
+a	O
+direct	O
+or	O
+indirect	O
+manner	O
+.	O
+Nevertheless	O
+,	O
+it	O
+becomes	O
+clear	O
+that	O
+composite	O
+cis	B-structure_element
+-	I-structure_element
+elements	I-structure_element
+,	O
+that	O
+is	O
+,	O
+the	O
+presence	O
+of	O
+several	O
+SLs	B-structure_element
+as	O
+in	O
+Ox40	B-protein
+or	O
+Icos	B-protein
+,	O
+could	O
+attract	O
+multiple	O
+trans	O
+-	O
+acting	O
+factors	O
+that	O
+may	O
+potentially	O
+co	O
+-	O
+regulate	O
+or	O
+even	O
+act	O
+cooperatively	O
+to	O
+control	O
+mRNA	B-chemical
+expression	O
+through	O
+posttranscriptional	O
+pathways	O
+of	O
+gene	O
+regulation	O
+.	O
+The	O
+novel	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+loop	B-structure_element
+motif	I-structure_element
+that	O
+we	O
+have	O
+identified	O
+as	O
+a	O
+bona	O
+fide	O
+target	O
+of	O
+Roquin	B-protein
+now	O
+expands	O
+this	O
+multilayer	O
+mode	O
+of	O
+co	O
+-	O
+regulation	O
+.	O
+We	O
+suggest	O
+that	O
+differential	O
+regulation	O
+of	O
+mRNA	B-chemical
+expression	O
+is	O
+not	O
+only	O
+achieved	O
+through	O
+multiple	O
+regulators	O
+with	O
+individual	O
+preferences	O
+for	O
+a	O
+given	O
+motif	O
+or	O
+variants	O
+thereof	O
+,	O
+but	O
+that	O
+regulators	O
+may	O
+also	O
+identify	O
+and	O
+use	O
+distinct	O
+motifs	O
+,	O
+as	O
+long	O
+as	O
+they	O
+exhibit	O
+some	O
+basic	O
+features	O
+regarding	O
+shape	O
+,	O
+size	O
+and	O
+sequence	O
+.	O
+The	O
+presence	O
+of	O
+distinct	O
+motifs	O
+in	O
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+offers	O
+a	O
+broader	O
+variability	O
+for	O
+gene	O
+regulation	O
+by	O
+RNA	B-chemical
+cis	B-structure_element
+elements	I-structure_element
+.	O
+Their	O
+accessibility	O
+can	O
+be	O
+modulated	O
+by	O
+trans	O
+-	O
+acting	O
+factors	O
+that	O
+may	O
+bind	O
+regulatory	O
+motifs	O
+,	O
+unfold	O
+higher	O
+-	O
+order	O
+structures	O
+in	O
+the	O
+RNA	B-chemical
+or	O
+maintain	O
+a	O
+preference	O
+for	O
+duplex	O
+structures	O
+as	O
+was	O
+shown	O
+recently	O
+for	O
+mRNAs	B-chemical
+that	O
+are	O
+recognized	O
+by	O
+Staufen	B-protein
+-	I-protein
+1	I-protein
+(	O
+ref	O
+.).	O
+In	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+the	O
+Ox40	B-protein
+mRNA	B-chemical
+,	O
+we	O
+find	O
+one	O
+ADE	B-structure_element
+-	O
+like	O
+and	O
+one	O
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+,	O
+with	O
+similar	O
+binding	O
+to	O
+the	O
+ROQ	B-structure_element
+domain	O
+.	O
+The	O
+exact	O
+stoichiometry	O
+of	O
+Roquin	B-protein
+bound	B-protein_state
+to	I-protein_state
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+is	O
+unknown	O
+.	O
+The	O
+recently	O
+identified	O
+secondary	B-site
+binding	I-site
+site	I-site
+for	O
+dsRNA	B-chemical
+in	O
+Roquin	B-protein
+(	O
+B	B-site
+-	I-site
+site	I-site
+)	O
+could	O
+potentially	O
+allow	O
+for	O
+simultaneous	O
+binding	O
+of	O
+dsRNA	B-chemical
+and	O
+thereby	O
+promote	O
+engagement	O
+of	O
+Roquin	B-protein
+and	O
+target	O
+RNAs	B-chemical
+before	O
+recognition	O
+of	O
+high	O
+-	O
+affinity	B-evidence
+SLs	B-structure_element
+.	O
+In	O
+this	O
+respect	O
+,	O
+it	O
+is	O
+interesting	O
+to	O
+note	O
+that	O
+symmetry	O
+-	O
+related	O
+RNA	B-chemical
+molecules	O
+of	O
+both	O
+Tnf	B-protein
+CDE	B-structure_element
+and	O
+ADE	B-structure_element
+SL	B-structure_element
+RNAs	B-chemical
+are	O
+found	O
+in	O
+the	O
+respective	O
+crystal	B-evidence
+lattice	I-evidence
+in	O
+a	O
+position	O
+that	O
+corresponds	O
+to	O
+the	O
+recognition	O
+of	O
+dsRNA	B-chemical
+in	O
+the	O
+B	B-site
+site	I-site
+.	O
+This	O
+opens	O
+the	O
+possibility	O
+that	O
+one	O
+Roquin	B-protein
+molecule	O
+may	O
+cluster	O
+two	O
+motifs	O
+in	O
+a	O
+given	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+and	O
+/	O
+or	O
+cluster	O
+motifs	O
+from	O
+distinct	O
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+to	O
+enhance	O
+downstream	O
+processing	O
+.	O
+Interestingly	O
+,	O
+two	O
+SL	B-structure_element
+RNA	B-chemical
+elements	O
+that	O
+resemble	O
+bona	O
+fide	O
+ligands	O
+of	O
+Roquin	B-protein
+have	O
+also	O
+been	O
+identified	O
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+the	O
+Nfkbid	B-protein
+mRNA	B-chemical
+.	O
+We	O
+therefore	O
+hypothesize	O
+that	O
+the	O
+combination	O
+of	O
+multiple	O
+binding	B-site
+sites	I-site
+may	O
+be	O
+more	O
+commonly	O
+used	O
+to	O
+enhance	O
+the	O
+functional	O
+activity	O
+of	O
+Roquin	B-protein
+.	O
+At	O
+the	O
+same	O
+time	O
+,	O
+the	O
+combination	O
+of	O
+cis	B-structure_element
+elements	I-structure_element
+may	O
+be	O
+important	O
+for	O
+differential	O
+gene	O
+regulation	O
+,	O
+as	O
+composite	O
+cis	B-structure_element
+elements	I-structure_element
+with	O
+lower	O
+affinity	B-evidence
+may	O
+be	O
+less	O
+sensitive	O
+to	O
+Roquin	B-protein
+.	O
+This	O
+will	O
+lead	O
+to	O
+less	O
+effective	O
+repression	O
+in	O
+T	O
+cells	O
+when	O
+antigen	O
+recognition	O
+is	O
+of	O
+moderate	O
+signal	O
+strength	O
+and	O
+only	O
+incomplete	O
+cleavage	O
+of	O
+Roquin	B-protein
+by	O
+MALT1	B-protein
+occurs	O
+.	O
+For	O
+understanding	O
+the	O
+intricate	O
+complexity	O
+of	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+regulation	O
+,	O
+future	O
+work	O
+will	O
+be	O
+necessary	O
+by	O
+combining	O
+large	O
+-	O
+scale	O
+approaches	O
+,	O
+such	O
+as	O
+cross	B-experimental_method
+-	I-experimental_method
+linking	I-experimental_method
+and	I-experimental_method
+immunoprecipitation	I-experimental_method
+experiments	I-experimental_method
+to	O
+identify	O
+RNA	B-site
+-	I-site
+binding	I-site
+sites	I-site
+,	O
+and	O
+structural	B-experimental_method
+biology	I-experimental_method
+to	O
+dissect	O
+the	O
+underlying	O
+molecular	O
+mechanisms	O
+.	O
+SELEX	B-experimental_method
+identifies	O
+a	O
+novel	O
+SL	B-structure_element
+RNA	B-chemical
+ligand	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+.	O
+(	O
+a	O
+)	O
+Enriched	O
+hexamers	O
+that	O
+were	O
+found	O
+by	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+N	O
+terminus	O
+(	O
+residues	O
+2	B-residue_range
+–	I-residue_range
+440	I-residue_range
+)	O
+or	O
+Roquin	B-mutant
+-	I-mutant
+1	I-mutant
+M199R	I-mutant
+N	O
+terminus	O
+(	O
+residues	O
+2	B-residue_range
+–	I-residue_range
+440	I-residue_range
+)	O
+(	O
+see	O
+also	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+).	O
+(	O
+b	O
+)	O
+An	O
+ADE	B-structure_element
+sequence	O
+motif	O
+in	O
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+closely	O
+resembles	O
+the	O
+MEME	B-experimental_method
+motif	O
+found	O
+in	O
+SELEX	B-experimental_method
+-	O
+enriched	O
+RNA	B-chemical
+sequences	O
+.	O
+(	O
+c	O
+)	O
+Conservation	O
+of	O
+the	O
+motif	O
+found	O
+in	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+for	O
+various	O
+species	O
+as	O
+indicated	O
+.	O
+rn5	B-gene
+is	O
+the	O
+fifth	O
+assembly	O
+version	O
+of	O
+the	O
+rat	B-taxonomy_domain
+(	O
+Rattus	B-species
+novegicus	I-species
+).	O
+(	O
+d	O
+)	O
+Schematic	O
+representation	O
+of	O
+the	O
+predicted	O
+SELEX	B-experimental_method
+-	O
+derived	O
+consensus	O
+SL	B-structure_element
+,	O
+ADE	B-structure_element
+and	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+hexaloop	B-structure_element
+SL	B-structure_element
+.	O
+The	O
+broken	O
+line	O
+between	O
+the	O
+G	O
+–	O
+G	O
+base	O
+pair	O
+in	O
+the	O
+ADE	B-structure_element
+SL	B-structure_element
+indicates	O
+a	O
+putative	O
+non	B-bond_interaction
+-	I-bond_interaction
+Watson	I-bond_interaction
+–	I-bond_interaction
+Crick	I-bond_interaction
+pairing	I-bond_interaction
+.	O
+The	O
+Ox40	B-protein
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+and	O
+the	O
+Tnf	B-protein
+CDE	B-structure_element
+SL	B-structure_element
+are	O
+shown	O
+for	O
+comparison	O
+.	O
+NMR	B-experimental_method
+analysis	O
+of	O
+the	O
+SL	B-structure_element
+RNAs	B-chemical
+used	O
+in	O
+this	O
+study	O
+.	O
+Imino	O
+proton	O
+regions	O
+of	O
+one	O
+-	O
+dimensional	O
+1H	B-experimental_method
+NMR	I-experimental_method
+spectra	B-evidence
+of	O
+(	O
+a	O
+)	O
+the	O
+ADE	B-structure_element
+SL	B-structure_element
+(	O
+b	O
+),	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+and	O
+(	O
+c	O
+)	O
+the	O
+Ox40	B-protein
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+are	O
+shown	O
+for	O
+free	B-protein_state
+RNAs	B-chemical
+(	O
+black	O
+)	O
+and	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+the	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+ROQ	B-structure_element
+domain	O
+(	O
+red	O
+).	O
+The	O
+respective	O
+SL	B-structure_element
+RNAs	B-chemical
+and	O
+their	O
+base	O
+pairs	O
+are	O
+indicated	O
+.	O
+Red	O
+asterisks	O
+indicate	O
+NMR	B-experimental_method
+signals	O
+of	O
+the	O
+protein	O
+.	O
+Green	O
+lines	O
+in	O
+the	O
+secondary	O
+structure	O
+schemes	O
+on	O
+the	O
+left	O
+refer	O
+to	O
+visible	O
+imino	O
+NMR	B-experimental_method
+signals	B-evidence
+and	O
+thus	O
+experimental	O
+confirmation	O
+of	O
+the	O
+base	O
+pairs	O
+indicated	O
+.	O
+The	O
+dotted	O
+green	O
+line	O
+between	O
+G6	B-residue_name_number
+and	O
+G15	B-residue_name_number
+in	O
+a	O
+highlights	O
+a	O
+G	B-residue_name
+–	O
+G	B-residue_name
+base	O
+pair	O
+.	O
+Structure	B-evidence
+of	O
+the	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+ROQ	B-structure_element
+domain	O
+bound	B-protein_state
+to	I-protein_state
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+RNA	B-chemical
+.	O
+(	O
+a	O
+)	O
+Cartoon	O
+presentation	O
+of	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+ROQ	B-structure_element
+domain	O
+(	O
+residues	O
+174	B-residue_range
+–	I-residue_range
+325	I-residue_range
+;	O
+blue	O
+)	O
+and	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNA	B-chemical
+(	O
+magenta	O
+).	O
+Selected	O
+RNA	B-chemical
+bases	O
+and	O
+protein	O
+secondary	O
+structure	O
+elements	O
+are	O
+labelled	O
+.	O
+(	O
+b	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+(	O
+bases	O
+in	O
+the	O
+RNA	B-chemical
+hexaloop	B-structure_element
+are	O
+shown	O
+in	O
+magenta	O
+)	O
+and	O
+(	O
+c	O
+)	O
+the	O
+previously	O
+reported	O
+structure	B-evidence
+of	O
+the	O
+ROQ	B-complex_assembly
+-	I-complex_assembly
+Tnf	I-complex_assembly
+CDE	I-complex_assembly
+complex	O
+(	O
+bases	O
+of	O
+the	O
+triloop	O
+RNA	B-chemical
+are	O
+shown	O
+in	O
+green	O
+).	O
+Only	O
+RNA	B-site
+-	I-site
+interacting	I-site
+residues	I-site
+that	O
+are	O
+different	O
+in	O
+both	O
+structures	B-evidence
+are	O
+shown	O
+.	O
+Both	O
+protein	O
+chains	O
+and	O
+remaining	O
+parts	O
+of	O
+both	O
+RNAs	B-chemical
+are	O
+shown	O
+in	O
+grey	O
+and	O
+protein	O
+residue	O
+side	O
+chains	O
+are	O
+shown	O
+in	O
+turquoise	O
+.	O
+(	O
+d	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+contacts	O
+between	O
+the	O
+ROQ	B-structure_element
+domain	O
+and	O
+nucleotides	O
+U11	B-residue_name_number
+and	O
+U13	B-residue_name_number
+of	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNA	B-chemical
+.	O
+The	O
+nucleotides	O
+interact	O
+with	O
+the	O
+C	O
+-	O
+terminal	O
+end	O
+of	O
+helix	B-structure_element
+α4	B-structure_element
+(	O
+Tyr250	B-residue_name_number
+and	O
+Ser253	B-residue_name_number
+)	O
+and	O
+the	O
+N	O
+-	O
+terminal	O
+part	O
+of	O
+strand	B-structure_element
+β3	B-structure_element
+(	O
+Phe255	B-residue_name_number
+and	O
+Val257	B-residue_name_number
+).	O
+The	O
+protein	O
+chain	O
+is	O
+shown	O
+in	O
+turquoise	O
+and	O
+the	O
+RNA	B-chemical
+is	O
+shown	O
+in	O
+grey	O
+.	O
+(	O
+e	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+contacts	O
+between	O
+the	O
+ROQ	B-structure_element
+domain	O
+and	O
+nucleotides	O
+U10	B-residue_name_number
+,	O
+U11	B-residue_name_number
+and	O
+U13	B-residue_name_number
+in	O
+the	O
+RNA	B-chemical
+hexaloop	B-structure_element
+.	O
+U11	B-residue_name_number
+and	O
+U13	B-residue_name_number
+contact	O
+the	O
+C	O
+-	O
+terminal	O
+end	O
+of	O
+helix	B-structure_element
+α4	B-structure_element
+:	O
+residues	O
+Tyr250	B-residue_name_number
+and	O
+Gln247	B-residue_name_number
+.	O
+The	O
+side	O
+chain	O
+of	O
+Tyr250	B-residue_name_number
+makes	O
+hydrophobic	B-bond_interaction
+interactions	I-bond_interaction
+with	O
+the	O
+pyrimidine	O
+side	O
+chain	O
+of	O
+U10	B-residue_name_number
+on	O
+one	O
+side	O
+and	O
+U11	B-residue_name_number
+on	O
+the	O
+other	O
+side	O
+.	O
+Lys259	B-residue_name_number
+interacts	O
+with	O
+the	O
+phosphate	O
+groups	O
+of	O
+U10	B-residue_name_number
+and	O
+U11	B-residue_name_number
+.	O
+(	O
+f	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+hydrophobic	B-bond_interaction
+interaction	I-bond_interaction
+between	O
+Val257	B-residue_name_number
+and	O
+U11	B-residue_name_number
+,	O
+as	O
+well	O
+as	O
+the	O
+double	O
+hydrogen	B-bond_interaction
+bond	I-bond_interaction
+of	O
+Lys259	B-residue_name_number
+with	O
+phosphate	O
+groups	O
+of	O
+U10	B-residue_name_number
+and	O
+U11	B-residue_name_number
+.	O
+NMR	B-experimental_method
+analysis	O
+of	O
+ROQ	B-structure_element
+domain	O
+interactions	O
+with	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+hexaloop	B-structure_element
+RNA	B-chemical
+.	O
+(	O
+a	O
+)	O
+Overlay	B-experimental_method
+of	O
+1H	B-experimental_method
+,	I-experimental_method
+15N	I-experimental_method
+HSQC	I-experimental_method
+spectra	B-evidence
+of	O
+either	O
+the	O
+free	B-protein_state
+ROQ	B-structure_element
+domain	O
+(	O
+171	B-residue_range
+–	I-residue_range
+326	I-residue_range
+,	O
+black	O
+)	O
+or	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+stoichiometric	O
+amounts	O
+of	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+(	O
+red	O
+).	O
+(	O
+b	O
+)	O
+Plot	O
+of	O
+chemical	B-evidence
+shift	I-evidence
+change	I-evidence
+versus	O
+residue	O
+number	O
+in	O
+the	O
+ROQ	B-structure_element
+domain	O
+(	O
+residues	O
+171	B-residue_range
+–	I-residue_range
+326	I-residue_range
+)	O
+from	O
+a	O
+.	O
+Grey	O
+negative	O
+bars	O
+indicate	O
+missing	O
+assignments	O
+in	O
+one	O
+of	O
+the	O
+spectra	B-evidence
+.	O
+Gaps	O
+indicate	O
+prolines	B-residue_name
+.	O
+(	O
+c	O
+)	O
+Overlay	B-experimental_method
+of	O
+the	O
+ROQ	B-structure_element
+domain	O
+alone	B-protein_state
+(	O
+black	O
+)	O
+or	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+(	O
+red	O
+)	O
+or	O
+the	O
+Ox40	B-protein
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+(	O
+green	O
+).	O
+Mutational	B-experimental_method
+analysis	I-experimental_method
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+-	O
+interactions	O
+with	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+and	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+.	O
+(	O
+a	O
+)	O
+EMSA	B-experimental_method
+assay	I-experimental_method
+comparing	O
+binding	O
+of	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+of	O
+the	O
+Y250A	B-mutant
+mutant	B-protein_state
+ROQ	B-structure_element
+domain	O
+for	O
+binding	O
+to	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+(	O
+left	O
+)	O
+or	O
+the	O
+previously	O
+described	O
+Tnf	B-protein
+CDE	B-structure_element
+SL	B-structure_element
+(	O
+right	O
+).	O
+A	O
+comparison	O
+of	O
+further	O
+mutants	O
+is	O
+shown	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+.	O
+(	O
+b	O
+)	O
+Schematic	O
+overview	O
+of	O
+the	O
+timeline	O
+used	O
+for	O
+the	O
+reconstitution	O
+experiment	O
+shown	O
+in	O
+c	O
+.	O
+(	O
+c	O
+)	O
+Flow	B-experimental_method
+cytometry	I-experimental_method
+of	O
+Ox40	B-protein
+and	O
+Icos	B-protein
+surface	O
+expression	O
+on	O
+CD4	O
++	O
+Th1	O
+cells	O
+from	O
+Rc3h1	B-gene
+/	O
+2fl	B-gene
+/	O
+fl	B-gene
+;	O
+Cd4	O
+-	O
+Cre	O
+-	O
+ERT2	O
+;	O
+rtTA	O
+mice	B-taxonomy_domain
+treated	O
+with	O
+tamoxifen	B-chemical
+(+	O
+tam	O
+)	O
+to	O
+induce	O
+Rc3h1	B-gene
+/	O
+2fl	B-gene
+/	O
+fl	B-gene
+deletion	B-experimental_method
+or	O
+left	O
+untreated	O
+(−	O
+tam	O
+).	O
+The	O
+cells	O
+were	O
+then	O
+either	O
+left	O
+untransduced	O
+(	O
+UT	O
+)	O
+or	O
+were	O
+transduced	O
+with	O
+retrovirus	B-taxonomy_domain
+containing	O
+a	O
+doxycycline	B-chemical
+-	O
+inducible	O
+cassette	O
+,	O
+to	O
+express	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+WT	B-protein_state
+,	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+Y250A	B-mutant
+or	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+K220A	B-mutant
+,	O
+K239A	B-mutant
+and	O
+R260A	B-mutant
+mutants	B-protein_state
+(	O
+see	O
+also	O
+Supplementary	O
+Fig	O
+.	O
+5	O
+).	O
+Functional	O
+importance	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+target	O
+motifs	O
+in	O
+cells	O
+.	O
+(	O
+a	O
+)	O
+Overview	O
+of	O
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+and	O
+truncated	B-protein_state
+/	O
+mutated	B-protein_state
+versions	O
+thereof	O
+as	O
+used	O
+for	O
+EMSA	B-experimental_method
+assays	O
+in	O
+b	O
+and	O
+the	O
+expression	O
+experiments	O
+of	O
+Ox40	B-protein
+in	O
+c	O
+and	O
+d	O
+.	O
+(	O
+b	O
+)	O
+EMSA	B-experimental_method
+experiments	O
+probing	O
+the	O
+interaction	O
+between	O
+the	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+N	O
+-	O
+terminal	O
+region	O
+(	O
+residues	O
+2	B-residue_range
+–	I-residue_range
+440	I-residue_range
+)	O
+and	O
+either	O
+the	O
+complete	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+or	O
+versions	O
+with	O
+mutations	B-experimental_method
+of	O
+the	O
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+,	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+or	O
+both	O
+SLs	B-structure_element
+(	O
+see	O
+a	O
+).	O
+It	O
+is	O
+noteworthy	O
+that	O
+the	O
+higher	O
+bands	O
+observed	O
+at	O
+large	O
+protein	O
+concentrations	O
+are	O
+probably	O
+additional	O
+nonspecific	O
+,	O
+lower	O
+-	O
+affinity	O
+interactions	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+with	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+or	O
+protein	O
+aggregates	O
+.	O
+(	O
+c	O
+)	O
+Relative	O
+Ox40	B-protein
+MFI	B-evidence
+normalized	I-evidence
+to	I-evidence
+expression	I-evidence
+levels	I-evidence
+from	O
+the	O
+Ox40	B-protein
+CDS	B-structure_element
+construct	O
+.	O
+Error	O
+bars	O
+show	O
+s	O
+.	O
+d	O
+.	O
+of	O
+seven	O
+(	O
+CDS	B-structure_element
+,	O
+1	B-residue_range
+–	I-residue_range
+40	I-residue_range
+,	O
+1	B-residue_range
+–	I-residue_range
+80	I-residue_range
+,	O
+1	B-residue_range
+–	I-residue_range
+120	I-residue_range
+and	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+),	O
+six	O
+(	O
+ADE	B-structure_element
+-	O
+like	O
+mut	B-protein_state
+and	O
+CDE	B-structure_element
+mut	B-protein_state
+)	O
+or	O
+three	O
+(	O
+double	B-protein_state
+mut	I-protein_state
+)	O
+independent	O
+experiments	O
+.	O
+Statistical	O
+significance	O
+was	O
+calculated	O
+by	O
+one	B-experimental_method
+-	I-experimental_method
+way	I-experimental_method
+analysis	I-experimental_method
+of	I-experimental_method
+variance	I-experimental_method
+(	O
+ANOVA	B-experimental_method
+)	O
+Kruskal	B-experimental_method
+–	I-experimental_method
+Wallis	I-experimental_method
+test	I-experimental_method
+followed	O
+by	O
+Dunn	B-experimental_method
+’	I-experimental_method
+s	I-experimental_method
+multiple	I-experimental_method
+comparison	I-experimental_method
+test	I-experimental_method
+(**	O
+P	O
+<	O
+0	O
+.	O
+01	O
+).	O
+(	O
+d	O
+)	O
+mRNA	B-evidence
+decay	I-evidence
+curves	I-evidence
+of	O
+Hela	O
+Tet	O
+-	O
+Off	O
+cells	O
+stably	O
+transduced	O
+with	O
+retroviruses	B-taxonomy_domain
+expressing	O
+Ox40	B-protein
+CDS	B-structure_element
+without	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+(	O
+CDS	B-structure_element
+,	O
+red	O
+line	O
+),	O
+Ox40	B-protein
+CDS	B-structure_element
+with	O
+its	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+(	O
+full	B-protein_state
+length	I-protein_state
+,	O
+black	O
+line	O
+),	O
+Ox40	B-protein
+full	B-protein_state
+length	I-protein_state
+with	O
+mutated	B-protein_state
+ADE	B-structure_element
+-	O
+like	O
+motif	O
+(	O
+ADE	B-structure_element
+-	O
+like	O
+mut	B-protein_state
+,	O
+grey	O
+line	O
+),	O
+Ox40	B-protein
+full	B-protein_state
+length	I-protein_state
+with	O
+mutated	B-protein_state
+CDE	B-structure_element
+-	O
+like	O
+motif	O
+(	O
+CDE	B-structure_element
+-	O
+like	O
+mut	B-protein_state
+,	O
+green	O
+line	O
+)	O
+or	O
+Ox40	B-protein
+full	B-protein_state
+length	I-protein_state
+with	O
+mutated	B-protein_state
+ADE	B-structure_element
+and	O
+CDE	B-structure_element
+motifs	O
+(	O
+Double	B-protein_state
+mut	I-protein_state
+,	O
+blue	O
+line	O
+).	O
+mRNA	B-evidence
+half	I-evidence
+-	I-evidence
+life	I-evidence
+times	I-evidence
+were	O
+calculated	O
+with	O
+Graph	O
+Pad	O
+Prism	O
+.	O
+Data	B-evidence
+collection	I-evidence
+and	I-evidence
+refinement	I-evidence
+statistics	I-evidence
+.	O
+ROQ	B-structure_element
+-	O
+Ox40ADE	B-protein
+-	O
+like	O
+SL	B-structure_element
+ROQ	B-structure_element
+-	O
+ADE	B-structure_element
+SL	B-structure_element
+Data	O
+collection	O
+space	O
+group	O
+P21212	O
+P212121	O
+Cell	O
+dimensions	O
+a	O
+,	O
+b	O
+,	O
+c	O
+(	O
+Å	O
+)	O
+89	O
+.	O
+66	O
+,	O
+115	O
+.	O
+79	O
+,	O
+42	O
+.	O
+61	O
+72	O
+.	O
+90	O
+,	O
+89	O
+.	O
+30	O
+,	O
+144	O
+.	O
+70	O
+α	O
+,	O
+β	O
+,	O
+γ	O
+(°)	O
+90	O
+,	O
+90	O
+,	O
+90	O
+90	O
+,	O
+90	O
+,	O
+90	O
+Resolution	O
+(	O
+Å	O
+)	O
+50	O
+–	O
+2	O
+.	O
+23	O
+(	O
+2	O
+.	O
+29	O
+–	O
+2	O
+.	O
+23	O
+)	O
+50	O
+–	O
+3	O
+.	O
+0	O
+(	O
+3	O
+.	O
+08	O
+–	O
+3	O
+.	O
+00	O
+)	O
+Rmerge	O
+5	O
+.	O
+9	O
+(	O
+68	O
+.	O
+3	O
+)	O
+14	O
+.	O
+8	O
+(	O
+93	O
+.	O
+8	O
+)	O
+I	O
+/	O
+σI	O
+14	O
+.	O
+9	O
+(	O
+2	O
+.	O
+1	O
+)	O
+16	O
+.	O
+7	O
+(	O
+3	O
+.	O
+1	O
+)	O
+Completeness	O
+(%)	O
+98	O
+.	O
+7	O
+(	O
+97	O
+.	O
+7	O
+)	O
+99	O
+.	O
+9	O
+(	O
+99	O
+.	O
+9	O
+)	O
+Redundancy	O
+3	O
+.	O
+9	O
+(	O
+3	O
+.	O
+7	O
+)	O
+13	O
+.	O
+2	O
+(	O
+12	O
+.	O
+7	O
+)	O
+Refinement	O
+Resolution	O
+(	O
+Å	O
+)	O
+2	O
+.	O
+23	O
+3	O
+.	O
+00	O
+No	O
+.	O
+reflections	O
+21	O
+,	O
+018	O
+18	O
+,	O
+598	O
+Rwork	O
+/	O
+Rfree	O
+21	O
+.	O
+8	O
+/	O
+25	O
+.	O
+7	O
+18	O
+.	O
+6	O
+/	O
+23	O
+.	O
+4	O
+No	O
+.	O
+atoms	O
+Protein	O
+2	O
+,	O
+404	O
+4	O
+,	O
+820	O
+Ligand	O
+/	O
+ion	O
+894	O
+1	O
+,	O
+708	O
+Water	O
+99	O
+49	O
+B	O
+-	O
+factor	O
+overall	O
+47	O
+.	O
+2	O
+60	O
+.	O
+4	O
+Root	B-evidence
+mean	I-evidence
+squared	I-evidence
+deviations	I-evidence
+Bond	O
+lengths	O
+(	O
+Å	O
+)	O
+0	O
+.	O
+006	O
+0	O
+.	O
+014	O
+Bond	O
+angles	O
+(°)	O
+1	O
+.	O
+07	O
+1	O
+.	O
+77	O
+Ramachandran	O
+plot	O
+Most	O
+favoured	O
+(%)	O
+98	O
+.	O
+6	O
+99	O
+.	O
+8	O
+Additional	O
+allowed	O
+(%)	O
+1	O
+.	O
+4	O
+0	O
+.	O
+2	O
+ADE	B-structure_element
+,	O
+alternative	B-structure_element
+decay	I-structure_element
+element	I-structure_element
+;	O
+CDE	B-structure_element
+,	O
+constitutive	B-structure_element
+decay	I-structure_element
+element	I-structure_element
+;	O
+SL	B-structure_element
+,	O
+stem	B-structure_element
+loop	I-structure_element
+.	O
+For	O
+each	O
+data	O
+set	O
+,	O
+only	O
+one	O
+crystal	B-evidence
+has	O
+been	O
+used	O
+.	O
+KD	B-evidence
+for	O
+selected	O
+RNAs	B-chemical
+obtained	O
+from	O
+SPR	B-experimental_method
+measurements	I-experimental_method
+with	O
+immobilized	O
+ROQ	B-structure_element
+domain	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+.	O

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+<collection><source>PMC</source><date>20201216</date><key>pmc.key</key><document><id>4806292</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/srep23641</infon><infon key="article-id_pii">srep23641</infon><infon key="article-id_pmc">4806292</infon><infon key="article-id_pmid">27009356</infon><infon key="elocation-id">23641</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:Klima;given-names:Martin</infon><infon key="name_1">surname:Tóth;given-names:Dániel J.</infon><infon key="name_10">surname:Humpolickova;given-names:Jana</infon><infon key="name_11">surname:Nencka;given-names:Radim</infon><infon key="name_12">surname:Veverka;given-names:Vaclav</infon><infon key="name_13">surname:Balla;given-names:Tamas</infon><infon key="name_14">surname:Boura;given-names:Evzen</infon><infon key="name_2">surname:Hexnerova;given-names:Rozalie</infon><infon key="name_3">surname:Baumlova;given-names:Adriana</infon><infon key="name_4">surname:Chalupska;given-names:Dominika</infon><infon key="name_5">surname:Tykvart;given-names:Jan</infon><infon key="name_6">surname:Rezabkova;given-names:Lenka</infon><infon key="name_7">surname:Sengupta;given-names:Nivedita</infon><infon key="name_8">surname:Man;given-names:Petr</infon><infon key="name_9">surname:Dubankova;given-names:Anna</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 and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>121</offset><text>Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1035</offset><text>Phosphatidylinositol 4-kinase beta (PI4KB, also known as PI4K IIIβ) is a soluble cytosolic protein yet its function is to phosphorylate membrane lipids. It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P). PI4P is an essential lipid found in various membrane compartments including the Golgi and trans-Golgi network (TGN), the plasma membrane and the endocytic compartments. In these locations, PI4P plays an important role in cell signaling and lipid transport, and serves as a precursor for higher phosphoinositides or as a docking site for clathrin adaptor or lipid transfer proteins. A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories. These structures are essential for effective viral replication. Recently, highly specific PI4KB inhibitors were developed as potential antivirals.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2112</offset><text>PI4K kinases must be recruited to the correct membrane type to fulfill their enzymatic functions. Type II PI4Ks (PI4K2A and PI4K2B) are heavily palmitoylated and thus behave as membrane proteins. In contrast, type III PI4Ks (PI4KA and PI4KB) are soluble cytosolic proteins that are recruited to appropriate membranes indirectly via protein-protein interactions. The recruitment of PI4KA to the plasma membrane by EFR3 and TTC7 is relatively well understood even at the structural level, but, the actual molecular mechanism of PI4KB recruitment to the Golgi is still poorly understood.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2697</offset><text>Acyl-coenzyme A binding domain containing protein 3 (ACBD3, also known as GCP60 and PAP7) is a Golgi resident protein. Its membrane localization is mediated by the interaction with the Golgi integral protein golgin B1/giantin. ACBD3 functions as an adaptor protein and signaling hub across cellular signaling pathways. ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis. ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2. ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3754</offset><text>We present a biochemical and structural characterization of the molecular complex composed of the ACBD3 protein and the PI4KB enzyme. We show that ACBD3 can recruit PI4KB to model membranes as well as redirect PI4KB to cellular membranes where it is not naturally found. Our data also show that ACBD3 regulates the enzymatic activity of PI4KB kinase through membrane recruitment rather than allostery.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>4156</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>4164</offset><text>ACBD3 and PI4KB interact with 1:1 stoichiometry with submicromolar affinity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4240</offset><text>In order to verify the interactions between ACBD3 and PI4KB we expressed and purified both proteins. To increase yields of bacterial expression the intrinsically disordered region of PI4KB (residues 423–522) was removed (Fig. 1A). This internal deletion does not significantly affect the kinase activity(SI Fig. 1A) or interaction with ACBD3 (SI Fig. 1B,C). In an in vitro binding assay, ACBD3 co-purified with the NiNTA-immobilized N-terminal His6GB1-tagged PI4KB (Fig. 1B, left panel), suggesting a direct interaction. Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain. We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5275</offset><text>Because it has been reported that ACBD3 can dimerize in a mammalian two-hybrid assay, we were interested in determining the stoichiometry of the ACBD3:PI4KB protein complex. The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively. This result suggests that both proteins are monomeric and the stoichiometry of the ACBD3: PI4KB protein complex is 1:1 (Fig. 1C, left panel). Similar results were obtained for the complex of the Q domain of ACBD3 and the N-terminal region of PI4KB (Fig. 1C, right panel). We also determined the strength of the interaction between recombinant full length ACBD3 and PI4KB using surface plasmon resonance (SPR). SPR measurements revealed a strong interaction with a Kd value of 320 +/−130 nM (Fig. 1D, SI Fig. 1D). We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>6446</offset><text>Structural analysis of the ACBD3:PI4KB complex</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6493</offset><text>Full length ACBD3 and PI4KB both contain large intrinsically disordered regions that impede crystallization. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of the complex to determine which parts of the complex are well folded (SI Fig. 2). However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6926</offset><text>For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization. As the N-terminal region protein complex was prepared by co-expression of both proteins, the samples consisted of an equimolar mixture of two uniformly 15N/13C labelled molecules. Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously. Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44. Over 93% of non-exchangeable side-chain signals were assigned for the free ACBD3 Q domain. Apart from the four N-terminal residues, the side-chain assignments were missing for Gln (Hg3), Gln (Ha/Hb/Hg), Gln44 (Ha/Hb/Hg) and Gln48 (Hg) mainly due to extensive overlaps within the spectral regions populated by highly abundant glutamine side-chain resonances. The protein complex yielded relatively well resolved spectra (SI Fig. 4) that resulted in assignment of backbone amide signals for all residues apart from Gln (ACBD3) and Ala2 (PI4KB). Assignments obtained for non-exchangeable side-chain signals were over 99% complete. The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation. Structural statistics for the final water-refined sets of structures are shown in SI Table 1.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8698</offset><text>This structure revealed that the Q domain forms a two helix hairpin. The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A). Preceding the kinase helix are three ordered residues (Val42, Ile43, and Asp44) that also contribute to the interaction (Fig. 2B). The remaining part of the PI4KB N-termini, however, is disordered (SI Fig. 5). Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B). Interestingly, we noted that the PI4KB helix is amphipathic and its hydrophobic surface leans on the Q domain (Fig. 2C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9580</offset><text>To corroborate the structural data, we introduced a number of point mutations and validated their effect on complex formation using an in vitro pull-down assay (Fig. 2D). Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10144</offset><text>ACBD3 efficiently recruits the PI4KB enzyme to membranes</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10201</offset><text>We next sought to determine if the ACBD3:PI4KB interaction drives membrane localization of the PI4KB enzyme. To do this, we first established an in vitro membrane recruitment system using Giant Unilamellar Vesicles (GUVs) containing the PI4KB substrate – the PI lipid. We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10759</offset><text>We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel). We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B). Given this result, overexpression of the Q domain should also interfere with the PI4KB dependent Golgi functions. Ceramide transport and accumulation in Golgi is a well-known PI4KB dependent process. We have used fluorescently labeled ceramide and analyzed its trafficking in non-transfected cells and cell overexpressing the Q domain. As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12033</offset><text>We further analyzed the function of ACBD3:PI4KB interaction in membrane recruitment of PI4KB in living cells using fluorescently tagged proteins. We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system. We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP. The ACBD3 Q domain was then fused to FKBP12 and mRFP (Fig. 3D). We analyzed localization of the ACBD3 Q domain and GFP – PI4KB before and after the addition of rapamycin. As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase. In every case the ACDB3 Q domain was rapidly (within 5 minutes) recruited to the mitochondrial membrane upon addition of rapamycin, but only the wild-type protein effectively directed the kinase to the mitochondria (Fig. 3E, Movie 1 and 2). Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel). However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13272</offset><text>ACBD3 increases PI4KB enzymatic activity by recruiting PI4KB to close vicinity of its substrate</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13368</offset><text>To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate. We observed no effect on the kinase activity of PI4KB (Fig. 4A) suggesting that ACBD3 does not directly affect the enzyme (e.g. induction of a conformation change). However, in vivo ACBD3 is located at the Golgi membranes, whereas in this experiment, ACBD3 was located in the solution and PI is provided as micelles. We therefore designed a more physiologically relevant experiment. For this, we again turned to the GUV system with ACBD3 localized to the GUV membrane. The GUVs contained 10% PI to serve as a substrate for PI4KB kinase. The buffer also contained CFP-SidC, which binds to PI4P with nanomolar affinity. This enabled visualization of the kinase reaction using a confocal microscope. We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3. Reaction was also performed in the absence of ATP as a negative control (Fig. 4B). These experiments showed that PI4KB enzymatic activity increases when ACBD3 is membrane localized (Fig. 4C, SI Fig. 6). We conclude that enzyme activation proceeds through a membrane recruitment mechanism.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>14664</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>14675</offset><text>Membrane recruitment of PI4KB enzyme is crucial to ensure its proper function at the Golgi and TGN. However, the molecular mechanism and structural basis for PI4KB interaction with the membrane is poorly understood. In principle, any of the binding partners of PI4KB could play a role in membrane recruitment. To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>15258</offset><text>The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase. Although Rab11 does not appear to be required for recruitment of PI4KB to the Golgi, PI4KB is required for Golgi recruitment of Rab11. Arf1, the other small GTP binding protein, is known to influence the activity and localization of PI4KB, but it does not appear to interact directly with PI4KB (our unpublished data). The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear. NCS-1 is an N-terminally myristoylated protein that participates in exocytosis. It is expressed only in certain cell types, suggesting that if it contributes to PI4KB membrane recruitment, it does so in a tissues specific manner. The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation. However, 14-3-3 proteins do not appear to interfere with membrane recruitment of this kinase. ACBD3 is a Golgi resident protein, conserved among vertebrates (SI Fig. 7), that interacts directly with PI4KB (see also SI Fig. 8 and SI Discussion), and whose genetic inactivation interferes with the Golgi localization of the kinase. For these reasons we focused on the interaction of the PI4KB enzyme with the Golgi resident ACBD3 protein in this study.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>16782</offset><text>Here we present the mechanism for membrane recruitment of PI4KB by the Golgi resident ACBD3 protein. We show that these proteins interact directly with a Kd value in the submicromolar range. The interaction is sufficient to recruit PI4KB to model membranes in vitro as well as to the mitochondria where PI4KB is never naturally found. To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain. The Q domain adopts a helical hairpin fold that is further stabilized upon binding the kinase helix (Fig. 2A). Our data strongly suggest that formation of the complex does not directly influence the catalytic abilities of the kinase but experiments with model membranes revealed that ACBD3 enhances catalytic activity of the kinase by a recruitment based mechanism; it recruits the kinase to the membrane and thus increases the local concentration of the substrate in the vicinity of the kinase. Based on our and previously published structures we built a pseudoatomic model of PI4KB multi-protein assembly on the membrane (Fig. 5) that illustrates how the enzyme is recruited and positioned towards its lipidic substrate and how it in turn recruits Rab11.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>18118</offset><text>+RNA viruses replicate at specific PI4P-enriched membranous compartments. These are called replication factories (because they enhance viral replication) or membranous webs (because of their appearance under the electron microscope). To generate replication factories, viruses hijack several host factors including the PI4K kinases to secure high content of the PI4P lipid. Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3. Our data suggest that they could do this via 3A:ACBD3:PI4KB complex formation. The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication. This could eventually have implications for therapeutic intervention to combat picornaviruses-mediated diseases ranging from polio to the common cold.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>19131</offset><text>Materials and Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>19153</offset><text>Plasmid construction, protein expression, and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>19212</offset><text>All proteins used in this study were recombinant and were expressed in E. coli using previously developed protocols. Briefly, full-length human ACBD3 (UniProtKB entry Q9H3P7) and PI4KB (UniProtKB entry Q9UBF8, isoform 1) lipid kinase and their deletion mutants were cloned into a previously modified pRSFD vector (Novagen) that already contained an N-terminal 6xHis tag followed by a GB1 solubility tag and TEV protease cleavage site. Mutations were generated using the Phusion Site-Directed Mutagenesis Kit (Thermo Scientific). The plasmids used are listed in the SI (SI Table 2). The proteins were expressed in E. coli BL21 Star cells as previously described. Upon overnight expression in autoinduction media bacterial cells were harvested and lysed in lysis buffer (50 mM Tris pH 8, 300 mM NaCl, 3 mM β-mercaptoethanol, 20 mM imidazole, 10% glycerol). The lysate was incubated with the Ni-NTA resin (Macherey-Nagel) and then extensively washed with the lysis buffer. The protein was eluted with the lysis buffer supplemented with 300 mM imidazole. When appropriate, tags were removed with TEV protease, and the protein was further purified using the size exclusion chromatography on Superdex 75 or Superdex 200 columns (GE Healthcare) in SEC buffer (10 mM Tris pH 8, 200 mM NaCl, 3 mM β-ME). Proteins were concentrated to 1–5 mg/ml (measured spectroscopically) and stored at −80 °C until needed.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>20638</offset><text>In vitro pull-downs</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20658</offset><text>Ni-NTA sepharose beads (Macherey-Nagel) were mixed with both binding partners (one of which was tagged with an N-terminal 6xHis tag) at a final concentration of 1 μM in a final volume of 200 μL binding buffer (30 mM Tris pH 8, 200 mM NaCl, 10 mM imidazole, and 1 mM TCEP). After 30 min incubation at 4 °C the beads were washed twice with 200 μL of the binding buffer, and total protein was directly eluted with the Laemmli sample buffer and analyzed by SDS-PAGE.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>21143</offset><text>SPR (Surface plasmon resonance) and AUC (Analytical ultracentrifugation)</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>21216</offset><text>PI4KB was chip-immobilized as detailed in the SI. Afterwards, the ACBD3 protein was injected in a series of concentrations for 3 min and then dissociation was monitored for another 5 min. The data were fit to a single-exponential model. Rate constants of association and dissociation were obtained by fitting the observed change in resonance signal using the following equations:</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>21600</offset><text>where c is the protein concentration, t is time, kon is the association rate constant, koff is the dissociation rate constant, D1 and D2 are the linear drift terms, and Ras, Rdis, R0, R1, and Rmax are corresponding changes in the relative response signal.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>21856</offset><text>AUC was used to perform sedimentation velocity experiments using a ProteomeLab XL-I Beckman Coulter analytical ultracentrifuge equipped with an AN50Ti rotor. All measurements were performed in 10 mM Tris pH 8, 200 mM NaCl, and 3 mM β-mercaptoethanol at 20 °C and 48000 rpm. All data were collected using an absorbance (230 nm and 280 nm) optical system. Data analysis was performed with the SEDFIT package and data were analyzed using a sedimentation coefficient distribution model c(s).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>22360</offset><text>In vitro kinase assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>22382</offset><text>In vitro kinase activity was measured using a bioluminescent ADP-Glo assay (Promega) as described previously. Briefly, reactions were carried out in a total volume of 5 μL in 384-well plates by diluting the indicated amounts of the PI4KB enzyme and/or ACBD3 protein into the kinase buffer (20 mM Tris pH 7.5, 5 mM MgCl2, 0.2% Triton-X100, 0.1 mg/mL BSA, 2 mM DTT, 50 μM phosphatidylinositol). Reaction was initiated by adding ATP to a final concentration of 100 μM. Samples were incubated for 60 min at 25 °C and the amount of hydrolyzed ATP was measured according to the manufacturer’s protocol using a TECAN infinite M 1000 plate reader.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>23046</offset><text>NMR spectroscopy</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>23063</offset><text>NMR spectra were acquired at 25 °C on a 600 MHz and 850 MHz Bruker Avance spectrometers, both of which were equipped with a triple-resonance (15N/13C/1H) cryoprobe. The sample volume was 0.35 mL, with a 280 μM concentration for the free Q domain and a 470 μM concentration for the ACBD3:PI4KB complex in the NMR buffer (25 mM sodium phosphate pH 6.5, 100 mM NaCl, 1 mM TCEP, 0.01% NaN3), 5% D2O/95% H2O. A series of double- and triple-resonance spectra were recorded to determine essentially complete sequence-specific resonance backbone and side-chain assignments. Constraints for 1H-1H distance required to calculate the structure of free Q domain and ACBD3:PI4KB complex were derived from 3D 15N/1H NOESY-HSQC and 13C/1H NOESY-HMQC, which were acquired using a NOE mixing time of 100 ms.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>23877</offset><text>The families of converged structures for the ACBD3:PI4KB complex and free Q domain were calculated using standard software as detailed in the SI. The structures with the lowest total energy were selected and validated. The statistics for the resulting structures are summarized in SI Table 1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24170</offset><text>Protein labeling with fluorescent dyes</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24209</offset><text>PI4KB was labeled on native cysteine residues. Briefly, pure recombinant protein was incubated overnight at 4 °C with a 3x molar excess of Alexa 488 C5 maleimide (Life Technologies). The reaction was quenched by adding 10 mM β-mercaptoethanol (βME) and the protein was repurified by size exclusion chromatography.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24530</offset><text>Giant Unilamellar Vesicle Preparation and Imaging</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24580</offset><text>Giant Unilamellar Vesicles (GUVs) composed of POPC (54.9 mol %), POPS (10 mol %), cholesterol (20 mol %), PI (10 mol %), DGS-NTA(Ni) [1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) ] (5 mol %) (Avanti Polar lipids), and ATTO647N-DOPE (0.1 mol %) (ATTO-TEC GmbH) were prepared by electroformation as described previously, please see SI.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24979</offset><text>Live Cell Imaging</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24997</offset><text>COS-7 cells were plated onto 29-mm-diameter poly-L-Lysine coated glass bottom dishes (In Vitro Scientific) at 100,000 cells/well density and transfected using the Lipofectamine2000 reagent (Invitrogen) with plasmid DNAs (0.5–1 mg/well) according to manufacturer’s instructions. The plasmids are described in SI Table 2. 24 hr post transfection, COS-7 cells were washed with a modified Krebs-Ringer solution (10 mM Na-HEPES pH 7.4, 120 mM NaCl, 4.7 mM KCl, 2 mM CaCl2, 0.7 mM MgSO4, 10 mM glucose) in the same dish and were imaged using an LSM 710 confocal microscope (Carl Zeiss MicroImaging) with a 63 × 1.4-numerical-aperture planapochromatic objective. For ceramide uptake experiments, COS-7 cells were loaded with 0.05 μM BODIPY® FL C5-Ceramide (Molecular Probes, ThermoFisher Scientific) complexed with BSA in modified Krebs-Ringer solution at room temperature for 20 min. Cells were then washed three times and imaged using the above mentioned settings.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>25988</offset><text>Immunofluorescent imaging</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26014</offset><text>COS-7 cells were plated onto 25-mm-diameter poly-L-Lysine coated circular glass coverslips in six-well plates (100,000 cells/well), and transfected using the Lipofectamine2000 reagent (Invitrogen) with plasmid DNAs (0.5–1 mg/well) according to manufacturer’s instructions. Twenty four hours post transfection, cells were washed with PBS, fixed with 4% paraformaldehyde, stained with mouse anti-PI4KB primary antibody (BD Transduction Laboratories, 1:500 dilution) and then after washing with PBS stained with Alexa Fluor 647 conjugated donkey anti-mouse secondary antibody (Molecular Probes, ThermoFisher Scientific, 1:500 dilution). Cover slips were mounted and observed with the above mentioned microscopy settings.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>26738</offset><text>HD exchange</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26750</offset><text>Hydrogen/deuterium exchange was performed as previously described with the following modifications. The exchange was done in 10 mM Tris-HCl pD 8.0, 200 mM NaCl at 20 °C. Protein concentration during the exchange was 1 μM. Aliquots (50 μL) were removed after 10, 20, 60, 120, 600, 1800, and 3600 s and the exchange was quenched by the addition of 50 μL of 0.25 M glycine-HCl pH 2.3 and rapid freezing in liquid nitrogen.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>27188</offset><text>Prior to the analysis each sample was quickly thawed and injected onto an immobilized rhizopuspepsin column (bed volume 66 μL). Digestion was driven by a flow of 0.4% formic acid in water at a flow rate of 100 μL/min (LC-20AD pump, Shimadzu). The resulting peptides were trapped and desalted online on a peptide microtrap (Optimize Technologies). After a desalting step (3 min), peptides were separated using a linear gradient of 10–25% buffer B for 2 min, followed by a quick jump to 99% buffer B (buffer A = 0.4% formic acid/2% acetonitrile in water; buffer B = 95% acetonitrile/0.4% formic acid in water). The outlet of the LC system was interfaced to an electrospray ionization source of a Fourier transform ion cyclotron resonance mass spectrometer (12 T SolariX XR, Bruker Daltonics). Exchange was followed on 32 peptides from PI4KB (N) and 26 peptides from ACBD3(Q), covering in both cases 100% of the protein sequence. Peptides were identified by LC-MS/MS and MASCOT search against a database containing the sequences of the studied proteins. Data from H/D exchange were analyzed by program DeutEx written in the laboratory (unpublished).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>28355</offset><text>Additional Information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28378</offset><text>Accession codes: The structures and assigned chemical shifts for the free Q domain and the ACBD3:PI4KB complex were deposited in PDB database under accession codes 2N72 and 2N73, and BMRB database under accession codes 25790 and 25791.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28614</offset><text>How to cite this article: Klima, M. et al. Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein. Sci. 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A.</infon><infon key="name_1">surname:Mezulis;given-names:S.</infon><infon key="name_2">surname:Yates;given-names:C. M.</infon><infon key="name_3">surname:Wass;given-names:M. N.</infon><infon key="name_4">surname:Sternberg;given-names:M. J.</infon><infon key="pub-id_doi">10.1038/nprot.2015.053</infon><infon key="pub-id_pmid">25950237</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>33401</offset><text>The Phyre2 web portal for protein modeling, prediction and analysis</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>33469</offset><text>The authors declare no competing financial interests.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>33523</offset><text>Author Contributions M.K. and A.D. carried out DNA cloning, M.K., A.B., D.C. and E.B. carried out protein expression and purification, M.K. performed pull-down assays, L.R. carried out analytical ultracentrifugation, M.K. and J.T. performed S.P.R. experiments, R.H. and V.V. carried out NMR experiments, structure refinement, and deposition, A.B. and P.M. performed HDX/MS experiments, D.C. carried out in vitro kinase assay, E.B. performed protein labeling, E.B. and J.H. carried out GUV preparation and imaging, D.T. and N.S. performed some of the cloning and the cell-based experiments, E.B. supervised the project, E.B., M.K., M.N., V.V. and T.B. wrote the manuscript, all authors contributed to data analysis and commented on the manuscript.</text></passage><passage><infon key="file">srep23641-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>34270</offset><text>Biochemical characterization of the ACBD3:PI4KB complex.</text></passage><passage><infon key="file">srep23641-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>34327</offset><text>(A) Schematic representation of the ACBD3 and PI4KB constructs used for the experiments. ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD). PI4KB is composed of the N-terminal region, helical domain, and kinase domain which can be divided into N- and C-terminal lobes. (B) In vitro pull-down assay. Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3. The inputs and bound proteins were analyzed on SDS gels stained with Coomassie Blue. The asterisks mark the bands corresponding to specific interactions. Cropped gels ran the same experimental conditions are shown. Please, see SI Fig. 9 for original full-length gels. (C) Analytical Ultracentrifugation. AUC analysis of the ACBD3:PI4KB full-length complex at the concentration of 5 μM (both proteins, left panel) and ACBD3 Q domain: PI4KB N terminal region complex at the concentration of 35 μM (both proteins, right panel). (D) Surface plasmon resonance. SPR analysis of the PI4KB binding to immobilized ACBD3. Sensorgrams for four concentrations of PI4KB are shown.</text></passage><passage><infon key="file">srep23641-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>35540</offset><text>Structural analysis of the ACBD3:PI4KB complex.</text></passage><passage><infon key="file">srep23641-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>35588</offset><text>(A) Overall structure of the ACBD3 Q domain by itself and in complex with the PI4KB N-terminal region. Superposition of the 30 converged structures obtained for the Q domain (top) and the 45 converged structures obtained for the complex (bottom), with only the folded part of PI4KB shown (see SI Fig. 2 for the complete view). (B) Detailed view of the complex. The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain. ACBD3 is shown in magenta and PI4KB in orange. (C) Top view of the kinase helix. The kinase helix is amphipathic and its hydrophobic surface overlaps with the ACBD3 binding surface (shown in magenta). Strong and weak hydrophobes are in green and cyan respectively, basic residues in blue, acidic residues in red and nonpolar hydrophilic residues in orange. (D) Pull-down assay with a NiNTA-immobilized N-terminally His6GB1-tagged PI4KB kinase and untagged ACBD3 protein. Wild type proteins and selected point mutants of both PI4KB and ACBD3 were used. Inputs and bound proteins were analyzed on SDS gels and stained with Coomassie Blue. Cropped gels ran the same experimental conditions are shown. Please, see SI Fig. 9 for original full-length gels.</text></passage><passage><infon key="file">srep23641-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>36896</offset><text>ACBD3 is sufficient to recruit the PI4KB kinase to membranes.</text></passage><passage><infon key="file">srep23641-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>36958</offset><text>(A) GUVs recruitment assay. Top – Virtually no membrane bound kinase was observed when 600 nM PI4KB was added to the GUVs. Bottom – in the presence of 600 nM GUV tethered ACBD3 a significant signal of the kinase is detected on the surface of GUVs. (B) Golgi displacement experiment. Upper panel: ACBD3 Q domain fused to GFP was overexpressed and the endogenous PI4KB was immunostained. Middle panel: The same experiment performed with GFP alone. Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. Middle panel – The same experiment performed with mRFP-FKBP alone. Lower panel – The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment. – The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex. Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment. Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin. Right – The same experiment performed using the H264A Q domain mutant.</text></passage><passage><infon key="file">srep23641-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>38600</offset><text>ACBD3 indirectly increases the activity of PI4KB.</text></passage><passage><infon key="file">srep23641-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>38650</offset><text>(A) Micelles-based kinase assay – PI in TX100 micelles was used in a luminescent kinase assay and the production of PI4P was measured. Bar graph presents the mean values of PI4P generated in the presence of the proteins as indicated, normalized to the amount of PI4P generated by PI4KB alone. Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay – GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor. (C)–Quantification of the GUV phosphorylation assay – Mean membrane fluorescence intensity of the PI4P reporter (SidC-label) under different protein/ATP conditions. The mean membrane intensity value is relative to the background signal and the difference between the membrane and background signal in the reference system lacking ATP. The error bars stand for SEM based on three independent experiments (also SI Fig. 6).</text></passage><passage><infon key="file">srep23641-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>39617</offset><text>Pseudoatomic model of the PI4KB multiprotein complex assembly.</text></passage><passage><infon key="file">srep23641-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>39680</offset><text>PI4KB in orange, Rab11 in purple, ACBD3 in blue. The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server. The GOLD domain is tethered to the membrane by GolginB1 (also known as Giantin) which is not shown for clarity. Intrinsically disordered linkers are modeled in an arbitrary but physically plausible conformation.</text></passage></document></collection>

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+<collection><source>PMC</source><date>20201217</date><key>pmc.key</key><document><id>4981400</id><infon key="license">CC BY</infon><passage><infon key="alt-title">Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA</infon><infon key="article-id_doi">10.1371/journal.pone.0160694</infon><infon key="article-id_pmc">4981400</infon><infon key="article-id_pmid">27513867</infon><infon key="article-id_publisher-id">PONE-D-16-20928</infon><infon key="elocation-id">e0160694</infon><infon key="issue">8</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:Zhang;given-names:Yinglu</infon><infon key="name_1">surname:Rataj;given-names:Katarzyna</infon><infon key="name_2">surname:Simpson;given-names:Gordon G.</infon><infon key="name_3">surname:Tong;given-names:Liang</infon><infon key="name_4">surname:Candela;given-names:Hector</infon><infon key="name_5">surname:Tong;given-names:Liang</infon><infon key="name_6">surname:Tong;given-names:Liang</infon><infon key="name_7">surname:Simpson;given-names:Gordon G.</infon><infon key="name_8">surname:Simpson;given-names:Gordon G.</infon><infon key="notes">All relevant data are within the paper and its Supporting Information files.</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 Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>80</offset><text>The Arabidopsis protein FPA controls flowering time by regulating the alternative 3′-end processing of the FLOWERING LOCUS (FLC) antisense RNA. FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain. The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower. We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 Å resolution. The overall structure is similar to that of the SPOC domain in human SMRT/HDAC1 Associated Repressor Protein (SHARP), although there are also substantial conformational differences between them. Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs. Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1277</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1290</offset><text>Eukaryotic messenger RNAs (mRNAs) are made as precursors through transcription by RNA polymerase II (Pol II), and these primary transcripts undergo extensive processing, including 3′-end cleavage and polyadenylation. In addition, alternative 3′-end cleavage and polyadenylation is an essential and ubiquitous process in eukaryotes. Misregulation of (alternative) 3′-end processing can lead to various genetic defects, cancer and other diseases. There is currently great interest in understanding the molecular mechanisms and functional impacts of alternative 3′-end processing.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1876</offset><text>Recently, the split ends (SPEN) family of proteins was identified as RNA binding proteins that regulate alternative 3′-end cleavage and polyadenylation. They are characterized by possessing N-terminal RNA recognition motifs (RRMs) and a conserved SPEN paralog and ortholog C-terminal (SPOC) domain (Fig 1A). The SPOC domain is believed to mediate protein-protein interactions and has diverse functions among SPEN family proteins, but the molecular mechanism of these functions is not well understood.</text></passage><passage><infon key="file">pone.0160694.g001.jpg</infon><infon key="id">pone.0160694.g001</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>2379</offset><text>Sequence conservation of SPOC domains.</text></passage><passage><infon key="file">pone.0160694.g001.jpg</infon><infon key="id">pone.0160694.g001</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>2418</offset><text>(A). Domain organization of A. thaliana FPA. (B). Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP. Residues in surface patch 1 are indicated with the orange dots, and those in surface patch 2 with the green dots. The secondary structure elements in the structure of FPA SPOC are labeled. Residues that are strictly conserved among the five proteins are shown in white with a red background, and those that are mostly conserved in red.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2931</offset><text>FPA, a SPEN family protein in Arabidopsis thaliana and other plants, was found to regulate the 3′-end alternative cleavage and polyadenylation of the antisense RNAs of FLOWERING LOCUS (FLC), a flowering repressor gene. FPA promotes the 3′-end processing of class I FLC antisense RNAs, which includes the proximal polyadenylation site. This is associated with histone demethylase activity and down-regulation of FLC transcription. However, the functional mechanism of this complex is still not clear.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3435</offset><text>Although a SPOC domain is found in all the SPEN family proteins, its sequence conservation is rather low. For example, the sequence identity between the SPOC domains of A. thaliana FPA and human SMRT/HDAC1 Associated Repressor Protein (SHARP) is only 19% (Fig 1B). Currently, the SHARP SPOC domain is the only one with structural information.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3778</offset><text>As a first step toward understanding the molecular basis for the regulation of alternative 3′-end processing and flowering by FPA, we have determined the crystal structure of the SPOC domain of A. thaliana FPA at 2.7 Å resolution. The overall structure is similar to that of the SHARP SPOC domain, although there are also substantial conformational differences between them. The structure reveals a surface patch that is conserved among FPA homologs.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>4232</offset><text>Results and Discussion</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>4255</offset><text>Structure of FPA SPOC domain</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4284</offset><text>The crystal structure of the SPOC domain of A. thaliana FPA has been determined at 2.7 Å resolution using the selenomethionyl single-wavelength anomalous dispersion method. The expression construct contained residues 433–565 of FPA, but only residues 439–460 and 465–565 are ordered in the crystal. The atomic model has good agreement with the X-ray diffraction data and the expected bond lengths, bond angles and other geometric parameters (Table 1). All the residues are located in the favored regions of the Ramachandran plot (data not shown). The structure has been deposited in the Protein Data Bank, with accession code 5KXF.</text></passage><passage><infon key="file">pone.0160694.t001.xml</infon><infon key="id">pone.0160694.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>4923</offset><text>Summary of crystallographic information.</text></passage><passage><infon key="file">pone.0160694.t001.xml</infon><infon key="id">pone.0160694.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;/colgroup&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution range (Å)&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t001fn001&quot;&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;50–2.7 (2.8–2.7)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Number of observations&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;78,008&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;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;10.5 (45.3)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;I/σI&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;24.1 (6.3)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Redundancy&lt;/td&gt;&lt;td align=&quot;center&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;Completeness (%)&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100 (100)&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; factor (%)&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;19.2 (25.0)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Free &lt;italic&gt;R&lt;/italic&gt; factor (%)&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;25.4 (35.4)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Rms deviation in bond lengths (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.017&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 deviation in bond angles (°)&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.9&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>4964</offset><text>Resolution range (Å)1	50–2.7 (2.8–2.7)	 	Number of observations	78,008	 	Rmerge (%)	10.5 (45.3)	 	I/σI	24.1 (6.3)	 	Redundancy		 	Completeness (%)	100 (100)	 	R factor (%)	19.2 (25.0)	 	Free R factor (%)	25.4 (35.4)	 	Rms deviation in bond lengths (Å)	0.017	 	Rms deviation in bond angles (°)	1.9	 	</text></passage><passage><infon key="file">pone.0160694.t001.xml</infon><infon key="id">pone.0160694.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>5272</offset><text>1The numbers in parentheses are for the highest resolution shell.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5338</offset><text>The crystal structure of the FPA SPOC domain contains a seven-stranded, mostly anti-parallel β-barrel (β1-β7) and three helices (αA-αC) (Fig 2A). Only two of the neighboring strands, β1 and β3, are parallel to each other. Helix αB covers one end of the barrel, while helices αA and αC are located next to each other at one side of the barrel (Fig 2B). The other end of the β-barrel is covered by the loop connecting strands β2 and β3, which contains the disordered 461–464 segment. The center of the barrel is filled with hydrophobic side chains and is not accessible to the solvent.</text></passage><passage><infon key="file">pone.0160694.g002.jpg</infon><infon key="id">pone.0160694.g002</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>5966</offset><text>Crystal structure of the SPOC domain of A. thaliana FPA.</text></passage><passage><infon key="file">pone.0160694.g002.jpg</infon><infon key="id">pone.0160694.g002</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>6023</offset><text>(A). Schematic drawing of the structure of FPA SPOC domain, colored from blue at the N terminus to red at the C terminus. The view is from the side of the β-barrel. The disordered segment (residues 460–465) is indicated with the dotted line. (B). Structure of the FPA SPOC domain, viewed from the end of the β-barrel, after 90° rotation around the horizontal axis from panel A. All structure figures were produced with PyMOL (www.pymol.org).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>6471</offset><text>Comparisons to structural homologs of the SPOC domain</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6525</offset><text>Only five structural homologs of the FPA SPOC domain were found in the Protein Data Bank with the DaliLite server, suggesting that the SPOC domain structure is relatively unique. The top hit is the SPOC domain of human SHARP (Fig 3A), with a Z score of 12.3. The other four structural homologs include the β-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D). The next structural homolog has a Z score of 3.0.</text></passage><passage><infon key="file">pone.0160694.g003.jpg</infon><infon key="id">pone.0160694.g003</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>7135</offset><text>Structural homologs of the FPA SPOC domain.</text></passage><passage><infon key="file">pone.0160694.g003.jpg</infon><infon key="id">pone.0160694.g003</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>7179</offset><text>(A). Overlay of the structures of the FPA SPOC domain (cyan) and the SHARP SPOC domain (gray). The bound position of a doubly-phosphorylated peptide from SMRT is shown in magenta. (B). Overlay of the structures of the FPA SPOC domain (cyan) and the Ku70 β-barrel domain (gray). Ku80 contains a homologous domain (green), which forms a hetero-dimer with that in Ku70. The two domains, and inserted segments on them, mediate the binding of dsDNA (orange). The red rectangle highlights the region of contact between the two β-barrel domains. (C). Overlay of the structures of the FPA SPOC domain (cyan) and the homologous domain in Chp1 (gray). The binding partner of Chp1, Tas3, is shown in green. The red rectangle indicates the region equivalent to the binding site of the SMART phosphopeptide in SHARP SPOC domain, where a loop of Tas3 is also located. (D). Overlay of the structures of the FPA SPOC domain (cyan) and the Med25 ACID (gray).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8127</offset><text>SHARP is a transcriptional co-repressor in the nuclear receptor and Notch/RBP-Jκ signaling pathways. The SPOC domain of SHARP interacts directly with silencing mediator for retinoid and thyroid receptor (SMRT), nuclear receptor co-repressor (N-CoR), HDAC, and other components to represses transcription. While the overall structure of the FPA SPOC domain is similar to that of the SHARP SPOC domain, there are noticeable differences in the positioning of the β-strands and the helices, and most of the loops have substantially different conformations as well (Fig 3A). In addition, the SHARP SPOC domain has three extra helices. One of them covers the other end of the β-barrel, and the other two shield an additional surface of the side of the β-barrel from solvent. A doubly-phosphorylated peptide from SMRT is bound to the side of the barrel, near strands β1 and β3 (Fig 3A). Such a binding mode probably would not be possible in FPA, as the peptide would clash with the β1-β2 loop.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9139</offset><text>The Ku70-Ku80 hetero-dimer is involved in DNA double-strand break repair and the β-barrel domain contributes to DNA binding. In fact, the β-barrel domains of Ku70 and Ku80 form a hetero-dimer, primarily through interactions between the loops connecting the third and fourth strands of the barrel (Fig 3B). The open ends of the two β-barrels face the DNA binding sites, and contact the phosphodiester backbone of the dsDNA. In addition, a long insert connecting strands β2 and β3 in the two domains form an arch-like structure, encircling the dsDNA.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9703</offset><text>Chp1 is a subunit of the RNA-induced initiation of transcriptional gene silencing (RITS) complex. The partner of Chp1, Tas3, is bound between the barrel domain and the second domain of Chp1, and the linker between the two domains is also crucial for this interaction (Fig 3C). It is probably unlikely that the β-barrel itself is sufficient to bind Tas3. Interestingly, a loop in Tas3 contacts strand β3 of the barrel domain, at a location somewhat similar to that of the N-terminal segment of the SMRT peptide in complex with SHARP SPOC domain (Fig 3A).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10263</offset><text>Mediator is a coactivator complex that promotes transcription by Pol II. The Med25 subunit ACID is the target of the potent activator VP16 of the herpes simplex virus. The structure of ACID contains a helix at the C-terminus as well as an extended β1-β2 loop. Nonetheless, the binding site for VP16 has been mapped to roughly the same surface patch, near strands β1 and β3, that is used by the SHARP and Tas3 SPOC domains for binding their partners.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10725</offset><text>A conserved surface patch in the FPA SPOC domain</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10774</offset><text>An analysis of the SPOC domain indicates a large surface patch near strands β1, β3, β5 and β6 that is conserved among plant FPA homologs (Fig 4A). This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B). The first surface patch is electropositive in nature (Fig 4C), and residues Arg477 and Tyr515 are also conserved in the SHARP SPOC domain (Fig 1B). In fact, one of the phosphorylated residues of the SMRT peptide interacts with this surface patch (Fig 3A), suggesting that the FPA SPOC domain might also interact with a phosphorylated segment here. In comparison, the second surface patch is more hydrophobic in nature (Fig 4C).</text></passage><passage><infon key="file">pone.0160694.g004.jpg</infon><infon key="id">pone.0160694.g004</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>11638</offset><text>A conserved surface patch of FPA SPOC domain.</text></passage><passage><infon key="file">pone.0160694.g004.jpg</infon><infon key="id">pone.0160694.g004</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>11684</offset><text>(A). Two views of the molecular surface of FPA SPOC domain colored based on sequence conservation among plant FPA homologs. Purple: most conserved; cyan: least conserved. (B). Residues in the conserved surface patch of FPA SPOC domain. The side chains of the residues are shown in stick models, colored orange in the first sub-patch and green in the second. (C). Molecular surface of FPA SPOC domain colored based on electrostatic potential. Blue: positively charged; red: negatively charged.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>12177</offset><text>Testing the requirement of specific conserved amino acids for FPA functions</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12253</offset><text>We next examined the potential impact of the conserved surface patch on FPA function in vivo. We mutated two residues, Arg477 and Tyr515, of the surface patch, which are also conserved in the SHARP SPOC domain (Fig 1B) and were found to be functionally important. The mutations were introduced into a transgene designed to express FPA from its native control elements (promoter, introns and 3′ UTR). The resulting transgenes were then stably transformed into an fpa-8 mutant background so that the impact of the mutations on FPA function could be assessed. Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3′-end formation, for example in FPA pre-mRNA (panel B in S1 Fig). We examined independent transgenic lines expressing each R477A and Y515A mutation. In each case, we confirmed that detectable levels of FPA protein expression were restored close to wild-type levels in protein blot analyses using antibodies that specifically recognize FPA (S2 Fig).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13409</offset><text>We then examined the impact of the surface patch mutations on FPA’s function in controlling RNA 3′-end formation by determining whether the mutant proteins functioned in FPA autoregulation and the repression of FLC expression. FPA autoregulates its expression by promoting cleavage and polyadenylation within intron 1 of its own pre-mRNA, resulting in a truncated transcript that does not encode functional protein. We used RNA gel blot analyses to reveal that in each of three independent transgenic lines for each single mutant, rescue of proximally polyadenylated FPA pre-mRNA can be detected (Fig 5A and 5B). We therefore conclude that neither of these mutations disrupted the ability of FPA to promote RNA 3′-end formation in its own transcript.</text></passage><passage><infon key="file">pone.0160694.g005.jpg</infon><infon key="id">pone.0160694.g005</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>14166</offset><text>Impact of individual FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA.</text></passage><passage><infon key="file">pone.0160694.g005.jpg</infon><infon key="id">pone.0160694.g005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>14261</offset><text>RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A
+(A), or FPA::FPA Y515A
+(B) using poly(A)+ purified mRNAs. A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs. RNA size (kb) marker (Ambion). TUBULIN was detected as an internal control. Proximally and distally polyadenylated FPA transcripts are marked with arrows. The ratio of distal:proximal polyadenylated forms is given under each lane. (C,D) Impact of individual FPA SPOC domain mutations on FLC transcript levels. qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, 35S::FPA:YFP and FPA::FPA R477A
+(C), FPA::FPA Y515A
+(D) plants. Transcript levels were normalized to the control UBC. Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15153</offset><text>We next examined whether the corresponding mutations disrupted the ability of FPA to control FLC expression. We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15573</offset><text>Since each surface patch mutation appeared to be insufficient to disrupt FPA functions on its own, we combined both mutations into the same transgene. We could again confirm that near wild-type levels of FPA protein were expressed from three independent transgenic lines expressing the FPA R477A;Y515A doubly mutated protein in an fpa-8 mutant background (S3 Fig). We found that FPA R477A;Y515A protein functioned like wild-type FPA to restore FPA pre-mRNA proximal polyadenylation (Fig 6A) and FLC expression to wild-type levels (Fig 6B).</text></passage><passage><infon key="file">pone.0160694.g006.jpg</infon><infon key="id">pone.0160694.g006</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>16113</offset><text>Impact of double FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA and FLC expression.</text></passage><passage><infon key="file">pone.0160694.g006.jpg</infon><infon key="id">pone.0160694.g006</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>16223</offset><text>(A) RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing FPA::FPA R477A;Y515A using poly(A)+ purified mRNAs. Black arrows indicate the proximally and distally polyadenylated FPA mRNAs. A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs. RNA size (kb) marker (Ambion). TUBULIN was detected as an internal control. The ratio of distal:proximal polyadenylated forms is given under each lane. (B). qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, and FPA::FPA R477A;Y515A plants. Transcript levels were normalized to the control UBC. Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16983</offset><text>Together our findings suggest that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation, or that this combination of mutations is not sufficient to critically disrupt the function of the FPA SPOC domain. Since the corresponding mutations in the SHARP SPOC domain do disrupt its recognition of unphosphorylated SMRT peptides, these observations may reinforce the idea that the features and functions of the FPA SPOC domain differ from those of the only other well-characterized SPOC domain.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>17516</offset><text>Materials and Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>17538</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>17574</offset><text>The SPOC domain (residue 433–565) of A. thaliana FPA was sub-cloned into the pET28a vector (Novagen). The recombinant protein, with an N-terminal hexa-histidine tag, was over-expressed in E. coli BL21 Star (DE3) cells (Novagen), which were induced with 0.4 mM IPTG and allowed to grow at 20°C for 14–18 h. The soluble protein was purified by nickel-charged immobilized-metal affinity chromatography and gel filtration chromatography. The purified protein was concentrated and stored at –80°C in a buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, 10 mM DTT and 5% (v/v) glycerol. The His-tag was not removed for crystallization.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>18213</offset><text>The selenomethionine labeled SPOC domain was expressed in E. coli B834(DE3) strain using LeMaster media and purified with the same protocol as the native protein.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>18376</offset><text>Protein crystallization</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>18400</offset><text>Crystals of the native SPOC domain of FPA were grown at 20°C with the sitting-drop vapor diffusion method. The protein solution was at 30 mg/ml concentration, and the reservoir solution contained 0.2 M MgSO4, and 20% (v/v) PEG 3350. Fully-grown crystals were obtained two days after set-up. Crystals of the selenomethionine labeled SPOC domain were grown using the same condition as the native protein. The crystals were cryo-protected in the crystallization solution supplemented with 20% (v/v) glycerol and flash-frozen in liquid nitrogen for data collection at 100K.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>18971</offset><text>Data collection and processing</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>19002</offset><text>A single-wavelength anomalous dispersion (SAD) X-ray diffraction data set on a selenomethionine labeled SPOC domain crystal was collected at the National Synchrotron Light Source (NSLS) beamline X29A using an ADSC Q315r CCD. The diffraction images were processed and scaled with the HKL package. The crystal belongs to space group P65, with unit cell parameters of a = b = 108.2 Å, and c = 34.2 Å.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>19402</offset><text>Structure determination and refinement</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>19441</offset><text>The structure of the SPOC domain was solved by the selenomethionyl SAD method with the program SHELX. The phases were used by program PHENIX for automatic model building. Manual model rebuilding was carried out with Coot. The structure refinement was performed with the program PHENIX, with translation, libration, and screw-rotation (TLS) parameters. The data processing and refinement statistics are summarized in Table 1. The Ramachandran plot showed that 95.8% of the residues are located in the most favored regions, and 4.2% are in additional allowed regions.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>20007</offset><text>Generation of constructs with mutated genomic FPA sequence</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20066</offset><text>A series of constructs containing a mutated FPA genomic sequence was prepared based on pGreen I 0029 vector. pGreen I 0029 vector with inserted FPA genomic sequence was prepared. In this vector FPA genomic sequence is flanked by 2620bp of the native sequence upstream to the start codon and 1178bp downstream to the stop codon. The vector contains kanamycin resistance genes for both the bacteria and plant hosts. In order to obtain a series of constructs with mutated FPA genomic sequence, FPA sequence in this construct was modified using site-directed mutagenesis. Primers used to prepare required constructs are listed in S1 Table. After the mutagenesis reaction the presence of only the desired mutations was confirmed by sequencing of the whole FPA genomic sequence and flanking regions.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>20860</offset><text>Generation of Arabidopsis thaliana transgenic plants</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20913</offset><text>All transgenic plants were prepared in fpa-8 mutant background, which is in Col-0 accession. The prepared vectors for Arabidopsis transformations were introduced into electro-competent Agrobacterium tumefaciens cells (C58 CV3101 strain harbouring pSoup vector). The floral dip method was used for plant transformation. Transgenic plants were selected using kanamycin as a selection marker. Presence of the desired mutations in plants was confirmed with specific dCaps markers.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>21390</offset><text>Plant growth conditions</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>21414</offset><text>Wild type Col-0 plants used in this study were obtained from the Nottingham Arabidopsis Stock Centre. Seed of fpa-8 and 35S::FPA:YFP were obtained from Professor Caroline Dean. Plants were grown in pots containing Universal Extra general purpose soil. The glasshouse temperature was maintained at 20°C and the 16 hour daylight was provided by high pressure sodium vapour lamps (Philips Powertone SON-T AGRO 400). In order to grow plants in sterile conditions, seeds were first surface sterilized by a 5 min treatment with sterilizing solution (3% v/v sodium hypochlorite, 0.02% v/v Triton X-100), followed by three washes with 0.02% v/v Triton X-100 and one wash with sterile water. The sterile seeds were sown on MS10 media supplemented with 0.8% w/v agar. MS10 medium was also supplemented with specific antibiotics if required. After sowing, the seeds were stratified at 4°C for two days in order to synchronize their germination. Plants were grown in the tissue culture room at the following conditions: temperature 22°C, 16 hours daylight provided by the Master TL-D 36W/840 (Philips) lamps.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>22514</offset><text>Plant protein analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>22537</offset><text>Total protein samples were prepared using extraction buffer containing: 40 mM Tris-HCl, pH 6.8; 0.1 mM EDTA, pH 8.0; 8 M urea; 1.43 M β-mercaptoethanol, 7% v/v Complete Protease Inhibitors (Roche) and 5 mM PMSF. Equal volumes of samples were separated on 8% SDS-PAGE. Proteins were transferred onto Protran nitrocellulose transfer membrane (Whatman) using wet Criterion blotter system (BioRad). The transfer was performed at room temperature for two hours at a stable voltage of 70 V. Membrane was blocked in 3% (w/v) Milk in TBS for 1h at room temperature followed by overnight incubation with anti-FPA antibody (dilution 1:100 in 3% (w/v) Milk in TBS). After washes the membrane was incubated for 75 min with goat anti-rabbit antibody (Thermo Scientific) (1:3000 dilution in 3% (w/v) Milk in TBS). Protein was detected using SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific). Blots were re-probed following treatment with low pH solution (25mM glycine-HCl, pH 2, 1% (w/v) SDS) followed by blocking for 1h at room temperature in 3% (w/v) Milk in TBS. The membrane was incubated overnight with anti-TUBB2A, tubulin, beta 2A antibody (ARP40177_P050 Aviva systems biology; (dilution 1:1000 in 3% (w/v) Milk in TBS). After washes the membrane was incubated for 75 min with goat anti-rabbit antibody (Thermo Scientific) [1:3000 dilution in 3% (w/v) Milk in TBS]. 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+<collection><source>PMC</source><date>20201219</date><key>pmc.key</key><document><id>4993997</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/srep31425</infon><infon key="article-id_pii">srep31425</infon><infon key="article-id_pmc">4993997</infon><infon key="article-id_pmid">27550639</infon><infon key="elocation-id">31425</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:Chen;given-names:Ji-Yun</infon><infon key="name_1">surname:Liu;given-names:Liang</infon><infon key="name_2">surname:Cao;given-names:Chun-Ling</infon><infon key="name_3">surname:Li;given-names:Mei-Jun</infon><infon key="name_4">surname:Tan;given-names:Kemin</infon><infon key="name_5">surname:Yang;given-names:Xiaohan</infon><infon key="name_6">surname:Yun;given-names:Cai-Hong</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>Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>96</offset><text>N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains. Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes. This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε-acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine. Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA). The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures. Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions. Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1386</offset><text>Acetylation is one of the most ubiquitous modifications that plays a vital role in many biological processes, such as transcriptional regulation, protein-protein interaction, enzyme activity, protein stability, antibiotic resistance, biological rhythm and so on. Protein acetylation can be grouped into lysine Nε-acetylation and peptide N-terminal acetylation (Nt-acetylation). Generally, Nε-acetylation refers to the transfer of an acetyl group from an acetyl coenzyme A (Ac-CoA) to the ε-amino group of lysine. This kind of modification is catalyzed by lysine acetyltransferases (KATs), some of which are named histone acetyltransferases (HATs) because early studies focused mostly on the post-transcriptional acetylation of histones.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2141</offset><text>Despite the prominent accomplishments in the field regarding Nε-acetylation by KATs for over 50 years, the significance of the more evolutionarily conserved Nt-acetylation is still inconclusive. Nt-acetylation is an abundant and evolutionarily conserved modification occurring in bacteria, archaea and eukaryotes. It is estimated that about 80–90% of soluble human proteins and 50–70% of yeast proteins are subjected to Nt-acetylation, where an acetyl moiety is transferred from Ac-CoA to the α-amino group of the first residue. Recently Nt-acetylome expands the Nt-acetylation to transmembrane proteins. Unlike Nε-acetylation that can be eliminated by deacetylases, Nt-acetylation is considered irreversible since no corresponding deacetylase is found to date. Although Nt-acetylation has been regarded as a co-translational modification traditionally, there is evidence that post-translational Nt-acetylation exists. During the past decades, a large number of Nt-acetylome researches have shed light on the functional roles of Nt-acetylation, including protein degradation, subcellular localization, protein-protein interaction, protein-membrane interaction, plant development, stress-response and protein stability.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3376</offset><text>The Nt-acetylation is carried out by N-terminal acetyltransferases (NATs) that belong to the GNAT superfamily. To date, six NATs (NatA/B/C/D/E/F) have been identified in eukaryotes. About 40 percent of Nt-acetylation of soluble proteins in cells is catalyzed by NatA complex which is composed of the catalytic subunit Naa10p and the auxiliary subunit Naa15p. NatE was found to physically interact with the NatA complex without any observation of impact on NatA-activity. Two other multimeric complexes of NATs are NatB and NatC which contain the catalytic subunits Naa20 and Naa30 and the auxiliary subunits Naa25 and Naa35/Naa38, respectively. Furthermore, only the catalytic subunits Naa40 and Naa60 were found for NatD and NatF, respectively. Besides Nt-acetylation, accumulating reports have proposed Nε-acetylation carried out by NATs.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4223</offset><text>There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family. Unlike other NATs that are highly conserved among lower and higher eukaryotes, NatF only exists in higher eukaryotes. Subsequent researches indicated that NatF displays its catalytic ability with both Nt-acetylation and lysine Nε-acetylation. As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91. Another important feature of NatF is that this protein is anchored on the Golgi apparatus through its C-terminal membrane-integrating region and takes part in the maintaining of Golgi integrity. With its unique intracellular organellar localization and substrate selectivity, NatF appears to provide more evolutionary information among the NAT family members.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5697</offset><text>It was recently found that NatF facilitates nucleosomes assembly and that NAA60 knockdown in MCF7-cell inhibits cell proliferation, sensitizes cells to DNA damage and induces cell apoptosis. In Drosophila cells, NAA60 knockdown induces chromosomal segregation defects during anaphase including lagging chromosomes and chromosomal bridges. Much recent attention has also been focused on the requirement of NatF for regulation of organellar structure. In HeLa cells, NAA60 knockdown causes Golgi apparatus fragmentation which can be rescued by overexpression Naa60. The systematic investigation of publicly available microarray data showed that NATs share distinct tissue-specific expression patterns in Drosophila and NatF shows a higher expression level in central nervous system of Drosophila.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6492</offset><text>In this study, we solved the structures of human Naa60 (NatF) in complex with coenzyme. The hNaa60 protein contains a unique amphipathic α-helix (α5) following its GNAT domain that might account for the Golgi localization of this protein. Crystal structures showed that the β7-β8 hairpin rotated about 50 degrees upon removing the C-terminal region of the protein and this movement substantially changed the geometry of the substrate-binding pocket. Remarkably, we find that Phe 34 may participate in the proper positioning of the coenzyme for the transfer reaction to occur. Further structure comparison and biochemical studies also identified other key structural elements essential for the enzyme activity of Naa60.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>7225</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>7233</offset><text>Overall structure of hNaa60</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7261</offset><text>In the effort to prepare the protein for structural studies, we tried a large number of hNaa60 constructs but all failed due to heavy precipitation or aggregation. Sequence alignment of Naa60 from different species revealed a Glu-Glu-Arg (EER) versus Val-Val-Pro (VVP) sequence difference near the N-terminus of the protein in Xenopus Laevis versus Homo sapiens (Fig. 1A). Considering that terminal residues may lack higher-order structure and hydrophobic residues in this region may expose to solvent and hence cause protein aggregation, we mutated residues 4–6 from VVP to EER for the purpose of improving solubility of this protein. According to previous studies, this N-terminal region should not interfere with hNaa60’s Golgi localization. We tried many hNaa60 constructs with the three-residues mutation but only the truncated variant 1-199 and the full-length protein behaved well. We obtained the crystal of the truncated variant 1-199 in complex with CoA first, and after extensive trials we got the crystal of the full-length protein (spanning residues 1-242) in complex with Ac-CoA (Fig. 1B,C). Hereafter, all deletions or point mutants of hNaa60 we describe here are with the EER mutation. The crystal structures of hNaa60(1-242)/Ac-CoA and hNaa60(1-199)/CoA were determined by molecular replacement and refined to 1.38 Å and 1.60 Å resolution, respectively (Table 1). The electron density maps were of sufficient quality to trace residues 1-211 of hNaa60(1-242) and residues 5-199 of hNaa60(1-199).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8783</offset><text>The structure of hNaa60 protein contains a central domain exhibiting a classic GCN5-related N-acetyltransferase (GNAT) folding, along with the extended N- and C-terminal regions (Fig. 1B,C). The central domain includes nine β strands (β1-β9) and four α-helixes (α1-α4) and is highly similar to the known hNaa50p and other reported NATs (Fig. 1D). However, in hNaa60, there is an extra 20-residue loop between β3 and β4 that forms a small subdomain with well-defined 3D structure (Fig. 1B–D). Furthermore, the β7-β8 strands form an approximately antiparallel β-hairpin structure remarkably different from that in hNaa50p (Fig. 1D). The N- and C-terminal regions form helical structures (α0 and α5) stretching out from the central GCN5-domain (Fig. 1C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9579</offset><text>Interestingly, we found that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) (Figure S1), indicating that residues 200–242 may have some auto-inhibitory effect on the activity of the enzyme. However, since this region was not visible in the hNaa60(1-242) crystal structure, we do not yet understand how this happens. Another possibility is that since hNaa60 is localized on Golgi apparatus, the observed low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies. For the convenience of studying the kinetics of mutants, the mutagenesis studies described hereafter were all based on hNaa60 (1-199).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10278</offset><text>An amphipathic α-helix in the C-terminal region may contribute to Golgi localization of hNaa60</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10377</offset><text>There is one hNaa60 molecule in the asymmetric unit in the hNaa60(1-242)/Ac-CoA structure. The C-terminal region extended from the GCN5-domain forms an amphipathic helix (α5) and interacts with a molecule in a neighbor asymmetric unit through hydrophobic interactions between α5-helix and a hydrophobic groove between the N-terminal β1 and β3 strands of the neighbor molecule (Fig. 2A). The C-terminal extension following α5-helix forms a β-turn that wraps around and interacts with the neighbor protein molecule through hydrophobic interactions, too. In the hNaa60(1-199)/CoA structure, a part of the α5-helix is deleted due to truncation of the C-terminal region (Fig. 1B). Interestingly, the remaining residues in α5-helix still form an amphipathic helix although the hydrophobic interaction with the N-terminal hydrophobic groove of a neighbor molecule is abolished and the helix is largely exposed in solvent due to different crystal packing (Fig. 2B).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11364</offset><text>A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>11975</offset><text>The β7-β8 hairpin showed alternative conformations in the hNaa60 crystal structures</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12065</offset><text>Superposition of hNaa60(1-242)/Ac-CoA, hNaa60(1-199)/CoA and hNaa50/CoA/peptide (PDB 3TFY) revealed considerable difference in the β7-β8 hairpin region despite the overall stability and similarity of the GNAT domain (Fig. 1D). In hNaa60(1-242), the β7-β8 hairpin is located in close proximity to the α1-α2 loop, creating a more compact substrate binding site than that in hNaa50, where this region adopts a more flexible loop conformation (β6-β7 loop). Upon removing the C-terminal region of hNaa60, we observed that hNaa60 (1-199) molecules pack in a different way involving the β7-β8 hairpin in the crystal, leading to about 50 degree rotation of the hairpin which moves away from the α1-α2 loop (Figs 1D and 2C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12821</offset><text>This conformational change substantially altered the geometry of the substrate binding site, which could potentially change the way in which the substrate accesses the active site of the enzyme. In hNaa60(1-242), the β7-β8 hairpin covers the active site in a way similar to that observed in hNaa50, presumably leaving only one way for the substrate to access the active site, i.e. to enter from the opposite end into the same tunnel where Ac-CoA/CoA binds (Fig. 2D), which may accommodate access of a NAT substrate only. KAT activity of hNaa60 toward histone H4 has been noted in previous study, and our enzyme kinetic data also indicated that hNaa60 can acetylate H3-H4 tetramer in vitro (Figure S3). Furthermore, we analyzed the acetylation status of histone H3-H4 tetramer using mass spectrometry and observed that multiple lysine residues in the protein showed significantly increased acetylation level and changed acetylation profile upon treatment with hNaa60(1-199) (Figure S4). We also conducted liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis on a synthetic peptide (NH2-MKGKEEKEGGAR-COOH) after treatment with hNaa60(1-199), and the data confirmed that both the N-terminal α-amine and lysine side-chain ε-amine were robustly acetylated after the treatment (Table S1). Despite these observations, the mechanism for this alternative activity remains unknown. Recent structural investigation of other NATs proposed that the β6-β7 loop, corresponding to the β7-β8 hairpin in hNaa60, and the α1-α2 loop flanking the substrate-binding site of NATs, prevent the lysine side-chain of the KAT substrates from inserting into the active site. Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D). Interestingly, in the hNaa60(1-199) crystal structure, the displaced β7-β8 hairpin opened a second way for the substrate to access the active center that would readily accommodate the binding of the H4 peptide (Fig. 2E), thus implied a potential explanation for KAT activity of this enzyme from a structural biological view. However, since hNaa60(1-242) and hNaa60(1-199) were crystallized in different crystal forms, the observed conformational change of the β7-β8 hairpin may simply be an artifact related to the different crystal packing. Whether the KAT substrates bind to the β7-β8 hairpin displaced conformation of the enzyme needs to be verified by further structural and functional studies.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>15485</offset><text>Phe 34 facilitates proper positioning of the cofactor for acetyl-transfer</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15559</offset><text>The electron density of Phe 34 side-chain is well defined in the hNaa60(1-242)/Ac-CoA structure, but becomes invisible in the hNaa60(1-199)/CoA structure, indicating displacement of the Phe 34 side-chain in the latter (Fig. 3A,B). A solvent-derived malonate molecule is found beside Phe 34 and the ethanethioate moiety of Ac-CoA in the high-resolution hNaa60(1-242)/Ac-CoA structure (Fig. 3A). Superposition of this structure on that of hNaa50p/CoA/peptide shows that the malonate molecule overlaps well on the N-terminal methionine of the substrate peptide and residue Phe 34 in hNaa60 overlaps well on Phe 27 in hNaa50 (Fig. 4A). Interestingly, in the structure of hNaa60(1-199)/CoA, the terminal thiol of CoA adopts alternative conformations. One is to approach the substrate amine (as indicated by the superimposed hNaa50/CoA/peptide structure), similar to the terminal ethanethioate of Ac-CoA in the structure of hNaa60(1-242)/Ac-CoA; the other is to approach the α1-α2 loop and away from the substrate amine (Fig. 3B). To rule out the possibility that the electron density we define as the alternative conformation of the thiol terminus is residual electron density of the displaced side-chain of Phe 34, we solved the crystal structure of hNaa60(1-199) F34A/CoA. The structure of this mutant is highly similar to hNaa60(1-199)/CoA and there is essentially the same electron density corresponding to the alternative conformation of the thiol (Fig. 3C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17026</offset><text>Phe 27 in hNaa50p (equivalent to Phe 34 in hNaa60) has been implicated to facilitate the binding of N-terminal methionine of the substrate peptide through hydrophobic interaction. However, in the hNaa60/Ac-CoA structure, a hydrophilic malonate molecule is found at the same location where the N-terminal methionine should bind as is indicated by the superposition (Fig. 3A), suggesting that Phe 34 may accommodate binding of hydrophilic substrate, too. Moreover, orientation of Phe 34 side-chain seems to be co-related to positioning of the terminus of the co-enzyme and important for placing it at a location in close proximity to the substrate amine. We hypothesize that if Phe 34 only works to facilitate the binding of the hydrophobic N-terminal Met residue, to mutate it from Phe to Ala would not abolish the catalytic activity of this enzyme, while if Phe 34 also plays an essential role to position the ethanethioate moiety of Ac-CoA, the mutation would be expected to abrogate the activity of the enzyme. Indeed, our enzyme kinetic data showed that hNaa60(1-199) F34A mutant showed no detectable activity (Fig. 5A). In order to rule out the possibility that the observed loss of activity may be related to bad folding of the mutant protein, we studied the circular dichroism (CD) spectrum of the protein (Fig. 5B) and determined its crystal structure (Fig. 3C). Both studies proved that the F34A mutant protein is well-folded. Many studies have addressed the crucial effect of α1-α2 loop on catalysis, showing that some residues located in this area are involved in the binding of substrates. We propose that Phe 34 may play a dual role both in interacting with the peptide substrate (recognition) and in positioning of the ethanethioate moiety of Ac-CoA to the right location to facilitate acetyl-transfer.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>18850</offset><text>Structural basis for hNaa60 substrate binding</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18896</offset><text>Several studies have demonstrated that the substrate specificities of hNaa60 and hNaa50 are highly overlapped. The structure of hNaa50p/CoA/peptide provides detailed information about the position of substrate N-terminal residues in the active site of hNaa50. Comparing the active site of hNaa60(1-242)/Ac-CoA with hNaa50p/CoA/peptide revealed that key catalytic and substrate binding residues are highly conserved in both proteins (Fig. 4A). With respect to catalysis, hNaa50p has been shown to employ residues Tyr 73 and His 112 to abstract proton from the α-amino group from the substrate’s first residue through a well-ordered water. A well-ordered water was also found between Tyr 97 and His 138 in hNaa60 (1-199)/CoA and hNaa60 (1-242)/Ac-CoA (Fig. 4B). To determine the function of Tyr 97 and His 138 in hNaa60 catalysis, we mutated these residues to alanine and phenylalanine, respectively, and confirmed that all these mutants used in our kinetic assays are well-folded by CD spectra (Fig. 5B). Purity of all proteins were also analyzed by SDS-PAGE (Figure S5). As show in Fig. 5A, the mutants Y97A, Y97F, H138A and H138F abolished the activity of hNaa60. In contrast, to mutate the nearby solvent exposed residue Glu 37 to Ala (E37A) has little impact on the activity of hNaa60 (Figs 4B and 5A). In conclusion, the structural and functional studies indicate that hNaa60 applies the same two base mechanism through Tyr 97, His 138 and a well-ordered water as was described for hNaa50.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20394</offset><text>The malonate molecule observed in the hNaa60(1-242)/Ac-CoA crystal structure may be indicative of the substrate binding position of hNaa60 since it is located in the active site and overlaps the N-terminal Met of the substrate peptide in the superposition with the hNaa50p/CoA/peptide structure (Fig. 4A). Residues Tyr 38, Asn 143 and Tyr 165 are located around the malonate and interact with it through direct hydrogen bonds or water bridge (Fig. 4C). Although malonate is negatively charged, which is different from that of lysine ε-amine or peptide N-terminal amine, similar hydrophilic interactions may take place when substrate amine presents in the same position, since Tyr 38, Asn 143 and Tyr 165 are not positively or negatively charged. In agreement with this hypothesis, it was found that the Y38A, N143A and Y165A mutants all showed remarkably reduced activities as compared to WT, implying that these residues may be critical for substrate binding (Figs 4C and 5A).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>21378</offset><text>The β3-β4 loop participates in the regulation of hNaa60-activity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21449</offset><text>Residues between β3 and β4 of hNaa60 form a unique 20-residue long loop (residues 73–92) that is a short turn in many other NAT members (Fig. 1D). Previous study indicated that auto-acetylation of hNaa60K79 could influence the activity of hNaa60; however, we were not able to determine if Lys 79 is acetylated in our crystal structures due to poor quality of the electron density of Lys 79 side-chain. We therefore used mass spectrometry to analyze if Lys 79 was acetylated in our bacterially purified proteins, and observed no modification on this residue (Figure S6). To assess the impact of hNaa60K79 auto-acetylation, we studied the kinetics of K79R and K79Q mutants which mimic the un-acetylated and acetylated form of Lys 79, respectively. Interestingly, both K79R and K79Q mutants led to an increase in the catalytic activity of hNaa60, while K79A mutant led to modest decrease of the activity (Fig. 5A). These data indicate that the acetylation of Lys 79 is not required for optimal catalytic activity of hNaa60 in vitro.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22485</offset><text>It is noted that the β3-β4 loop of hNaa60 acts like a door leaf to partly cover the substrate-binding pathway. We hence hypothesize that the β3-β4 loop may interfere with the access of the peptide substrates and that the solvent-exposing Lys 79 may play a potential role to remove the door leaf when it hovers in solvent (Fig. 4D). Acidic residues Glu 80, Asp 81 and Asp 83 interact with His 138, His 159 and His 158 to maintain the conformation of the β3-β4 loop, thus contribute to control the substrate binding (Fig. 4D). To verify this hypothesis, we mutated Glu 80, Asp 81 and Asp 83 to Ala respectively. In line with our hypothesis, E80A, D81A and D83A mutants exhibit at least 2-fold increase in hNaa60-activity (Fig. 5A). Interestingly, the structure of an ancestral NAT from S. solfataricus also exhibits a 10-residue long extension between β3 and β4, and the structure and biochemical studies showed that the extension of SsNat has the ability to stabilize structure of the active site and potentiate SsNat-activity.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>23536</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>23547</offset><text>Nt-acetylation, which is carried out by the NAT family acetyltransferases, is an ancient and essential modification of proteins. Although many NATs are highly conserved from lower to higher eukaryotes and the substrate bias of them appears to be partially overlapped, there is a significant increase in the overall level of N-terminal acetylation from lower to higher eukaryotes. In this study we provide structural insights into Naa60 found only in multicellular eukaryotes.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>24023</offset><text>The N-terminus of hNaa60 harbors three hydrophobic residues (VVP) that makes it very difficult to express and purify the protein. This problem was solved by replacing residues 4–6 from VVP to EER that are found in Naa60 from Xenopus Laevis. Since Naa60 from human and from Xenopus Laevis are highly homologous (Fig. 1A), we speculate that these two proteins should have the same biological function. Therefore it is deduced that the VVP to EER replacement on the N-terminus of hNaa60 may not interfere with its function. However, in the hNaa60(1-242) structure the N-terminus adopts an α-helical structure which will probably be kinked if residue 6 is proline (Fig. 1C), and in the hNaa60(1-199) structure the N-terminus adopts a different semi-helical structure (Fig. 1B) likely due to different crystal packing. Hence it is not clear if the N-terminal end of wild-type hNaa60 is an α-helix, and what roles the hydrophobic residues 4–6 play in structure and function of wild-type hNaa60. In addition to the three-residue mutation (VVP to EER), we also tried many other hNaa60 constructs, but only the full-length protein and the truncated variant 1-199 behaved well. The finding that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) is intriguing. We speculate that low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies or there remains some undiscovered auto-inhibitory regulation in the full-length protein.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>25544</offset><text>The hNaa60 protein was proven to be localized on Golgi apparatus. Aksnes and colleagues predicted putative transmembrane domains and two putative sites of S-palmitoylation, by bioinformatics means, to account for Golgi localization of the protein. They then mutated all five cysteine residues of hNaa60’s to serine, including the two putative S-palmitoylation sites. However, these mutations did not abolish Naa60 membrane localization, indicating that S-palmitoylation is unlikely to (solely) account for targeting hNaa60 on Golgi. Furthermore, adding residues 217–242 of hNaa60 (containing residues 217–236, one of the putative transmembrane domains) to the C terminus of eGFP were not sufficient to localize the protein on Golgi apparatus, while eGFP-hNaa60182-242 was sufficient to, suggesting that residues 182–216 are important for Golgi localization of hNaa60. We found that residues 190–202 formed an amphipathic helix with an array of hydrophobic residues located on one side. This observation is reminiscent of the protein/membrane interaction through amphipathic helices in the cases of KalSec14, Atg3, PB1-F2 etc. In this model an amphipathic helix can immerse its hydrophobic side into the lipid bilayer through hydrophobic interactions. Therefore we propose that the amphipathic helix α5 may contribute to Golgi localization of hNaa60. This model, though may need further studies, is supported by the Aksnes studies.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>26986</offset><text>Previous studies indicated that members of NAT family are bi-functional NAT and KAT enzymes. However, known structures of NATs do not well support this hypothesis, since the β6-β7 hairpin/loop of most of NATs is involved in the formation of a tunnel-like substrate-binding site with the α1-α2 loop, which would be good for the NAT but not KAT activity of the enzyme. Kinetic studies have been conducted to compare the NAT and KAT activity of hNaa50 in vitro, and indicate that the NAT activity of Naa50 is much higher than KAT activity. However, the substrate used in this study for assessing KAT activity was a small peptide which could not really mimic the 3D structure of a folded protein substrate in vivo. Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro. Conformational change of the β7-β8 hairpin (corresponding to the β6-β7 loop of other NATs) is noted in our structures (Figs 1D and 2C), which might provide an explanation to the NAT/KAT dual-activity in a structural biological view, but we were unable to rule out the possibility that the observed conformational change of this hairpin might be an artifact related to crystal packing or truncation of the C-terminal end of the protein. Further studies are therefore needed to reveal the mechanism for the KAT activity of this enzyme.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>28518</offset><text>The relationship between enzyme, co-enzyme and substrates has been documented for several years. In early years, researchers found adjustment of GCN5 histone acetyltransferase structure when it binds CoA molecule. The complexed form of NatA is more suitable for catalytic activation, since the α1-α2 loop undergoes a conformation change to participate in the formation of substrate-binding site when the auxiliary subunit Naa15 interacts with Naa10 (the catalytic subunit of NatA). In the structure of hNaa50/CoA/peptide, Phe 27 in the α1-α2 loop appears to make hydrophobic interaction with the N-terminal Met of substrate peptide. However, the hNaa60(1-242)/Ac-CoA crystal structure indicated that its counterpart in hNaa60, Phe 34, could also accommodate the binding of a hydrophilic malonate that occupied the substrate binding site although it maintained the same conformation as that observed in hNaa50. Interestingly, the terminal thiol of CoA adopted alternative conformations in the structure of hNaa60(1-199)/CoA. One was to approach the substrate amine; the other was to approach the α1-α2 loop and away from the substrate amine. Same alternative conformations of CoA were observed in the hNaa60(1-199)(F34A) crystal structure, and our kinetic data showed that the F34A mutation abolished the activity of the enzyme. Taken together, our data indicated that Phe 34 in hNaa60 may play a role in placing co-enzyme at the right location to facilitate the acetyl-transfer. However, these data did not rule out that possibility that Phe 34 may coordinate the binding of the N-terminal Met through hydrophobic interaction as was proposed by previous studies.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>30205</offset><text>Furthermore, we showed that hNaa60 adopts the classical two base mechanism to catalyze acetyl-transfer. Although sequence identity between hNaa60 and hNaa50 is low, key residues in the active site of both enzymes are highly conserved. This can reasonably explain the high overlapping substrates specificities between hNaa60 and hNaa50. Another structural feature of hNaa60 that distinguishes it from other NATs is the β3-β4 long loop which appears to inhibit the catalytic activity of hNaa60. However, this loop also seems to stabilize the whole hNaa60 structure, because deletion mutations of this region led to protein precipitation and aggregation (Figure S7). A previous study suggested that the auto-acetylation of Lys 79 was important for hNaa60-activity, whereas the point mutation K79R did not decrease the activity of hNaa60 in our study. Meanwhile, no electron density of acetyl group was found on Lys 79 in our structures and mass spectrometry analysis. Hence, it appears that the auto-acetylation of hNaa60 is not an essential modification for its activity for the protein we used here. As for the reason why K79R in Yang’s previous studies reduced the activity of the enzyme, but in our studies it didn’t, we suspect that the stability of this mutant may play some role. K79R is less stable than the wild-type enzyme as was judged by its poorer gel-filtration behavior and tendency to precipitate. In our studies we have paid special attention and carefully handled this protein to ensure that we did get enough of the protein in good condition for kinetic assays. The intracellular environment is more complicated than our in vitro assay and the substrate specificity of hNaa60 most focuses on transmembrane proteins. The interaction between hNaa60 and its substrates may involve the protein-membrane interaction which would further increase the complexity. It is not clear if the structure of hNaa60 is different in vivo or if other potential partner proteins may help to regulate its activity. Nevertheless, our study may be an inspiration for further studies on the functions and regulation of this youngest member of the NAT family.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>32362</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>32370</offset><text>Cloning, expression and purification of Homo sapiens Naa60 (hNaa60)</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>32438</offset><text>The cDNA encoding hNaa60 residues 1–242 (full-length) or residues 1–199 were amplified by PCR and inserted into the pET23a vector, which had been modified to provide an N-terminal 6xHis-tag followed by a tobacco etch virus (TEV) protease cleavage site. The VVP to EER (residues 4–6) mutation and other mutations for functional studies were introduced using the quick change method. The protein was expressed in Escherichia coli BL21 (DE3) or Escherichia coli BL21 (DE3) pLysS at 16 °C for 15 h in the presence of 0.1 mM IPTG. Cells were harvested at 4 °C by centrifugation (4,000 g for 10 min) and resuspended in buffer A containing 20 mM Tris, pH 8.0, 500 mM NaCl, 50 mM imidazole, 10% glycerol, 1 mM protease inhibitor PMSF (Phenylmethylsulfonyl fluoride) and 1 mM Tris (2-carboxyethyl)phosphine (TCEP) hydrochloride. Cells were lysed by sonication and the lysate was cleared by centrifugation (18,000 g at 4 °C for 20 min). Then the supernatant was loaded onto a 5-mL Chelating Sepharose column (GE Healthcare) charged with Ni2+ and washed with buffer B (20 mM Tris, pH 8.0, 500 mM NaCl, 50 mM imidazole, 1% glycerol and 1 mM TCEP). The protein was eluted with buffer C (20 mM Tris, pH 8.0, 500 mM NaCl, 300 mM imidazole, 1% glycerol and 1 mM TCEP). The eluent was digested by His-tagged TEV protease and concentrated by ultrafiltration at the same time. After 3 hours, the concentrated eluent was diluted 10 times with buffer D (20 mM Tris, pH 8.0, 500 mM NaCl, 1% glycerol and 1 mM TCEP) and the diluent was passed through the nickel column once again to remove the His-tagged TEV protease and the un-cleaved His-hNaa60 protein. The flow-through was concentrated to 500 μl and loaded onto a Superose 6 or Superdex 200 10/300 gel-filtration column (GE Healthcare) equilibrated with buffer E (20 mM Tris, pH 8.0, 150 mM NaCl, 1% glycerol and 1 mM TCEP). Fractions containing the protein were collected and concentrated to a final concentration of 10 mg/ml for crystallization or acetyltransferases assays.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>34517</offset><text>Circular Dichroism (CD) Spectroscopy</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34554</offset><text>CD spectra of the proteins were obtained using a Jasco J-810 circular dichroism spectropolarimeter scanning from 190 to 250 nm with a 1 mm quartz cuvette. The wild-type and mutant proteins were examined at 4.5 μM concentration in 20 mM Tris, pH 8.0, 150 mM NaCl, 1% glycerol and 1 mM TCEP at room temperature. All samples were centrifuged at 10,000 g for 5 min before analysis.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>34949</offset><text>Crystallization, data collection and structure determination</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35010</offset><text>The purified hNaa60(1-242), hNaa60(1-199) or F34A(1-199) protein was mixed with acetyl coenzyme A (Ac-CoA) or coenzyme A (CoA) (Sigma), respectively, at a 1:5 molar ratio before crystallization. All crystals were made by the hanging-drop vapor diffusion method. The crystallization reservoir solution for hNaa60(1-242) was 10 mM Tris pH 8.0, 75 mM NaCl, 0.5% glycerol, 3% v/v Tacsimate pH 4.0 (Hampton Research) and 7.5% w/v polyethylene glycol 3350 (PEG 3350), and for hNaa60(1-199) was 0.2 M L-Proline, 0.1 M HEPES pH 7.5, 10% w/v PEG 3350. Crystals of F34A mutation were obtained in 0.2 M Lithium Sulfate monohydrate, 0.1 M Tris pH 8.5, 20% w/v PEG 3350. The crystals were flash-frozen in liquid nitrogen in a cryo-protectant made of the reservoir solution supplemented with 25% glycerol.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35814</offset><text>The diffraction data were collected at the Shanghai SSRF BL18U1 beamline or at the Argonne National Laboratory APS ID19 beamline at 100 K. The data were processed with HKL3000. The hNaa60(1-199) structure was determined by molecular replacement with Phaser using a previously reported GNAT family acetyltransferase structure (PDB 2AE6) as the search model. The hNaa60(1-242) structure was solved by molecular replacement using hNaa60(1-199) structure as the search model. To improve the model quality, the programs ARP/wARP in CCP4 or simulated-annealing in CNS were used. Iterative cycles of manual refitting and crystallographic refinement were performed using COOT and Phenix. Ac-CoA/CoA and malonate were modeled into the closely fitting positive Fo-Fc electron density and then included in following refinement cycles. Topology and parameter files for Ac-CoA/CoA and malonate were generated using PRODRG. All figures for the molecular models were prepared using the PyMOL program. Statistics of diffraction data processing and structure refinement are shown in Table 1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>36891</offset><text>Acetyltransferase assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>36915</offset><text>Acetyltransferase assay of hNaa60 was conducted as described previously. Briefly, a reaction cocktail containing 100 mM Tris-HCl buffer, pH 8.5, 0.07% alkylated BSA, 0.01% NP-40, 1 mM EDTA, 150 μM Ac-CoA (Sigma) was prepared and varied concentrations of the substrate peptide (0–400 μM) (NH2-MKGKEEKEGGAR-COOH) was added in a 1.5-mL microfuge tube, and then the respective enzyme was added to initiate the reaction with a final assay volume of 100 μL. The reaction was carried out for 20 minutes at 37 °C. Aliquots (40 μL) of the reaction were then removed and quenched with 40 μL of ice-cold isopropanol in individual wells of a 96-well black microplate (Corning), and then mixed with 80 μl of 25 μM 7-diethylamino-3-(49 maleimidylphenyl)-4-methylcoumarin (CPM) (Sigma) in 100 mM Tris-HCl (pH 8.5) and 1% Triton X-100 and allowed to react in darkness for 10 minutes prior to reading. The fluorescence signal was monitored using a Varioskan Flash plate reader (Thermo Scientific) at Exmax = 385 nm and Emmax = 465 nm. Substrate inhibition appeared at high concentrations of substrate peptide prevented our kinetics assays from reaching saturation of the enzyme. Therefore, we determined the value of kcat/Km by fitting our data to the equation: v = (kcat/Km)[ET][S] when the substrate concentration was far less than Km. The assays were done in triplicate. The slope of the line indicates the kcat/Km value of the enzyme (Figure S1).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>38401</offset><text>Additional Information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38424</offset><text>How to cite this article: Chen, J.-Y. et al. Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase. Sci. 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Section D, Biological crystallography</infon><infon key="type">ref</infon><infon key="volume">62</infon><infon key="year">2006</infon><offset>42209</offset><text>Scaling and assessment of data quality</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>42248</offset><text>Author Contributions J.-Y.C., L.L., C.-L.C., M.-J.L. and X.Y. designed and performed the experiments. K.T. collected diffraction data at APS. C.-H.Y. conceived and instructed the project. All authors are involved in data analysis. J.-Y.C. and C.-H.Y. wrote the manuscript.</text></passage><passage><infon key="file">srep31425-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>42521</offset><text>Overall structure of Naa60.</text></passage><passage><infon key="file">srep31425-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>42549</offset><text>(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus). Alignment was generated using NPS@ and ESPript.3.0 (http://espript.ibcp.fr/ESPript/ESPript/). Residues 4–6 are highlighted in red box. (B) The structure of hNaa60(1-199)/CoA complex is shown as a yellow cartoon model. The CoA molecule is shown as sticks. (C) The structure of hNaa60(1-242)/Ac-CoA complex is presented as a cartoon model in cyan. The Ac-CoA and malonate molecules are shown as cyan and purple sticks, respectively. The secondary structures are labeled starting with α0. (D) Superposition of hNaa60(1-242) (cyan), hNaa60(1-199) (yellow) and hNaa50 (pink, PDB 3TFY). The Ac-CoA of hNaa60(1-242)/Ac-CoA complex is represented as cyan sticks.</text></passage><passage><infon key="file">srep31425-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>43389</offset><text>Amphipathicity of the α5 helix and alternative conformations of the β7-β8 hairpin.</text></passage><passage><infon key="file">srep31425-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>43482</offset><text>(A) The α5 helix of hNaa60(1-242) in one asymmetric unit (slate) interacts with another hNaa60 molecule in a neighboring asymmetric unit (cyan). A close view of the interaction is shown in red box. Side-chains of hydrophobic residues on α5 helix and the neighboring molecule participating in the interaction are shown as yellow and green sticks, respectively. (B) The α5 helix of hNaa60(1-199) in one asymmetric unit (yellow) interacts with another hNaa60 molecule in the neighboring asymmetric units (green). A close view of the interaction is shown in the red box. Side-chains of hydrophobic residues on α5 helix and the neighboring molecule (green) participating in the interaction are shown as yellow and green sticks, respectively. The third molecule (pink) does not directly interact with the α5 helix. (C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the β7-β8 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E). The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D). The α-amine of the NAT substrate and ε-amine of the KAT substrate (along with the lysine side-chain) subject to acetylation are shown as sticks.</text></passage><passage><infon key="file">srep31425-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>44987</offset><text>Electron density map of the active site.</text></passage><passage><infon key="file">srep31425-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>45028</offset><text>The 2Fo-Fc maps contoured at 1.0σ are shown for hNaa60(1-242)/Ac-CoA (A), hNaa60(1-199)/CoA (B) and hNaa60(1-199) F34A/CoA (C). The putative substrate peptide binding site is indicated by the peptide (shown as pink sticks) from the hNaa50/CoA/peptide complex structure after superimposing hNaa50 on the hNaa60 structures determined in this study. The black arrow indicates the α-amine of the first Met (M1) (all panels). The purple arrow indicates the acetyl moiety of Ac-CoA (A). The red arrow indicates the alternative conformation of the thiol moiety of the co-enzyme when Phe 34 side-chain is displaced (B) or mutated to Ala (C).</text></passage><passage><infon key="file">srep31425-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>45670</offset><text>Structural basis for hNaa60 catalytic activity.</text></passage><passage><infon key="file">srep31425-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>45718</offset><text>(A) Superposition of hNaa60 active site (cyan) on that of hNaa50 (pink, PDB 3TFY). Side-chains of key catalytic and substrate-binding residues are highlighted as sticks. The malonate molecule in the hNaa60(1-242)/Ac-CoA structure and the peptide in the hNaa50/CoA/peptide structure are shown as purple and yellow sticks respectively. (B) A close view of the active site of hNaa60. Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks. The water molecules participating in catalysis in the hNaa60 and hNaa50 structures are showed as green and red spheres, separately. (C) The interaction between the malonate molecule and surrounding residues observed in the hNaa60(1-242)/Ac-CoA structure. The yellow dotted lines indicate the hydrogen bonds. (D) A zoomed view of β3-β4 loop of hNaa60. Key residues discussed in the text (cyan), the malonate (purple) and Ac-CoA (gray) are shown as sticks. The yellow dotted lines indicate the salt bridges.</text></passage><passage><infon key="file">srep31425-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>46876</offset><text>Catalytic activity of hNaa60 and mutant proteins.</text></passage><passage><infon key="file">srep31425-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>46926</offset><text>(A) Catalytic efficiency (shown as kcat/Km values) of hNaa60 (1-199) WT and mutants. Error bars indicate the Standard Error (SE). (B) CD spectra of wild-type and mutant proteins from 250 nm to 190 nm. The sample concentration was 4.5 μM in 20 mM Tris, pH 8.0, 150 mM NaCl, 1% glycerol and 1 mM TCEP at room temperature.</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>47259</offset><text>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;/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;Structure and PDB ID&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;hNaa60(1-242)/Ac-CoA 5HGZ&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;hNaa60(1-199)/CoA 5HH0&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;hNaa60(1-199)F34A/CoA 5HH1&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign=&quot;top&quot;&gt;&lt;tr&gt;&lt;td colspan=&quot;4&quot; align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Data collection&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn1&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; Space group&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;P2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;italic&gt;2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;italic&gt;2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;P2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;italic&gt;2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;italic&gt;2&lt;/italic&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;P2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;italic&gt;2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;italic&gt;2&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;4&quot; align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Cell dimensions&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;53.3, 57.4, 68.8&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;67.8, 73.8, 43.2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;66.7, 74.0, 43.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; α,β,γ (°)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90.0, 90.0, 90.0&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90.0, 90.0, 90.0&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90.0, 90.0, 90.0&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 (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;50–1.38 (1.42–1.38)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;50–1.60 (1.66–1.60)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;50–1.80 (1.86–1.80)&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;p.i.m.&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;3.0 (34.4)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;2.1 (32.5)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;2.6 (47.8)&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;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;21.5 (2.0)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;31.8 (2.0)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;28.0 (2.4)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Completeness (%)&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;99.6 (98.5)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.9 (99.7)&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;6.9 (5.0)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;6.9 (6.2)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;6.3 (5.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;4&quot; align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Refinement&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 (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;25.81–1.38&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;33.55–1.60&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;43.52–1.80&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&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;43660&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;28588&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;20490&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;0.182/0.192&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.181/0.184&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.189/0.209&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;4&quot; align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;No. atoms&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;1717&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1576&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1566&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;116&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;96&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;96&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;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;289&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;258&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;168&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;4&quot; 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;/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;23.8&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;32.0&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;37.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Ligand/ion&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;22.2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;34.6&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;43.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Water&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;35.1&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;46.4&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;49.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;4&quot; align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;R.m.s. deviations&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;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.018&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.017&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.015&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;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.529&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.651&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.581&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;4&quot; align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Ramachandran Plot&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Favoured region&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;98.98%&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;98.93%&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;98.96%&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Allowed region&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.02%&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;1.04%&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Outliers&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.00%&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.00%&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.00%&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>47302</offset><text>Structure and PDB ID	hNaa60(1-242)/Ac-CoA 5HGZ	hNaa60(1-199)/CoA 5HH0	hNaa60(1-199)F34A/CoA 5HH1	 	Data collection*	 	 Space group	P212121	P21212	P21212	 	Cell dimensions	 	 a, b, c (Å)	53.3, 57.4, 68.8	67.8, 73.8, 43.2	66.7, 74.0, 43.5	 	 α,β,γ (°)	90.0, 90.0, 90.0	90.0, 90.0, 90.0	90.0, 90.0, 90.0	 	Resolution (Å)	50–1.38 (1.42–1.38)	50–1.60 (1.66–1.60)	50–1.80 (1.86–1.80)	 	Rp.i.m.(%)**	3.0 (34.4)	2.1 (32.5)	2.6 (47.8)	 	I/σ	21.5 (2.0)	31.8 (2.0)	28.0 (2.4)	 	Completeness (%)	99.8 (99.1)	99.6 (98.5)	99.9 (99.7)	 	Redundancy	6.9 (5.0)	6.9 (6.2)	6.3 (5.9)	 	Refinement	 	 Resolution (Å)	25.81–1.38	33.55–1.60	43.52–1.80	 	 No. reflections	43660	28588	20490	 	 Rwork/Rfree	0.182/0.192	0.181/0.184	0.189/0.209	 	No. atoms	 	 Protein	1717	1576	1566	 	 Ligand/ion	116	96	96	 	 Water	289	258	168	 	B-factors	 	 Protein	23.8	32.0	37.4	 	 Ligand/ion	22.2	34.6	43.7	 	 Water	35.1	46.4	49.1	 	R.m.s. deviations	 	 Bond lengths (Å)	0.018	0.017	0.015	 	 Bond angles (°)	1.529	1.651	1.581	 	Ramachandran Plot	 	 Favoured region	98.98%	98.93%	98.96%	 	 Allowed region	1.02%	1.07%	1.04%	 	 Outliers	0.00%	0.00%	0.00%	 	</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>48477</offset><text>*Values in parentheses are for highest-resolution shell. One crystal was used for each data set.</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>48574</offset><text>**Rp.i.m., a redundancy-independent R factor was used to evaluate the diffraction data quality as was proposed by Evans.</text></passage></document></collection>

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+<collection><source>PMC</source><date>20201220</date><key>pmc.key</key><document><id>5014086</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1016/j.str.2016.06.020</infon><infon key="article-id_pmc">5014086</infon><infon key="article-id_pmid">27524201</infon><infon key="article-id_publisher-id">S0969-2126(16)30167-8</infon><infon key="fpage">1599</infon><infon key="issue">9</infon><infon key="license">This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).</infon><infon key="lpage">1605</infon><infon key="name_0">surname:Zebisch;given-names:Matthias</infon><infon key="name_1">surname:Jackson;given-names:Verity A.</infon><infon key="name_2">surname:Zhao;given-names:Yuguang</infon><infon key="name_3">surname:Jones;given-names:E. Yvonne</infon><infon key="notes">Published: August 11, 2016</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">24</infon><infon key="year">2016</infon><offset>0</offset><text>Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>115</offset><text>Summary</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>123</offset><text>Kremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling. We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains. The structures reveal an unpredicted homology of the WSC domain to hepatocyte growth factor. We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD. DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC. Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>1127</offset><text>Graphical Abstract</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>1146</offset><text>Highlights</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1157</offset><text>The structure of the KREMEN 1 ectodomain is solved from three crystal forms</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1233</offset><text>Kringle, WSC, and CUB subdomains interact tightly to form a single structural unit</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1316</offset><text>The interface to DKKs is formed from the Kringle and WSC domains</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1381</offset><text>The CUB domain is found to interact directly with LRP6PE1PE2</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1442</offset><text>Zebisch et al. describe the ectodomain structure of KREMEN 1, a receptor for Wnt antagonists of the DKK family. Apo structures and a complex with functional fragments of DKK1 and LRP6 shed light on the function of this dual-mode regulator of Wnt signaling.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1700</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1713</offset><text>Signaling by Wnt morphogens is renowned for its fundamental roles in embryonic development, tissue homeostasis, and stem cell maintenance. Due to these functions, generation, delivery, and interpretation of Wnt signals are all heavily regulated in the animal body. Vertebrate Dickkopf proteins (Dkk1, 2, and 4) are one of many secreted antagonists of Wnt and function by blocking access to the Wnt co-receptor LRP5/6. Kremen proteins (Krm1 and Krm2) have been identified as additional high-affinity transmembrane receptors for Dkk. Krm and Dkk synergize in Wnt inhibition during Xenopus embryogenesis to regulate anterior-posterior patterning. Mechanistically it is thought that, in the presence of Dkk, Krm forms a ternary complex with Lrp6, which is then rapidly endocytosed. This amplifies the intrinsic Wnt antagonistic activity of Dkk by efficiently depleting the cell surface of the Wnt co-receptor. In accordance with this, Krm1−/− and Krm2−/− double knockout mice show a high bone mass phenotype typical of increased Wnt signaling, as well as growth of ectopic forelimb digits. Growth of ectopic digits is further enhanced upon additional loss of dkk expression. The Wnt antagonistic activity of Krm1 is also linked to its importance for correct thymus epithelium formation in mice. The importance of intact KRM1 for normal human development and health is highlighted by the recent finding that a homozygous mutation in the ectodomain of KRM1 leads to severe ectodermal dysplasia including oligodontia. Interestingly, the Wnt antagonistic activity of Krm is context dependent, and Krm proteins are actually dual-mode Wnt regulators. In the absence of Dkk, Krm1 and 2 change their function from inhibition to enhancement of Lrp6-mediated signaling. By direct binding to Lrp6 via the ectodomains, Krm proteins promote Lrp6 cell-surface localization and hence increase receptor availability. Further increasing the complexity of Krm functionality, it was recently found that Krm1 (but not Krm2) can also act independently of LRP5/6 and Wnt as a dependence receptor, triggering apoptosis unless bound to Dkk.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3834</offset><text>Structurally, Krm1 and 2 are type I transmembrane proteins with a 40 kDa ectodomain and a flexible cytoplasmic tail consisting of 60–75 residues. The ectodomain consists of three similarly sized structural domains of around 10 kDa each: the N-terminal Kringle domain (KR) is followed by a WSC domain of unknown fold. The third structural domain is a CUB domain. An approximately 70-residue linker connects the CUB domain to the transmembrane span. An intact KR-WSC-CUB domain triplet and membrane attachment is required for Wnt antagonism. The transmembrane span and cytoplasmic tail can be replaced with a GPI linker without impact on Wnt antagonism.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4490</offset><text>We sought to provide structural insights into the multi-functionality of this cell-surface receptor. The structures presented here reveal the unknown fold of the WSC domain and the tight interactions of all three domains. We further succeeded in determination of a low-resolution LRP6PE3PE4-DKK1CRD2-KRM1ECD complex, defining the architecture of the Wnt inhibitory complex that leads to Lrp6 cell-surface depletion.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>4907</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4915</offset><text>The recombinant production of the extracellular domain of Krm for structural studies proved challenging (see Experimental Procedures). We succeeded in purifying KRM1ECD complexes with DKK1fl, DKK1Linker-CRD2, and DKK1CRD2 that were monodisperse and stable in gel filtration, hence indicating at least micromolar affinity (data not shown). Several crystal forms were obtained from these complexes, however, crystals always contained only KRM1 protein.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5366</offset><text>We solved the structure of KRM1ECD in three crystal forms at 1.9, 2.8, and 3.2 Å resolution (Table 1). The high-resolution structure is a near full-length model (Figure 1). The small, flexible, and charged 98AEHED102 loop could only be modeled in a slightly lower resolution structure and in crystal form III. The KR, WSC, and CUB are arranged in a roughly triangular fashion with tight interactions between all three domains. The KR domain, which bears two of the four glycosylation sites, contains the canonical three disulfide bridges (C32-C114, C55-C95, C84-C109) and, like other Kringle domains, is low in secondary structure elements. The structurally most similar Kringle domain is that of human plasminogen (PDB: 1PKR) with an root-mean-square deviation (RMSD) of 1.7 Å for 73 aligned Cα (Figure 1B). The KRM1 structure reveals the fold of the WSC domain for the first time. The structure is best described as a sandwich of a β1-β5-β3-β4-β2 antiparallel β sheet and a single α helix. The structure is also rich in loops and is stabilized by four disulfide bridges (C122-C186, C147-C167, C151-C169, C190-C198). Using the PDBeFold server, we detected a surprising yet significant homology to PAN module domains. The closest structural relative is hepatocyte growth factor (HGF, PDB: 1GP9), which superposes with an RMSD of 2.3 Å for 58 aligned Cα (Figure 1B). The CUB domain bears two glycosylation sites. Although present, the quality of the electron density around N217 did not allow modeling of the sugar moiety. In crystal form I, a calcium ion is present at the canonical position coordinated by the carboxylates of D263, D266 (bidentate), and D306, as well as the carbonyl of N309 and a water molecule. The coordination sphere deviates significantly from perfectly octahedral (not shown). This might result in the site having a low affinity and may explain why calcium is not present in the two low-resolution crystal forms. Loss of calcium has led to loop rearrangements and partial disorder in these crystal forms. The closest structural relative is the CUB_C domain of Tsg-6 (PDB: 2WNO), which superposes with KRMCUB with an RMSD of 1.6 Å for 104 Cα (Figure 1B).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7578</offset><text>A superposition of the three KRM1 structures reveals no major structural differences (Figure 1C) as anticipated from the plethora of interactions between the three domains. Minor differences are caused by the collapse of the Ca2+ binding site in crystal forms II and III and loop flexibility in the KR domain. The F207S mutation recently found to cause ectodermal dysplasia in Palestinian families maps to the hydrophobic core of the protein at the interface of the three subdomains (Figure 1A). Such a mutation is bound to severely destabilize the protein structure of KRM1, leading to disturbance of its Wnt antagonistic, Wnt stimulatory, and Wnt independent activity.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8251</offset><text>Low-Resolution Insight into Ternary Complex Formation</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8305</offset><text>Co-crystallization of LRP6PE3PE4 with DKK1CRD2, and LRP6PE1 with an N-terminal peptide of DKK1 has provided valuable structural insight into direct Wnt inhibition by Dkk ligands. One face of the rather flat DKK1CRD2 fragment binds to the third β propeller of LRP6. Mutational analyses further implied that the LRP6PE3-averted face of DKK1CRD2 bears the Krm binding site, hence suggesting how Dkk can recruit both receptors into a ternary complex.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8755</offset><text>To obtain direct insight into ternary complex formation by Lrp5/6, Dkk, and Krm, we subjected an LRP6PE3PE4-DKK1fl-KRM1ECD complex to crystallization trials. Diffraction data collected from the resulting crystals were highly anisotropic with diffraction extending in the best directions to 3.5 Å and 3.7 Å but only to 6.4 Å in the third direction. Despite the lack of high-resolution diffraction, the general architecture of the ternary complex is revealed (Figure 2A). DKK1CRD2 binds to the top face of the LRP6 PE3 β propeller as described earlier for the binary complex. KRM1ECD does indeed bind on the opposite side of DKK1CRD2 with only its KR and WSC domains engaged in binding (Figure 2A). Although present in the complex subjected to crystallization, we observe no density that could correspond to CRD1 or the domain linker (L). We confirm that the CRD2 of DKK1 is required and sufficient for binding to KRM1: In surface plasmon resonance (SPR), we measured low micromolar affinity between full-length DKK1 and immobilized KRM1ECD (Figure 2B). A SUMO fusion of DKK1L-CRD2 displayed a similar (slightly higher) affinity. In contrast, a SUMO fusion of DKK1CRD1-L did not display binding for concentrations tested up to 325 μM (Figure 2B).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10014</offset><text>Overall, the DKK1-KRM1 interface is characterized by a large number of polar interactions but only few hydrophobic contacts (Figure 2C). The crystal structure gives an explanation for DKK1 loss-of-binding mutations identified previously: R191 of DKK1 forms a double salt bridge to D125 and E162 of KRM1 (Figure 2C). A charge reversal as in the mouse Dkk1 (mDkk1) R197E variant would severely disrupt the binding. Similarly, the K226 side chain of DKK1, which points to a small hydrophobic pocket on the surface of KRM1 formed by Y108, W94, and W106, forms salt bridges with the side chains of KRM1 D88 and D90. Again, a charge reversal as shown before for mDkk1 K232E would be incompatible with binding. The side chain of DKK1 S192 was also predicted to be involved in Krm binding. Indeed, we found (Figure 2C) that the side chain of D201 of KRM1 forms two hydrogen bonds to the side-chain hydroxyl and the backbone amide of S192 (mouse, S198). Additional polar interactions are formed between the N140, S163, and Y165 side chains of KRM1 and DKK1 backbone carbonyls of W206, L190, and C189, respectively. The carbonyl of DKK1 R224 is hydrogen bonded to Y105 and W106 of KRM1. We suspect that the Dkk charge reversal mutations performed in the murine background and shown to diminish Krm binding K211E and R203E (mouse K217E and R209E) do so likely indirectly by disruption of the Dkk fold. We further validated the DKK1 binding site on KRM1 by introducing glycosylation sites at the KR (90DVS92→NVS) and WSC (189VCF191→NCS) domains pointing toward DKK (Figures 2A and 2D). Introduction of N-linked glycans in protein-protein-binding sites is an established way of disrupting protein-binding interfaces. Both ectodomain mutants were secreted comparably with the wild-type, indicating correct folding, but failed to achieve any detectable binding in SPR using full-length DKK1 as analyte. In contrast, a mutant carrying an additional N-glycan outside the interface at the CUB domain (309NQA311→NQS), was wild-type-like in DKK1 binding (Figure 2D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>12076</offset><text>Identification of a Direct LRP6-KRM1 Binding Site</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12126</offset><text>The LRP6PE3PE4-DKK1CRD2-KRM1ECD complex structure reveals no direct interactions between KRM1 and LRP6. We constructed in silico a ternary complex with a close to full-length LRP6 ectodomain (PE1PE2PE3PE4 horse shoe) similar to but without refinement against electron microscopy (EM) or small-angle X-ray scattering data. An auxiliary PE3PE4 fragment was superimposed via PE4 onto PE3 of the crystal structure, and the LRP6PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment (Figure 3A).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12642</offset><text>For this crude approximation of a true ternary complex, we noted very close proximity between the Ca2+-binding region of KRM1 and the top face of the PE2 β propeller of LRP6. The solvent-exposed residues R307, I308, and N309 of the central Ca2+-binding β connection loop of KRM1 would be almost ideally positioned for binding to this face, which is commonly used as a binding site on β propellers. Peptides containing arginine/lysine, isoleucine, and asparagine (consensus sequence N-X-I-(G)-R/K) are also employed by DKK1 and SOST to bind to LRP6 (albeit to propeller 1; Figure 3B). To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment. Indeed, we found that in the absence of Dkk, KRM1ECD bound with considerable affinity to LRP6PE1PE2 (Figure 3C). In contrast, no saturable binding was observed between KRM1 and LRP6PE3PE4. Introduction of an N-glycosylation site at N309 in KRM1ECD abolished LRP6PE1PE2 binding (Figure 3C), while binding to DKK1 was unaffected (Figure 2D). We conclude that the predicted binding site between KRM1CUB and LRP6PE2 is a strong candidate for mediating the direct Lrp6-Krm interaction, which is thought to increase Wnt responsiveness by stabilizing Lrp6 at the cell surface. Further experiments are required to pinpoint the exact binding site. Although LRP6PE1 appears somewhat out of reach in the modeled ternary complex, it cannot be excluded as the Krm binding site in the ternary complex and LRP6-Krm binary complex. The presence of DKK may govern which propeller (PE1 versus PE2) of LRP6 is available for Krm binding.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14374</offset><text>Apparent binding across the proposed KRM1CUB-LRP6PE2 interface is expected to be higher once Krm is also cross-linked to LRP6PE3 via DKK1CRD2 (Figure 3D). Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3. It is therefore possible that the interplay of Krm and Dkk binding can promote changes in LRP6 ectodomain conformation with functional consequences; however, such ideas await investigation.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15012</offset><text>Taken together, the structural and biophysical studies we report here extend our mechanistic understanding of Wnt signal regulation. We describe the ectodomain structure of the dual Wnt regulator Krm1, providing an explanation for the detrimental effect on health and development of a homozygous KRM1 mutation. We also reveal the interaction mode of Krm-Dkk and the architecture of the ternary complex formed by Lrp5/6, Dkk, and Krm. Furthermore, the ternary crystal structure has guided in silico and biophysical analyses to suggest a direct LRP6-KRM1 interaction site. Our findings provide a solid foundation for additional studies to probe how ternary complex formation triggers internalization, whereas Krm binding in the absence of Dkk stabilizes the Wnt co-receptor at the cell surface.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>15806</offset><text>Experimental Procedures</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>15830</offset><text>Large-Scale Mammalian Expression and Protein Purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>15888</offset><text>KrmECD fragments were cloned into pHLsec or variants thereof. Full ectodomain variants (e.g., KRM1 isoform 3, P30-T377) were well secreted into the conditioned medium (CM) of HEK293T cells, but exhibited extensive O-glycosylation (as judged from smeary bands in western blot), which would be detrimental to crystallization. Fragments truncated to the KR-WSC-CUB core gave sharp bands but were barely secreted. We therefore engineered an A23-G373 (isoform 1 numbering used throughout the article) full ectodomain construct (KRM1ECD-TEV) with a C-terminal His10 tag that contained a TEV protease cleavage site after E324. The expected sequence of the secreted protein is ETG-23APSPGLGPGPE31 … 320AVKEE324-GSENLYFQGGS-325LPQ … VPG373-THHHHHHHHHH (the isoform-2-specific PG insertion and the TEV site are underlined). This construct was well secreted and could be processed using TEV protease. However, 80%–90% of the protein eluted as aggregates from a size-exclusion column even before TEV treatment. The same applied to analog constructs for Krm1 from zebrafish, frog, and mouse. No monomeric protein at all could be obtained for several Krm2 constructs from multiple species. A KRM1ECD-TEV expressing stable GntI-deficient HEK293S cell line was generated by excision of an EcoRI-XhoI fragment, sub-cloning into pNeo-Sec-1, and selection of neomycin-resistant cells. The stable cell line showed expression levels superior to transiently transfected cells (not shown).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>17365</offset><text>Human LRP6PE1PE2, LRP6PE3PE4, and full-length DKK1 were produced in a similar way as described. Shorter constructs of DKK1 lacking the N-terminal flexible region and CRD1 were not secreted from HEK cells. However, using the approach of an N-terminal fusion to a modified SUMO protein as described earlier, we succeeded in secretory expression of a SUMO-DKK1Linker-CRD2 construct encompassing residues S141-H266. A variant of this containing a TEV cleavage site just before T181, SUMO-DKK1Linker-TEV-CRD2, was also well expressed and allowed removal of the flexible linker region.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>17945</offset><text>To obtain complexes of KRM1ECD-TEV, we (co-)transfected the stable cell line with DKK and LRP6PE3PE4 constructs described earlier. Binary and ternary KRM1ECD-DKK1fl and KRM1ECD-DKK1fl-LRP6PE3PE4 complexes were stable in gel-filtration eluting as distinct monodisperse peaks.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>18220</offset><text>Crystallization and Data Collection</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>18256</offset><text>All samples subjected to crystallization were purified from CM by affinity and size-exclusion chromatography. After treatment with TEV protease and endoglycosidase F1 overnight using mass equivalents of 1%, samples were subjected to size-exclusion chromatography in 10 mM HEPES/NaOH (pH 7.5), 150 mM NaCl. The crystals giving rise to the 1.9 Å dataset for KRM1 in crystal form I were obtained from a KRM1ECD-DKK1Linker-CRD2 complex concentrated to 12 mg/mL. Out of this complex, KRM1ECD crystallized alone in 2.0 M ammonium sulfate, 5% (v/v) iso-propanol. For cryoprotection, crystals were transferred to mother liquor mixed 1:1 with 3.4 M sodium malonate (pH 7.0). The slightly less well-ordered crystal of crystal form I and crystals of form II were obtained from a KRM1ECD-DKK1CRD2 complex using the SUMO-DKK1Linker-TEV-CRD2 construct and releasing SUMO and the DKK linker region by TEV and 3C protease treatment. Crystals of form I (2.1 Å) appeared from protein at 12 mg/mL in 1.0 M (NH4)H2PO4, 0.100 M sodium citrate (pH 5.6) and were cryoprotected by transfer to 2.9 M sodium malonate (pH 5.0). Crystals of form II grew from protein concentrated to 17 mg/mL in 1.0 M MgSO4, 0.1 M trisodium citrate (final pH 5.6). For cryoprotection, crystals were transferred to mother liquor mixed 1:3 with 3.0 M ammonium sulfate, 18% glycerol. Crystal form III appeared after 11 months in a dried-out drop of condition H5 of the Morpheus screen. The protein concentration had been 9 mg/mL. For cryoprotection, fresh liquid from Morpheus/H5 was added. The ternary complex structure was obtained from an LRP6PE3PE4-DKK1fl-KRM1ECD complex at 9 mg/mL that grew in condition E10 of the PACTpremier screen (pH approximately 6.8) over the course of 2–11 months. For cryoprotection, 10% PEG200 was added. By mistake, the crystals were incubated for 1 hr with 1 mM platinum compound in this cryosolution before cryocooling.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>20188</offset><text>Structure Determination</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20212</offset><text>Diffraction data were collected at DIAMOND synchrotron light source at the beamlines detailed in Table 1. The structure was initially solved from crystal form III by molecular replacement (MR) with PHASER, placing models for the CUB domain (PDB: 2WNO, CUB_C domain of Tsg-6, 37% sequence identity), and the KR domain (PDB: 1PKR, Kringle 1 of plasminogen; 39% sequence identity). Traceable density for the WSC domain became immediately evident. The KRM1 structure was then built and refined by cycling between the various crystal forms.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20748</offset><text>For the ternary complex, we obtained only a low-resolution, highly anisotropic dataset extending to Bragg spacings of 3.5 Å, 6.4 Å, and 3.7 Å along the three principle directions (&lt;I/σI&gt; = 2). All data to 3.5 Å were used during structure determination by MR. LRP6PE3PE4 (PDB: 4A0P) and KRM1ECD (both stripped of glycosylation sites) could be placed independently by PHASER, giving Z scores of &gt;10 and log likelihood gains (LLG) of &gt;200. The combined LLG was 673, increasing to 901 after rigid-body refinement. Strong electron density became apparent at glycosylation sites and close to methionines (see platinum soak above), further supporting the MR solution. Additional strong density was evident between LRP6 and KRM1, suggesting the presence of DKK1. A model of the DKK1CRD2 (PDB: 3S2K and 3S8V) could then be placed with PHASER by testing all rotation function peaks. This increased the LLG from 901 to 973 indicating a correct solution. The individually placed LRP6 and DKK models were then replaced with chains B and C from the LRP6-DKK complex in PDB: 3S2K. The structure was subjected to rigid-body refinement using single structural domains as individually positioned bodies.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>21945</offset><text>We then performed restrained refinement of the coordinates against the ellipsoidally truncated and anisotropically scaled diffraction data as obtained from the diffraction anisotropy server at UCLA. The resolution cutoffs were 3.5 Å, 6.4 Å, and 3.7 Å. Strong geometric restraints generated by PROSMART from the available high-resolution reference structures were used during refinement. No manual model building was attempted. Restrained refinement was followed by ten cycles of structure idealization. The final model had Rwork/Rfree errors of 32.5%/36.1% against the anisotropy-corrected data and 32.1%/35.5% against the unmodified but ellipsoidally truncated diffraction data.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>22631</offset><text>Surface Plasmon Resonance</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>22657</offset><text>Equilibrium experiments were performed as described before with the addition of 2 mM CaCl2 for experiments investigating the direct LRP6PE1PE2-KRM1ECD interaction.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">title_1</infon><offset>22822</offset><text>Author Contributions</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">paragraph</infon><offset>22843</offset><text>M.Z. and V.A.J. performed experiments with support from Y.Z., who generated the stable cell line. 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Cysteines as ball and sticks, glycosylation sites as sticks. The bound calcium is shown as a gray sphere. The site of the F207S mutation associated with ectodermal dysplasia in humans is shown as mesh.</text></passage><passage><infon key="file">gr1.jpg</infon><infon key="id">fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27356</offset><text>(B) Superposition of the three KRM1ECD subdomains (solid) with their next structurally characterized homologs (half transparent).</text></passage><passage><infon key="file">gr1.jpg</infon><infon key="id">fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27486</offset><text>(C) Superposition of KRM1ECD from the three crystal forms. Alignment scores for each pairing are indicated on the dashed triangle.</text></passage><passage><infon key="file">gr2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27617</offset><text>Insight into Ternary Complex Formation</text></passage><passage><infon key="file">gr2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27656</offset><text>(A) The structure of the ternary LRP6PE3PE4-DKK1CRD2-KRM1ECD complex. DKK1 (orange) is sandwiched between the PE3 module of LRP6 (blue) and the KR-WSC domain pair of KRM1 (green). Colored symbols indicate introduced N-glycan attachment sites (see D).</text></passage><passage><infon key="file">gr2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27907</offset><text>(B) SPR data comparing binding of full-length DKK1 and SUMO fusions of DKK1 truncations for binding to immobilized wild-type KRM1ECD.</text></passage><passage><infon key="file">gr2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28041</offset><text>(C) Close-up view of the DKK1CRD2-KRM1ECD interface. Residues involved in interface formation are shown as sticks; those mentioned in the text are labeled. Salt bridges are in pink and hydrogen bonds in black. Model bias cannot be excluded as single atoms and bonds are not resolved at 6.4–3.5 Å. See also Figure S1.</text></passage><passage><infon key="file">gr2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28364</offset><text>(D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange). See also symbols in (A).</text></passage><passage><infon key="file">gr3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28614</offset><text>LRP6-KRM1 Direct Interaction and Summary</text></passage><passage><infon key="file">gr3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28655</offset><text>(A) In a construction of a ternary complex with all four β propellers of LRP6 intact, the CUB domain points via its Ca2+-binding region toward the top face of the second β propeller.</text></passage><passage><infon key="file">gr3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28844</offset><text>(B) Close-up view of the potential interaction site. In addition, LRP6PE2 has been superimposed with DKK1 (yellow) and SOST (pink) peptide complexes of LRP6PE1.</text></passage><passage><infon key="file">gr3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>29005</offset><text>(C) SPR measurements comparing LRP6PE1PE2 binding with wild-type KRM1ECD and the GlycoCUB mutant bearing an N-glycan at N309.</text></passage><passage><infon key="file">gr3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>29131</offset><text>(D) Schematic representation of structural and biophysical findings and their implications for Wnt-dependent (left, middle) and independent (right) signaling. Conformational differences in the depictions of LRP6 are included purely for ease of representation.</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>29391</offset><text>Diffraction and Refinement Statistics</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 xmlns:xlink=&quot;http://www.w3.org/1999/xlink&quot; frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;thead&gt;&lt;tr&gt;&lt;th/&gt;&lt;th&gt;KRM1&lt;sub&gt;ECD&lt;/sub&gt;&lt;/th&gt;&lt;th&gt;KRM1&lt;sub&gt;ECD&lt;/sub&gt;&lt;/th&gt;&lt;th&gt;KRM1&lt;sub&gt;ECD&lt;/sub&gt;&lt;/th&gt;&lt;th&gt;KRM1&lt;sub&gt;ECD&lt;/sub&gt;&lt;/th&gt;&lt;th&gt;LRP6&lt;sub&gt;PE3PE4&lt;/sub&gt;-DKK&lt;sub&gt;CRD2&lt;/sub&gt;-KRM1&lt;sub&gt;ECD&lt;/sub&gt;&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td&gt;Crystal form&lt;/td&gt;&lt;td&gt;I&lt;/td&gt;&lt;td&gt;I&lt;/td&gt;&lt;td&gt;II&lt;/td&gt;&lt;td&gt;III&lt;/td&gt;&lt;td&gt;I&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;X-ray source&lt;/td&gt;&lt;td&gt;Diamond i04&lt;/td&gt;&lt;td&gt;Diamond i03&lt;/td&gt;&lt;td&gt;Diamond i03&lt;/td&gt;&lt;td&gt;Diamond i04&lt;/td&gt;&lt;td&gt;Diamond i04&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Wavelength (Å)&lt;/td&gt;&lt;td&gt;0.9793&lt;/td&gt;&lt;td&gt;0.9700&lt;/td&gt;&lt;td&gt;0.9700&lt;/td&gt;&lt;td&gt;0.9795&lt;/td&gt;&lt;td&gt;0.9795&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Space group&lt;/td&gt;&lt;td&gt;&lt;italic&gt;P&lt;/italic&gt;3&lt;sub&gt;1&lt;/sub&gt;21&lt;/td&gt;&lt;td&gt;&lt;italic&gt;P&lt;/italic&gt;3&lt;sub&gt;1&lt;/sub&gt;21&lt;/td&gt;&lt;td&gt;&lt;italic&gt;P&lt;/italic&gt;4&lt;sub&gt;3&lt;/sub&gt;&lt;/td&gt;&lt;td&gt;&lt;italic&gt;P&lt;/italic&gt;4&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;/td&gt;&lt;td&gt;&lt;italic&gt;C&lt;/italic&gt;222&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Unit cell a/α (Å/°)&lt;/td&gt;&lt;td&gt;50.9/90&lt;/td&gt;&lt;td&gt;50.5/90&lt;/td&gt;&lt;td&gt;65.8/90&lt;/td&gt;&lt;td&gt;67.8/90&lt;/td&gt;&lt;td&gt;86.9/90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;b/β (Å/°)&lt;/td&gt;&lt;td&gt;50.9/90&lt;/td&gt;&lt;td&gt;50.5/90&lt;/td&gt;&lt;td&gt;65.8/90&lt;/td&gt;&lt;td&gt;67.8/90&lt;/td&gt;&lt;td&gt;100.1/90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;c/γ (Å/°)&lt;/td&gt;&lt;td&gt;188.4/120&lt;/td&gt;&lt;td&gt;187.4/120&lt;/td&gt;&lt;td&gt;75.0/90&lt;/td&gt;&lt;td&gt;198.2/90&lt;/td&gt;&lt;td&gt;270.7/90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Wilson B factor (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/td&gt;&lt;td&gt;31&lt;/td&gt;&lt;td&gt;41&lt;/td&gt;&lt;td&gt;76&lt;/td&gt;&lt;td&gt;77&lt;/td&gt;&lt;td&gt;NA&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Resolution range (Å)&lt;/td&gt;&lt;td&gt;47.10–1.90 (1.95–1.90)&lt;/td&gt;&lt;td&gt;62.47–2.10 (2.16–2.10)&lt;/td&gt;&lt;td&gt;75.00–2.80 (2.99–2.80)&lt;/td&gt;&lt;td&gt;67.80–3.20 (3.42–3.20)&lt;/td&gt;&lt;td&gt;67.68–3.50 (7.16–6.40, 3.92–3.50)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Unique reflections&lt;/td&gt;&lt;td&gt;23,300 (1,524)&lt;/td&gt;&lt;td&gt;17,089 (1,428)&lt;/td&gt;&lt;td&gt;7,964 (1,448)&lt;/td&gt;&lt;td&gt;8,171 (1,343)&lt;/td&gt;&lt;td&gt;8,070 (723, 645)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Average multiplicity&lt;/td&gt;&lt;td&gt;9.1 (9.2)&lt;/td&gt;&lt;td&gt;5.2 (5.3)&lt;/td&gt;&lt;td&gt;3.7 (3.7)&lt;/td&gt;&lt;td&gt;22.7 (12.6)&lt;/td&gt;&lt;td&gt;3.8 (3.5, 4.4)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Completeness (%)&lt;/td&gt;&lt;td&gt;99.8 (98.5)&lt;/td&gt;&lt;td&gt;100 (100)&lt;/td&gt;&lt;td&gt;99.8 (100)&lt;/td&gt;&lt;td&gt;98.8 (93.4)&lt;/td&gt;&lt;td&gt;51.6 (98.5, 14.1)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&amp;lt;&lt;italic&gt;I&lt;/italic&gt;/&lt;italic&gt;σI&lt;/italic&gt;&amp;gt;&lt;/td&gt;&lt;td&gt;11.4 (1.7)&lt;/td&gt;&lt;td&gt;12.0 (1.7)&lt;/td&gt;&lt;td&gt;14.9 (1.5)&lt;/td&gt;&lt;td&gt;13.1 (1.9)&lt;/td&gt;&lt;td&gt;4.6 (4.1, 2.2)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;merge&lt;/sub&gt; (%)&lt;/td&gt;&lt;td&gt;14.8 (158.3)&lt;/td&gt;&lt;td&gt;9.3 (98.0)&lt;/td&gt;&lt;td&gt;6.2 (98.9)&lt;/td&gt;&lt;td&gt;29.8 (142.2)&lt;/td&gt;&lt;td&gt;44.9 (40.5, 114.2)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;pim&lt;/sub&gt; (%)&lt;/td&gt;&lt;td&gt;15.7 (55.3)&lt;/td&gt;&lt;td&gt;10.3 (109.0)&lt;/td&gt;&lt;td&gt;3.7 (53.8)&lt;/td&gt;&lt;td&gt;6.3 (40.0)&lt;/td&gt;&lt;td&gt;24.7 (23.9, 59.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;bold&gt;Refinement&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;work&lt;/sub&gt; (%)&lt;/td&gt;&lt;td&gt;17.9&lt;/td&gt;&lt;td&gt;18.4&lt;/td&gt;&lt;td&gt;21.6&lt;/td&gt;&lt;td&gt;20.2&lt;/td&gt;&lt;td&gt;32.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt; (%)&lt;/td&gt;&lt;td&gt;22.7&lt;/td&gt;&lt;td&gt;23.2&lt;/td&gt;&lt;td&gt;30.7&lt;/td&gt;&lt;td&gt;27.1&lt;/td&gt;&lt;td&gt;35.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;bold&gt;No. of Non-Hydrogen Atoms&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Protein&lt;/td&gt;&lt;td&gt;2,260&lt;/td&gt;&lt;td&gt;2,301&lt;/td&gt;&lt;td&gt;2,102&lt;/td&gt;&lt;td&gt;2,305&lt;/td&gt;&lt;td&gt;7,730&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;N-glycans&lt;/td&gt;&lt;td&gt;42&lt;/td&gt;&lt;td&gt;42&lt;/td&gt;&lt;td&gt;28&lt;/td&gt;&lt;td&gt;28&lt;/td&gt;&lt;td&gt;0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Water&lt;/td&gt;&lt;td&gt;79&lt;/td&gt;&lt;td&gt;54&lt;/td&gt;&lt;td&gt;0&lt;/td&gt;&lt;td&gt;2&lt;/td&gt;&lt;td&gt;0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Ligands&lt;/td&gt;&lt;td&gt;6&lt;/td&gt;&lt;td&gt;6&lt;/td&gt;&lt;td&gt;2&lt;/td&gt;&lt;td&gt;5&lt;/td&gt;&lt;td&gt;0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;bold&gt;Average B factor (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Protein&lt;/td&gt;&lt;td&gt;63&lt;/td&gt;&lt;td&gt;65&lt;/td&gt;&lt;td&gt;108&lt;/td&gt;&lt;td&gt;84&lt;/td&gt;&lt;td&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;N-glycans&lt;/td&gt;&lt;td&gt;35&lt;/td&gt;&lt;td&gt;46&lt;/td&gt;&lt;td&gt;102&lt;/td&gt;&lt;td&gt;18&lt;/td&gt;&lt;td&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Water&lt;/td&gt;&lt;td&gt;68&lt;/td&gt;&lt;td&gt;85&lt;/td&gt;&lt;td&gt;–&lt;/td&gt;&lt;td&gt;75&lt;/td&gt;&lt;td&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Ligands&lt;/td&gt;&lt;td&gt;36&lt;/td&gt;&lt;td&gt;47&lt;/td&gt;&lt;td&gt;91&lt;/td&gt;&lt;td&gt;75&lt;/td&gt;&lt;td&gt;66&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;bold&gt;RMSD from Ideality&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Bond lengths (Å)&lt;/td&gt;&lt;td&gt;0.020&lt;/td&gt;&lt;td&gt;0.016&lt;/td&gt;&lt;td&gt;0.019&lt;/td&gt;&lt;td&gt;0.016&lt;/td&gt;&lt;td&gt;0.004&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Bond angles (°)&lt;/td&gt;&lt;td&gt;2.050&lt;/td&gt;&lt;td&gt;1.748&lt;/td&gt;&lt;td&gt;1.952&lt;/td&gt;&lt;td&gt;1.796&lt;/td&gt;&lt;td&gt;0.770&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;bold&gt;Ramachandran Plot&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Favored (%)&lt;/td&gt;&lt;td&gt;96.8&lt;/td&gt;&lt;td&gt;95.5&lt;/td&gt;&lt;td&gt;96.9&lt;/td&gt;&lt;td&gt;94.9&lt;/td&gt;&lt;td&gt;92.3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Allowed (%)&lt;/td&gt;&lt;td&gt;99.7&lt;/td&gt;&lt;td&gt;100.0&lt;/td&gt;&lt;td&gt;100.0&lt;/td&gt;&lt;td&gt;99.7&lt;/td&gt;&lt;td&gt;99.8&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Number of outliers&lt;/td&gt;&lt;td&gt;1&lt;/td&gt;&lt;td&gt;0&lt;/td&gt;&lt;td&gt;0&lt;/td&gt;&lt;td&gt;1&lt;/td&gt;&lt;td&gt;2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;PDB code&lt;/td&gt;&lt;td&gt;&lt;ext-link ext-link-type=&quot;uri&quot; xlink:href=&quot;pdb:5FWS&quot; id=&quot;intref0085&quot;&gt;5FWS&lt;/ext-link&gt;&lt;/td&gt;&lt;td&gt;&lt;ext-link ext-link-type=&quot;uri&quot; xlink:href=&quot;pdb:5FWT&quot; id=&quot;intref0090&quot;&gt;5FWT&lt;/ext-link&gt;&lt;/td&gt;&lt;td&gt;&lt;ext-link ext-link-type=&quot;uri&quot; xlink:href=&quot;pdb:5FWU&quot; id=&quot;intref0095&quot;&gt;5FWU&lt;/ext-link&gt;&lt;/td&gt;&lt;td&gt;&lt;ext-link ext-link-type=&quot;uri&quot; xlink:href=&quot;pdb:5FWV&quot; id=&quot;intref0100&quot;&gt;5FWV&lt;/ext-link&gt;&lt;/td&gt;&lt;td&gt;&lt;ext-link ext-link-type=&quot;uri&quot; xlink:href=&quot;pdb:5FWW&quot; id=&quot;intref0105&quot;&gt;5FWW&lt;/ext-link&gt;&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>29429</offset><text>	KRM1ECD	KRM1ECD	KRM1ECD	KRM1ECD	LRP6PE3PE4-DKKCRD2-KRM1ECD	 	Crystal form	I	I	II	III	I	 	X-ray source	Diamond i04	Diamond i03	Diamond i03	Diamond i04	Diamond i04	 	Wavelength (Å)	0.9793	0.9700	0.9700	0.9795	0.9795	 	Space group	P3121	P3121	P43	P41212	C2221	 	Unit cell a/α (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	86.9/90	 	b/β (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	100.1/90	 	c/γ (Å/°)	188.4/120	187.4/120	75.0/90	198.2/90	270.7/90	 	Wilson B factor (Å2)	31	41	76	77	NA	 	Resolution range (Å)	47.10–1.90 (1.95–1.90)	62.47–2.10 (2.16–2.10)	75.00–2.80 (2.99–2.80)	67.80–3.20 (3.42–3.20)	67.68–3.50 (7.16–6.40, 3.92–3.50)	 	Unique reflections	23,300 (1,524)	17,089 (1,428)	7,964 (1,448)	8,171 (1,343)	8,070 (723, 645)	 	Average multiplicity	9.1 (9.2)	5.2 (5.3)	3.7 (3.7)	22.7 (12.6)	3.8 (3.5, 4.4)	 	Completeness (%)	99.8 (98.5)	100 (100)	99.8 (100)	98.8 (93.4)	51.6 (98.5, 14.1)	 	&lt;I/σI&gt;	11.4 (1.7)	12.0 (1.7)	14.9 (1.5)	13.1 (1.9)	4.6 (4.1, 2.2)	 	Rmerge (%)	14.8 (158.3)	9.3 (98.0)	6.2 (98.9)	29.8 (142.2)	44.9 (40.5, 114.2)	 	Rpim (%)	15.7 (55.3)	10.3 (109.0)	3.7 (53.8)	6.3 (40.0)	24.7 (23.9, 59.9)	 		 	Refinement	 		 	Rwork (%)	17.9	18.4	21.6	20.2	32.1	 	Rfree (%)	22.7	23.2	30.7	27.1	35.5	 		 	No. of Non-Hydrogen Atoms	 		 	Protein	2,260	2,301	2,102	2,305	7,730	 	N-glycans	42	42	28	28	0	 	Water	79	54	0	2	0	 	Ligands	6	6	2	5	0	 		 	Average B factor (Å2)	 		 	Protein	63	65	108	84	–	 	N-glycans	35	46	102	18	–	 	Water	68	85	–	75	–	 	Ligands	36	47	91	75	66	 		 	RMSD from Ideality	 		 	Bond lengths (Å)	0.020	0.016	0.019	0.016	0.004	 	Bond angles (°)	2.050	1.748	1.952	1.796	0.770	 		 	Ramachandran Plot	 		 	Favored (%)	96.8	95.5	96.9	94.9	92.3	 	Allowed (%)	99.7	100.0	100.0	99.7	99.8	 	Number of outliers	1	0	0	1	2	 	PDB code	5FWS	5FWT	5FWU	5FWV	5FWW	 	</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>31236</offset><text>Values in parentheses refer to the highest-resolution shell. An additional shell given for the ternary complex corresponds to the last shell with near-complete diffraction data. NA, not announced.</text></passage></document></collection>

raw_BioC_XML/PMC5063996_raw.xml ADDED Viewed

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