PMC 20140719 pmc.key 4850288 CC BY no 0 0 P. merdae C11 Cysteine Peptidase 10.1074/jbc.M115.706143 4850288 26940874 M115.706143 9482 18 C-terminal domain (carboxyl tail domain, CTD) crystal structure cysteine protease enzyme proteolysis active site domain kinteoplast Author's Choice—Final version free via Creative Commons CC-BY license. 9491 surname:McLuskey;given-names:Karen surname:Grewal;given-names:Jaspreet S. surname:Das;given-names:Debanu surname:Godzik;given-names:Adam surname:Lesley;given-names:Scott A. surname:Deacon;given-names:Ashley M. surname:Coombs;given-names:Graham H. surname:Elsliger;given-names:Marc-André surname:Wilson;given-names:Ian A. surname:Mottram;given-names:Jeremy C. TITLE front 291 2016 0 Crystal Structure and Activity Studies of the C11 Cysteine Peptidase from Parabacteroides merdae in the Human Gut Microbiome* evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:28:56Z Crystal Structure experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T13:29:22Z Activity Studies protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:32:18Z C11 protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:29:34Z Cysteine Peptidase species MESH: melaniev@ebi.ac.uk 2023-03-16T13:29:41Z Parabacteroides merdae species MESH: melaniev@ebi.ac.uk 2023-03-16T13:29:46Z Human ABSTRACT abstract 126 Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. However, for the clostripain family (denoted C11), little is currently known. Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/β/α sandwich and an unusual C-terminal domain. It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other families in the clan. These studies also revealed a well ordered break in the polypeptide chain at Lys147, resulting in a large conformational rearrangement close to the active site. Biochemical and kinetic analysis revealed Lys147 to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca2+ for activity. Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms. protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:31:49Z Clan CD cysteine peptidases protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:31:52Z peptidases taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:31:59Z mammalian protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:32:04Z caspases protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:32:11Z clostripain family protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:32:18Z C11 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:28:56Z crystal structure protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:32:18Z C11 species MESH: melaniev@ebi.ac.uk 2023-03-16T13:29:46Z human taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:32:46Z bacterium species MESH: melaniev@ebi.ac.uk 2023-03-16T13:29:41Z Parabacteroides merdae protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:57Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:57Z PmC11 oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:33:21Z monomeric protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:29:34Z cysteine peptidase structure_element SO: melaniev@ebi.ac.uk 2023-03-16T13:33:11Z extended caspase-like α/β/α sandwich structure_element SO: melaniev@ebi.ac.uk 2023-03-16T13:33:06Z C-terminal domain protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:41:33Z clan CD cysteine peptidases residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:33:27Z Lys147 site SO: melaniev@ebi.ac.uk 2023-03-16T13:34:26Z active site experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T13:34:21Z Biochemical and kinetic analysis residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:33:27Z Lys147 site SO: melaniev@ebi.ac.uk 2023-03-16T13:33:50Z intramolecular processing site ptm MESH: melaniev@ebi.ac.uk 2023-03-16T13:34:11Z cleavage protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:34:16Z full activation protein PR: melaniev@ebi.ac.uk 2023-03-16T13:34:18Z enzyme protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:57Z PmC11 site SO: melaniev@ebi.ac.uk 2023-03-16T13:34:33Z acidic binding pocket residue_name SO: melaniev@ebi.ac.uk 2023-03-16T14:54:55Z Arg residue_name SO: melaniev@ebi.ac.uk 2023-03-16T14:55:14Z Lys residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:48:22Z P1 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T13:34:44Z Ca2+ protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:57Z PmC11 protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:37:55Z C11 peptidases INTRO title_1 1565 Introduction INTRO paragraph 1578 Cysteine peptidases play crucial roles in the virulence of bacterial and other eukaryotic pathogens. In the MEROPS peptidase database, clan CD contains groups (or families) of cysteine peptidases that share some highly conserved structural elements. Clan CD families are typically described using the name of their archetypal, or founding, member and also given an identification number preceded by a “C,” to denote cysteine peptidase. Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80). No structural information is available for clostripain (C11), separase (C50), or PrtH-peptidase (C85). protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:39:31Z Cysteine peptidases taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:39:44Z bacterial taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:39:50Z eukaryotic protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:39:59Z clan CD protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:39:31Z cysteine peptidases protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:48:11Z highly conserved protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:40:10Z Clan CD families protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:29:34Z cysteine peptidase evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:40:27Z crystal structures protein PR: melaniev@ebi.ac.uk 2023-03-16T13:44:14Z legumain protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:40:35Z C13 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:44:28Z caspase protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:49:48Z C14a protein PR: melaniev@ebi.ac.uk 2023-03-16T13:46:27Z paracaspase protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:40:42Z C14b(P protein PR: melaniev@ebi.ac.uk 2023-03-16T13:46:38Z metacaspase protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:40:47Z C14b(M protein PR: melaniev@ebi.ac.uk 2023-03-16T13:46:53Z gingipain protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:40:52Z C25 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T13:40:54Z cysteine peptidase domain structure_element SO: melaniev@ebi.ac.uk 2023-03-16T13:40:57Z CPD protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:48:35Z C80 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:50:12Z clostripain protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:32:18Z C11 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:47:03Z separase protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:48:40Z C50 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:47:15Z PrtH-peptidase protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:48:43Z C85 INTRO paragraph 2445 Clan CD enzymes have a highly conserved His/Cys catalytic dyad and exhibit strict specificity for the P1 residue of their substrates. However, despite these similarities, clan CD forms a functionally diverse group of enzymes: the overall structural diversity between (and at times within) the various families provides these peptidases with a wide variety of substrate specificities and activation mechanisms. Several members are initially expressed as proenzymes, demonstrating self-inhibition prior to full activation. protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:48:05Z Clan CD enzymes protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:48:11Z highly conserved site SO: melaniev@ebi.ac.uk 2023-03-16T13:48:17Z His/Cys catalytic dyad residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:48:22Z P1 protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:39:59Z clan CD protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:48:29Z peptidases INTRO paragraph 2966 The archetypal and arguably most notable family in the clan is that of the mammalian caspases (C14a), although clan CD members are distributed throughout the entire phylogenetic kingdom and are often required in fundamental biological processes. Interestingly, little is known about the structure or function of the C11 proteins, despite their widespread distribution and its archetypal member, clostripain from Clostridium histolyticum, first reported in the literature in 1938. Clostripain has been described as an arginine-specific peptidase with a requirement for Ca2+ and loss of an internal nonapeptide for full activation; lack of structural information on the family appears to have prohibited further investigation. taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:31:59Z mammalian protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:32:04Z caspases protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:49:48Z C14a protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:39:59Z clan CD protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:32:18Z C11 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:50:12Z clostripain species MESH: melaniev@ebi.ac.uk 2023-03-16T13:50:06Z Clostridium histolyticum protein PR: melaniev@ebi.ac.uk 2023-03-16T13:50:12Z Clostripain protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:50:19Z arginine-specific peptidase chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T13:50:22Z Ca2+ structure_element SO: melaniev@ebi.ac.uk 2023-03-16T13:50:26Z internal nonapeptide protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:50:54Z full activation INTRO paragraph 3691 As part of an ongoing project to characterize commensal bacteria in the microbiome that inhabit the human gut, the structure of C11 peptidase, PmC11, from Parabacteroides merdae was determined using the Joint Center for Structural Genomics (JCSG)4 HTP structural biology pipeline. The structure was analyzed, and the enzyme was biochemically characterized to provide the first structure/function correlation for a C11 peptidase. taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:51:49Z bacteria species MESH: melaniev@ebi.ac.uk 2023-03-16T13:29:46Z human evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:51:44Z structure protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:51:40Z C11 peptidase protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:57Z PmC11 species MESH: melaniev@ebi.ac.uk 2023-03-16T13:29:41Z Parabacteroides merdae experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T13:51:56Z structure was analyzed experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T13:51:58Z biochemically characterized protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:51:40Z C11 peptidase METHODS title_1 4120 Experimental Procedures METHODS paragraph 4144 Cloning, expression, purification, crystallization, and structure determination of PmC11 were carried out using standard JCSG protocols as follows. METHODS title_4 4292 Cloning METHODS paragraph 4300 Clones were generated using the polymerase incomplete primer extension (PIPE) cloning method. The gene encoding PmC11 (SP5111E) was amplified by polymerase chain reaction (PCR) from P. merdae genomic DNA using PfuTurbo DNA polymerase (Stratagene), using I-PIPE primers that included sequences for the predicted 5′ and 3′ ends (shown below). The expression vector, pSpeedET, which encodes an amino-terminal tobacco etch virus protease-cleavable expression and purification tag (MGSDKIHHHHHHENLYFQ/G), was PCR amplified with V-PIPE (Vector) primers. V-PIPE and I-PIPE PCR products were mixed to anneal the amplified DNA fragments together. Escherichia coli GeneHogs (Invitrogen) competent cells were transformed with the I-PIPE/V-PIPE mixture and dispensed on selective LB-agar plates. The cloning junctions were confirmed by DNA sequencing. The plasmid encoding the full-length protein was deposited in the PSI:Biology Materials Repository at the DNASU plasmid repository (PmCD00547516). For structure determination, to obtain soluble protein using the PIPE, method the gene segment encoding residues Met1-Asn22 was deleted because these residues were predicted to correspond to a signal peptide using SignalP. METHODS title_4 5514 Protein Expression and Selenomethionine Incorporation METHODS paragraph 5568 The expression plasmid for the truncated PmC11 construct was transformed into E. coli GeneHogs competent cells and grown in minimal media supplemented with selenomethionine and 30 μg ml−1 of kanamycin at 37 °C using a GNF fermentor. A methionine auxotrophic strain was not required as selenomethionine is incorporated via the inhibition of methionine biosynthesis. Protein expression was induced using 0.1% (w/v) l-arabinose and the cells were left to grow for a further 3 h at 37 °C. At the end of the cell culture, lysozyme was added to all samples to a final concentration of 250 μg ml−1 and the cells were harvested and stored at −20 °C, until required. Primers used in this section are as follows: I-PIPE (forward): CTGTACTTCCAGGGCGAGACTCCGGAACCCCGGACAACCCGC; I-PIPE (reverse): AATTAAGTCGCGTTATTCATAAACTGCCTTATACCAGCCGAC; V-PIPE (forward): TAACGCGACTTAATTAACTCGTTTAAACGGTCTCCAGC; and V-PIPE (reverse): GCCCTGGAAGTACAGGTTTTCGTGATGATGATGATGAT. METHODS title_4 6526 Protein Purification for Crystallization METHODS paragraph 6567 Cells were resuspended, homogenized, and lysed by sonication in 40 mm Tris (pH 8.0), 300 mm NaCl, 10 mm imidazole, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Lysis Buffer 1) containing 0.4 mm MgSO4 and 1 μl of 250 unit/μl−1 of benzonase (Sigma). The cell lysate was then clarified by centrifugation (32,500 × g for 25 min at 4 °C) before being passed over Ni2+-chelating resin equilibrated in Lysis Buffer 1 and washed in the same buffer supplemented with 40 mm imidazole and 10% (v/v) glycerol. The protein was subsequently eluted in 20 mm Tris (pH 8.0), 150 mm NaCl, 10% (v/v) glycerol, 1 mm TCEP, and 300 mm imidazole, and the fractions containing the protein were pooled. METHODS paragraph 7267 To remove the His tag, PmC11 was exchanged into 20 mm Tris (pH 8.0), 150 mm NaCl, 30 mm imidazole, and 1 mm TCEP using a PD-10 column (GE Healthcare), followed by incubation with 1 mg of His-tagged tobacco etch virus protease per 15 mg of protein for 2 h at room temperature and subsequent overnight incubation at 4 °C. The sample was centrifuged to remove any precipitated material (13,000 × g for 10 min at 4 °C) and the supernatant loaded onto Ni2+-chelating resin equilibrated with 20 mm Tris (pH 8.0), 150 mm NaCl, 30 mm imidazole, and 1 mm TCEP and washed with the same buffer. The flow-through and wash fractions were collected and concentrated to 13.3 mg ml−1 using Amicon Ultra-15 5K centrifugal concentrators (Millipore). METHODS title_4 8004 Crystallization and Data Collection METHODS paragraph 8040 PmC11 was crystallized using the nanodroplet vapor diffusion method using standard JCSG crystallization protocols. Drops were comprised of 200 nl of protein solution mixed with 200 nl of crystallization solution in 96-well sitting-drop plates, equilibrated against a 50-μl reservoir. Crystals of PmC11 were grown at 4 °C in mother liquor consisting of 0.2 m NH4H2PO4, 20% PEG-3350 (JCSG Core Suite I). Crystals were flash cooled in liquid nitrogen using 10% ethylene glycol as a cryoprotectant prior to data collection and initial screening for diffraction was carried out using the Stanford Automated Mounting system at the Stanford Synchrotron Radiation Lightsource (SSRL, Menlo Park, CA). Single wavelength anomalous dispersion data were collected using a wavelength of 0.9793 Å, at the Advanced Light Source (ALS, beamline 8.2.2, Berkeley, CA) on an ADSC Quantum 315 CCD detector. The data were indexed and integrated with XDS and scaled using XSCALE. The diffraction data were indexed in space group P21 with a = 39.11, b = 108.68, c = 77.97 Å, and β = 94.32°. The unit cell contained two molecules in the asymmetric unit resulting in a solvent content of 39% (Matthews' coefficient (Vm) of 2.4 Å3 Da−1). METHODS title_4 9262 Structure Determination METHODS paragraph 9286 The PmC11 structure was determined by the single wavelength anomalous dispersion method using an x-ray wavelength corresponding to the peak of the selenium K edge. Initial phases were derived using the autoSHARP interface, which included density modification with SOLOMON. Good quality electron density was obtained at 1.7-Å resolution, allowing an initial model to be obtained by automated model building with ARP/wARP. Model completion and refinement were iteratively performed with COOT and REFMAC to produce a final model with an Rcryst and Rfree of 14.3 and 17.5%, respectively. The refinement included experimental phase restraints in the form of Hendrickson-Lattman coefficients, TLS refinement with one TLS group per molecule in the asymmetric unit, and NCS restraints. The refined structure contains residues 24–375 and 28–375 for the two molecules in the crystallographic asymmetric unit. Structural validation was carried using the JCSG Quality Control Server that analyzes both the coordinates and data using a variety of structural validation tools to confirm the stereochemical quality of the model (ADIT, MOLPROBITY, and WHATIF 5.0) and agreement between model and data (SGCHECK and RESOLVE). All of the main-chain torsion angles were in the allowed regions of the Ramachandran plot and the MolProbity overall clash score for the structure was 2.09 (within the 99th percentile for its resolution). The atomic coordinates and structure factors for PmC11 have been deposited in the Protein Data Bank (PDB) with the accession code 3UWS. Data collection, model, and refinement statistics are reported in Table 1. T1.xml T1 TABLE table_caption 10916 Crystallographic statistics for PDB code 3UWS T1.xml T1 TABLE table_caption 10962 Values in parentheses are for the highest resolution shell. T1.xml T1 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups"><tbody valign="top"><tr><td align="left" rowspan="1" colspan="1"><bold>Data collection</bold></td><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">    Wavelength (Å)</td><td align="left" rowspan="1" colspan="1">0.9793</td></tr><tr><td align="left" rowspan="1" colspan="1">    Space group</td><td align="left" rowspan="1" colspan="1">P2₁</td></tr><tr><td align="left" rowspan="1" colspan="1">    Unit cell dimensions <italic>a</italic>, <italic>b</italic>, <italic>c</italic> (Å); β^°</td><td align="left" rowspan="1" colspan="1">39.11, 108.68, 77.97; β = 94.32°</td></tr><tr><td align="left" rowspan="1" colspan="1">    Resolution range (Å)</td><td align="left" rowspan="1" colspan="1">28.73–1.70 (1.79–1.70)</td></tr><tr><td align="left" rowspan="1" colspan="1">    Unique reflections</td><td align="left" rowspan="1" colspan="1">70,913</td></tr><tr><td align="left" rowspan="1" colspan="1">    <italic>R</italic>_merge<xref ref-type="table-fn" rid="TF1-1"><italic>^a</italic></xref> on <italic>I</italic> (%)</td><td align="left" rowspan="1" colspan="1">10.2 (49.0)</td></tr><tr><td align="left" rowspan="1" colspan="1">    <italic>R</italic>_meas<xref ref-type="table-fn" rid="TF1-2"><italic>^b</italic></xref> on <italic>I</italic> (%)</td><td align="left" rowspan="1" colspan="1">11.0 (52.7)</td></tr><tr><td align="left" rowspan="1" colspan="1">    <italic>R</italic>_pim<xref ref-type="table-fn" rid="TF1-3"><italic>^c</italic></xref> on <italic>I</italic> (%)</td><td align="left" rowspan="1" colspan="1">4.1 (19.2)</td></tr><tr><td align="left" rowspan="1" colspan="1">    <italic>I</italic>/σ<italic>_I</italic></td><td align="left" rowspan="1" colspan="1">15.6 (4.6)</td></tr><tr><td align="left" rowspan="1" colspan="1">    Wilson B (Ų)</td><td align="left" rowspan="1" colspan="1">15.9</td></tr><tr><td align="left" rowspan="1" colspan="1">    Completeness (%)</td><td align="left" rowspan="1" colspan="1">99.6 (99.8)</td></tr><tr><td align="left" rowspan="1" colspan="1">    Multiplicity</td><td align="left" rowspan="1" colspan="1">7.3 (7.5)</td></tr><tr><td colspan="2" rowspan="1"><hr/></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Model and refinement</bold></td><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">    Reflections (total/test)</td><td align="left" rowspan="1" colspan="1">70,883/3,577</td></tr><tr><td align="left" rowspan="1" colspan="1">    <italic>R</italic>_cryst/<italic>R</italic>_free<xref ref-type="table-fn" rid="TF1-4"><italic>^d</italic></xref> (%)</td><td align="left" rowspan="1" colspan="1">14.3/17.5</td></tr><tr><td align="left" rowspan="1" colspan="1">    No. protein residues/atoms</td><td align="left" rowspan="1" colspan="1">700/5612</td></tr><tr><td align="left" rowspan="1" colspan="1">    No. of water/EDO molecules</td><td align="left" rowspan="1" colspan="1">690/7</td></tr><tr><td align="left" rowspan="1" colspan="1">    ESU<xref ref-type="table-fn" rid="TF1-5"><italic>^e</italic></xref> based on <italic>R</italic>_free (Å)</td><td align="left" rowspan="1" colspan="1">0.095</td></tr><tr><td align="left" rowspan="1" colspan="1">    B-values (Ų)</td><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">        Average isotropic B (overall)</td><td align="left" rowspan="1" colspan="1">20.0</td></tr><tr><td align="left" rowspan="1" colspan="1">        Protein overall</td><td align="left" rowspan="1" colspan="1">18.8</td></tr><tr><td align="left" rowspan="1" colspan="1">        All main/side chains</td><td align="left" rowspan="1" colspan="1">16.7/20.8</td></tr><tr><td align="left" rowspan="1" colspan="1">        Solvent/EDO</td><td align="left" rowspan="1" colspan="1">29.4/35.6</td></tr><tr><td align="left" rowspan="1" colspan="1">    RMSD<xref ref-type="table-fn" rid="TF1-7"><italic>^g</italic></xref></td><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">        Bond lengths (Å)</td><td align="left" rowspan="1" colspan="1">0.01</td></tr><tr><td align="left" rowspan="1" colspan="1">        Bond angles (°)</td><td align="left" rowspan="1" colspan="1">1.6</td></tr><tr><td align="left" rowspan="1" colspan="1">    Ramachandran analysis (%)</td><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">        Favored regions</td><td align="left" rowspan="1" colspan="1">97.0</td></tr><tr><td align="left" rowspan="1" colspan="1">        Allowed regions</td><td align="left" rowspan="1" colspan="1">3.0</td></tr><tr><td align="left" rowspan="1" colspan="1">        Outliers</td><td align="left" rowspan="1" colspan="1">0.0</td></tr></tbody></table> 11022 Data collection     Wavelength (Å) 0.9793     Space group P21     Unit cell dimensions a, b, c (Å); β° 39.11, 108.68, 77.97; β = 94.32°     Resolution range (Å) 28.73–1.70 (1.79–1.70)     Unique reflections 70,913     Rmergea on I (%) 10.2 (49.0)     Rmeasb on I (%) 11.0 (52.7)     Rpimc on I (%) 4.1 (19.2)     I/σI 15.6 (4.6)     Wilson B (Å2) 15.9     Completeness (%) 99.6 (99.8)     Multiplicity 7.3 (7.5) Model and refinement     Reflections (total/test) 70,883/3,577     Rcryst/Rfreed (%) 14.3/17.5     No. protein residues/atoms 700/5612     No. of water/EDO molecules 690/7     ESUe based on Rfree (Å) 0.095     B-values (Å2)         Average isotropic B (overall) 20.0         Protein overall 18.8         All main/side chains 16.7/20.8         Solvent/EDO 29.4/35.6     RMSDg         Bond lengths (Å) 0.01         Bond angles (°) 1.6     Ramachandran analysis (%)         Favored regions 97.0         Allowed regions 3.0         Outliers 0.0 T1.xml T1 TABLE table_footnote 12367 a Rmerge = ΣhklΣi|Ii(hkl) − 〈I(hkl)〉|/Σhkl Σi(hkl). T1.xml T1 TABLE table_footnote 12435 b Rmeas = Σhkl[N/(N-1)]1/2Σi|Ii(hkl) − 〈I(hkl)〉|/ΣhklΣiIi(hkl). T1.xml T1 TABLE table_footnote 12515 c Rpim (precision-indicating Rmerge) = Σhkl[(1/(N-1)]1/2 Σi|Ii (hkl) − 〈I(hkl)〉|/ΣhklΣi Ii(hkl), where n is the multiplicity of reflection hkl, and Ii(hkl) and 〈I(hkl)〉 are the intensity of the ith measurement and the average intensity of reflection hkl, respectively. T1.xml T1 TABLE table_footnote 12800 d Rcryst and Rfree = Σ‖Fobs| − |Fcalc‖/Σ|Fobs| for reflections in the working and test sets, respectively, where Fobs and Fcalc are the observed and calculated structure-factor amplitudes, respectively. Rfree is the same as Rcryst but for 5% of the total reflections chosen at random and omitted from structural refinement. T1.xml T1 TABLE table_footnote 13134 e ESU is the estimated standard uncertainties of atoms. T1.xml T1 TABLE table_footnote 13190 f The average isotropic B includes TLS and residual B components. T1.xml T1 TABLE table_footnote 13256 g RMSD, root-mean-square deviation. METHODS title_4 13292 Structural Analysis METHODS paragraph 13312 The primary sequence alignment with assigned secondary structure was prepared using CLUSTAL OMEGA and ALINE. The topology diagram was produced with TOPDRAW and all three-dimensional structural figures were prepared with PyMol with the electrostatic surface potential calculated with APBS and contoured at ±5 kT/e. Architectural comparisons with known structures revealed that PmC11 was most structurally similar to caspase-7, gingipain-K, and legumain (PBD codes 4hq0, 4tkx, and 4aw9, respectively). The statistical significance of the structural alignment between PmC11 and both caspase-7 and gingipain-K is equivalent (Z-score of 9.2) with legumain giving a very similar result (Z-score of 9.1). Of note, the β-strand topology of the CDP domains of Clostridium difficile toxin B (family C80; TcdB; PDB code 3pee) is identical to that observed in the PmC11 β-sheet, but the Z-score from DaliLite was notably less at 7.6. It is possible that the PmC11 structure is more closely related to the C80 family than other families in clan CD, and appear to reside on the same branch of the phylogenetic tree based on structure. METHODS title_4 14440 Protein Production for Biochemical Assays METHODS paragraph 14482 The PmCD00547516 plasmid described above was obtained from the PSI:Biology Materials Repository and used to generate a cleavage site mutant PmC11K147A and an active-site mutant PmC11C179A using the QuikChange Site-directed Mutagenesis kit (Stratagene) as per the manufacturer's instructions using the following primers: K147A mutant (forward): CAGAATAAGCTGGCAGCGTTCGGACAGGACG, and K147A mutant (reverse): CGTCCTGTCCGAACGCTGCCAGCTTATTCTG; C179A mutant (forward): CCTGTTCGATGCCGCCTACATGGCAAGC, and C179A mutant (reverse): GCTTGCCATGTAGGCGGCATCGAACAGG. The expression plasmids containing PmC11 were transformed into E. coli BL21 Star (DE3) and grown in Luria-Bertani media containing 30 μg ml−1 of kanamycin at 37 °C until an optical density (600 nm) of ∼0.6 was reached. l-Arabinose was added to a final concentration of 0.2% (w/v) and the cells incubated overnight at 25 °C. METHODS paragraph 15363 Compared with the protein production for crystallography, a slightly modified purification protocol was employed for biochemical assays. Initially, the cells were resuspended in 20 mm sodium phosphate (pH 7.5), 150 mm NaCl (Lysis Buffer 2) containing an EDTA-free protease inhibitor mixture (cOmplete, Roche Applied Science). Cells were disrupted by three passages (15 KPSI) through a One-Shot cell disruptor (Constant Systems) followed by centrifugation at 20,000 × g for 20 min at 4 °C. The supernatant was collected and sterile-filtered (0.2 μm) before being applied to a 5-ml HisTrap HP column (GE Healthcare) equilibrated in Lysis Buffer 2 containing 25 mm imidazole, and the protein was eluted in the same buffer containing 250 mm imidazole. The peak fractions were pooled and buffer exchanged into the assay buffer (20 mm Tris, 150 mm NaCl, pH 8.0) using a PD-10 column. When required, purified PmC11 was concentrated using Vivaspin 2 30-K centrifugal concentrators (Sartorius). Protein concentration was routinely measured using Bradford's reagent (Bio-Rad) with a BSA standard. METHODS title_4 16453 Fluorogenic Substrate Activity Assays METHODS paragraph 16491 The release of the fluorescent group AMC (7-amino-4-methylcoumarin) from potential peptide substrates was used to assess the activity of PmC11. Peptidase activity was tested using 20 μg of PmC11 and 100 μm substrate (unless otherwise stated) in assay buffer to a final reaction volume of 200 μl and all samples were incubated (without substrate) at 37 °C for 16 h prior to carrying out the assay. The substrate and plate reader were brought to 37 °C for 20 min prior to the addition of the PmC11 and samples prepared without PmC11 were used as blanks (negative controls). The curves were plotted using the blank-corrected fluorescence units against the time of acquisition (in min). The assays were carried out in black 96-well flat-bottomed plates (Greiner). AMC fluorescence was measured using a PHERAstar FS plate reader (BMG Labtech) with excitation and emission wavelengths of 355 and 460 nm, respectively. METHODS paragraph 17408 To investigate the substrate specificity of PmC11, substrates Z-GGR-AMC, Bz-R-AMC, Z-GP-AMC, Z-HGP-AMC, Ac-DEVD-AMC (all Bachem), BOC-VLK-AMC, and BOC-K-AMC (both PeptaNova) were prepared at 100 mm in 100% dimethyl sulfoxide. The amount of AMC (micromoles) released was calculated by generating an AMC standard curve (as described in Ref.) and the specific activity of PmC11 was calculated as picomoles of AMC released per min per mg of the protein preparation. METHODS paragraph 17870 The reaction rates (Vmax) and Km values were determined for mutants PmC11K147A and PmC11C179A by carrying out the activity assay at varying concentrations of Bz-R-AMC between 0 and 600 μm. The blank-corrected relative fluorescence units were plotted against time (min) with ΔFU/T giving the reaction rate. The Km and Vmax of PmC11 and PmC11K147A against an R-AMC substrate were determined from the Lineweaver-Burk plot as described, calculated using GraphPad Prism6 software. All experiments were carried out in triplicate. METHODS title_4 18399 Effect of VRPR-FMK on PmC11 METHODS paragraph 18427 To test the effect of the inhibitor on the activity of PmC11, 25 μm Z-VRPR-FMK (100 mm stock in 100% dimethyl sulfoxide, Enzo Life Sciences), 20 μg of PmC11, 100 μm R-AMC substrate, 1 mm EGTA were prepared in the assay buffer and the activity assay carried out as described above. A gel-shift assay, to observe Z-VRPR-FMK binding to PmC11, was also set up using 20 μg of PmC11, 25 μm inhibitor, 1 mm EGTA in assay buffer. The reactions were incubated at 37 °C for 20 min before being stopped by the addition SDS-PAGE sample buffer. Samples were analyzed by loading the reaction mixture on a 10% NuPAGE BisTris gel using MES buffer. METHODS title_4 19065 Effect of Cations on PmC11 METHODS paragraph 19092 The enzyme activity of PmC11 was tested in the presence of various divalent cations: Mg2+, Ca2+, Mn2+, Co2+, Fe2+, Zn2+, and Cu2+. The final concentration of the salts (MgSO4, CaCl2, MnCl2, CoCl2, FeSO4, ZnCl2, and CuSO4) was 1 mm and the control was set up without divalent ions but with addition of 1 mm EGTA. The assay was set up using 20 mg of PmC11, 1 mm salts, 100 μm R-AMC substrate, and the assay buffer, and incubated at 37 °C for 16 h. The activity assay was carried out as described above. METHODS title_4 19595 Size Exclusion Chromatography METHODS paragraph 19625 Affinity-purified PmC11 was loaded onto a HiLoad 16/60 Superdex 200 gel filtration column (GE Healthcare) equilibrated in the assay buffer. The apparent molecular weight of PmC11 was determined from calibration curves based on protein standards of known molecular weights. METHODS title_4 19898 Autoprocessing Profile of PmC11 METHODS paragraph 19930 Autoprocessing of PmC11 was evaluated by incubating the enzyme at 37 °C and removing samples at 1-h intervals from 0 to 16 h and placing into SDS-PAGE loading buffer to stop the processing. Samples were then analyzed on a 4–12% NuPAGE (Thermo Fisher) Novex BisTris gel run in MES buffer. METHODS title_4 20221 Autoprocessing Cleavage Site Analysis METHODS paragraph 20259 To investigate whether processing is a result of intra- or inter-molecular cleavage, the PmC11C179A mutant was incubated with increasing concentrations of activated PmC11 (0, 0.1, 0.2, 0.5, 1, 2, and 5 μg). The final assay volume was 40 μl and the proteins were incubated at 37 °C for 16 h in the PmC11 assay buffer. To stop the reaction, NuPAGE sample buffer was added to the protein samples and 20 μl was analyzed on 10% NuPAGE Novex BisTris gel using MES buffer. These studies revealed no apparent cleavage of PmC11C179A by the active enzyme at low concentrations of PmC11 and that only limited cleavage was observed when the ratio of active enzyme (PmC11: PmC11C179A) was increased to ∼1:10 and 1:4. RESULTS title_1 20969 Results RESULTS title_4 20977 Structure of PmC11 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:54:14Z Structure protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:57Z PmC11 RESULTS paragraph 20996 The crystal structure of the catalytically active form of PmC11 revealed an extended caspase-like α/β/α sandwich architecture comprised of a central nine-stranded β-sheet, with an unusual C-terminal domain (CTD), starting at Lys250. A single cleavage was observed in the polypeptide chain at Lys147 (Fig. 1, A and B), where both ends of the cleavage site are fully visible and well ordered in the electron density. The central nine-stranded β-sheet (β1–β9) of PmC11 consists of six parallel and three anti-parallel β-strands with 4↑3↓2↑1↑5↑6↑7↓8↓9↑ topology (Fig. 1A) and the overall structure includes 14 α-helices with six (α1–α2 and α4–α7) closely surrounding the β-sheet in an approximately parallel orientation. Helices α1, α7, and α6 are located on one side of the β-sheet with α2, α4, and α5 on the opposite side (Fig. 1A). Helix α3 sits at the end of the loop following β5 (L5), just preceding the Lys147 cleavage site, with both L5 and α3 pointing away from the central β-sheet and toward the CTD, which starts with α8. The structure also includes two short β-hairpins (βA–βB and βD–βE) and a small β-sheet (βC–βF), which is formed from two distinct regions of the sequence (βC precedes α11, α12 and β9, whereas βF follows the βD-βE hairpin) in the middle of the CTD (Fig. 1B). evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:28:56Z crystal structure protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:37:40Z catalytically active protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:57Z PmC11 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T15:37:30Z extended caspase-like α/β/α sandwich structure_element SO: melaniev@ebi.ac.uk 2023-03-16T15:37:44Z nine-stranded β-sheet structure_element SO: melaniev@ebi.ac.uk 2023-03-16T13:33:06Z C-terminal domain structure_element SO: melaniev@ebi.ac.uk 2023-03-16T13:58:04Z CTD residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:57:54Z Lys250 ptm MESH: melaniev@ebi.ac.uk 2023-03-16T15:38:37Z single cleavage residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:33:27Z Lys147 site SO: melaniev@ebi.ac.uk 2023-03-16T13:57:19Z cleavage site evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:57:49Z electron density structure_element SO: melaniev@ebi.ac.uk 2023-03-16T15:37:50Z nine-stranded β-sheet structure_element SO: melaniev@ebi.ac.uk 2023-03-16T13:58:11Z β1–β9 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:57Z PmC11 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:00:05Z parallel structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:00:22Z anti-parallel β-strands evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:00:29Z structure structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:01:00Z α-helices structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:01:26Z α1–α2 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:01:28Z α4–α7 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:01:40Z β-sheet structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:01:42Z Helices structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:01:45Z α1 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:01:47Z α7 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:01:49Z α6 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:01:56Z β-sheet structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:01:58Z α2 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:02:00Z α4 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:13:55Z α5 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:02:30Z Helix structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:02:40Z α3 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:02:48Z loop structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:02:50Z β5 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:03:12Z L5 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:33:27Z Lys147 site SO: melaniev@ebi.ac.uk 2023-03-16T13:57:19Z cleavage site structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:03:12Z L5 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:03:18Z α3 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:03:26Z β-sheet structure_element SO: melaniev@ebi.ac.uk 2023-03-16T13:58:04Z CTD structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:14:05Z α8 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:03:32Z structure structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:04:11Z β-hairpins structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:04:13Z βA–βB structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:21:20Z βD–βE structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:04:18Z small β-sheet structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:04:20Z βC–βF structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:04:28Z βC structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:14:10Z α11 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:05:30Z α12 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:21:41Z β9 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:05:37Z βF structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:05:43Z βD-βE structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:05:46Z hairpin structure_element SO: melaniev@ebi.ac.uk 2023-03-16T13:58:04Z CTD zbc0191642560001.jpg F1 FIG fig_caption 22424 Crystal structure of a C11 peptidase from P. merdae. A, primary sequence alignment of PmC11 (Uniprot ID A7A9N3) and clostripain (Uniprot ID P09870) from C. histolyticum with identical residues highlighted in gray shading. The secondary structure of PmC11 from the crystal structure is mapped onto its sequence with the position of the PmC11 catalytic dyad, autocatalytic cleavage site (Lys147), and S1 binding pocket Asp (Asp177) highlighted by a red star, a red downturned triangle, and a red upturned triangle, respectively. Connecting loops are colored gray, the main β-sheet is in orange, with other strands in olive, α-helices are in blue, and the nonapeptide linker of clostripain that is excised upon autocleavage is underlined in red. Sequences around the catalytic site of clostripain and PmC11 align well. B, topology diagram of PmC11 colored as in A except that additional (non-core) β-strands are in yellow. Helices found on either side of the central β-sheet are shown above and below the sheet, respectively. The position of the catalytic dyad (H, C) and the processing site (Lys147) are highlighted. Helices (1–14) and β-strands (1–9 and A-F) are numbered from the N terminus. The core caspase-fold is highlighted in a box. C, tertiary structure of PmC11. The N and C termini (N and C) of PmC11 along with the central β-sheet (1–9), helix α5, and helices α8, α11, and α13 from the C-terminal domain, are all labeled. Loops are colored gray, the main β-sheet is in orange, with other β-strands in yellow, and α-helices are in blue. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:28:56Z Crystal structure protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:51:40Z C11 peptidase species MESH: melaniev@ebi.ac.uk 2023-03-16T14:10:05Z P. merdae experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:10:10Z primary sequence alignment protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:57Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:50:12Z clostripain species MESH: melaniev@ebi.ac.uk 2023-03-16T14:10:15Z C. histolyticum protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:57Z PmC11 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:28:56Z crystal structure protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:57Z PmC11 site SO: melaniev@ebi.ac.uk 2023-03-16T14:10:24Z catalytic dyad site SO: melaniev@ebi.ac.uk 2023-03-16T14:10:52Z autocatalytic cleavage site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:33:27Z Lys147 site SO: melaniev@ebi.ac.uk 2023-03-16T14:10:59Z S1 binding pocket residue_name SO: melaniev@ebi.ac.uk 2023-03-16T14:11:03Z Asp residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:11:08Z Asp177 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:11:15Z loops structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:11:17Z β-sheet structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:11:20Z α-helices structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:11:23Z nonapeptide linker protein PR: melaniev@ebi.ac.uk 2023-03-16T13:50:12Z clostripain ptm MESH: melaniev@ebi.ac.uk 2023-03-16T14:11:44Z autocleavage site SO: melaniev@ebi.ac.uk 2023-03-16T14:11:52Z catalytic site protein PR: melaniev@ebi.ac.uk 2023-03-16T13:50:12Z clostripain protein PR: melaniev@ebi.ac.uk 2023-03-16T14:12:20Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:57Z PmC11 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:12:32Z β-strands structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:12:34Z Helices structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:12:46Z β-sheet structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:12:48Z sheet site SO: melaniev@ebi.ac.uk 2023-03-16T14:10:24Z catalytic dyad residue_name SO: melaniev@ebi.ac.uk 2023-03-16T14:12:51Z H residue_name SO: melaniev@ebi.ac.uk 2023-03-16T14:12:53Z C site SO: melaniev@ebi.ac.uk 2023-03-16T14:13:13Z processing site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:33:27Z Lys147 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:13:24Z Helices structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:13:27Z β-strands structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:12:57Z core caspase-fold protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:57Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:13:47Z β-sheet structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:13:50Z helix structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:13:55Z α5 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:13:59Z helices structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:14:05Z α8 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:14:10Z α11 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:14:15Z α13 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T13:33:06Z C-terminal domain structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:14:19Z Loops structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:14:21Z β-sheet structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:14:24Z β-strands structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:14:26Z α-helices RESULTS paragraph 24015 The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. Of the interacting secondary structure elements, α5 is perhaps the most interesting. This helix makes a total of eight hydrogen bonds with the CTD, including one salt bridge (Arg191-Asp255) and is surrounded by the CTD on one side and the main core of the enzyme on the other, acting like a linchpin holding both components together (Fig. 1C). structure_element SO: melaniev@ebi.ac.uk 2023-03-16T13:58:04Z CTD protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:20:20Z tight helical bundle structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:20:51Z helices structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:21:03Z α8–α14 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:21:06Z strands structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:21:09Z βC structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:21:12Z βF structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:21:15Z β-hairpin structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:21:20Z βD–βE structure_element SO: melaniev@ebi.ac.uk 2023-03-16T13:58:04Z CTD structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:03:18Z α3 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:13:55Z α5 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:21:41Z β9 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:22:08Z loops structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:22:18Z β8 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:13:55Z α5 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:22:34Z This helix structure_element SO: melaniev@ebi.ac.uk 2023-03-16T13:58:04Z CTD residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:22:52Z Arg191 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:22:58Z Asp255 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T13:58:04Z CTD structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:23:07Z main core RESULTS title_4 24647 Structural Comparisons RESULTS paragraph 24670 PmC11 is, as expected, most structurally similar to other members of clan CD with the top hits in a search of known structures being caspase-7, gingipain-K, and legumain (PBD codes 4hq0, 4tkx, and 4aw9, respectively) (Table 2). The C-terminal domain is unique to PmC11 within clan CD and structure comparisons for this domain alone does not produce any hits in the PDB (DaliLite, PDBeFold), suggesting a completely novel fold. As the archetypal and arguably most well studied member of clan CD, the caspases were used as the basis to investigate the structure/function relationships in PmC11, with caspase-7 as the representative member. Six of the central β-strands in PmC11 (β1–β2 and β5–β8) share the same topology as the six-stranded β-sheet found in caspases, with strands β3, β4, and β9 located on the outside of this core structure (Fig. 1B, box). His133 and Cys179 were found at locations structurally homologous to the caspase catalytic dyad, and other clan CD structures, at the C termini of strands β5 and β6, respectively (Figs. 1, A and B, and 2A). A multiple sequence alignment of C11 proteins revealed that these residues are highly conserved (data not shown). protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:39:59Z clan CD evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:25:20Z structures protein PR: melaniev@ebi.ac.uk 2023-03-16T14:25:28Z caspase-7 protein PR: melaniev@ebi.ac.uk 2023-03-16T14:25:31Z gingipain-K protein PR: melaniev@ebi.ac.uk 2023-03-16T13:44:14Z legumain structure_element SO: melaniev@ebi.ac.uk 2023-03-16T13:33:06Z C-terminal domain protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:39:59Z clan CD experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:25:41Z structure comparisons structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:25:45Z this domain alone experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:26:12Z DaliLite experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:26:25Z PDBeFold protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:39:59Z clan CD protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:32:04Z caspases protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T14:26:53Z caspase-7 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:27:24Z β-strands protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:27:20Z β1–β2 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:27:31Z β5–β8 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:27:38Z six-stranded β-sheet protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:32:04Z caspases structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:27:43Z strands structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:27:46Z β3 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:27:49Z β4 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:21:41Z β9 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:28:03Z core structure residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:28:08Z His133 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:28:13Z Cys179 protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:36:41Z caspase site SO: melaniev@ebi.ac.uk 2023-03-16T14:10:24Z catalytic dyad protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:39:59Z clan CD evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:28:26Z structures structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:28:37Z strands structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:28:40Z β5 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:28:43Z β6 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:28:46Z multiple sequence alignment protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:32:18Z C11 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:48:11Z highly conserved T2.xml T2 TABLE table_caption 25879 Summary of PDBeFOLD superposition of structures found to be most similar to PmC11 in the PBD based on DaliLite experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:29:05Z PDBeFOLD superposition protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:29:14Z DaliLite T2.xml T2 TABLE table_caption 25991 The results are ordered in terms of structural homology (QH), where %SSEPC-X is the percentage of the SSEs in the PmC11 that can be identified in the target X (where X = caspase-7, legumain, gingipain, and TcdB-CPD; % SSEX-PC is the percentage of SSEs in X (as above) that can be identified in PmC11 (as above); % sequence ID is the percentage sequence identity after structural alignment; Nalign is the number of matched residues; and r.m.s. deviation the root mean squared deviation on the Cα positions of the matched residues. T2.xml T2 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table xmlns:xlink="http://www.w3.org/1999/xlink" frame="hsides" rules="groups"><thead valign="bottom"><tr><th align="center" rowspan="1" colspan="1">Enzyme</th><th align="center" rowspan="1" colspan="1">Family</th><th align="center" rowspan="1" colspan="1">PDB code</th><th align="center" rowspan="1" colspan="1">Q^H</th><th align="center" rowspan="1" colspan="1">Z-score</th><th align="center" rowspan="1" colspan="1">%SSE^PC-X</th><th align="center" rowspan="1" colspan="1">%SSE^X-PC</th><th align="center" rowspan="1" colspan="1">% Seq. ID</th><th align="center" rowspan="1" colspan="1"><italic>N</italic>_align</th><th align="center" rowspan="1" colspan="1">RMSD (Å)</th><th align="center" rowspan="1" colspan="1"><italic>N</italic>_Strands</th></tr></thead><tbody valign="top"><tr><td align="left" rowspan="1" colspan="1">PmC11</td><td align="left" rowspan="1" colspan="1">C11</td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="3UWS">3UWS</ext-link></td><td align="left" rowspan="1" colspan="1">1.00</td><td align="left" rowspan="1" colspan="1">33.4</td><td align="left" rowspan="1" colspan="1">100</td><td align="left" rowspan="1" colspan="1">100</td><td align="left" rowspan="1" colspan="1">100</td><td align="left" rowspan="1" colspan="1">352</td><td align="left" rowspan="1" colspan="1">0.00</td><td align="left" rowspan="1" colspan="1">9</td></tr><tr><td align="left" rowspan="1" colspan="1">Caspase-7</td><td align="left" rowspan="1" colspan="1">C14A</td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="4HQ0">4HQ0</ext-link></td><td align="left" rowspan="1" colspan="1">0.16</td><td align="left" rowspan="1" colspan="1">4.3</td><td align="left" rowspan="1" colspan="1">38</td><td align="left" rowspan="1" colspan="1">79</td><td align="left" rowspan="1" colspan="1">14</td><td align="left" rowspan="1" colspan="1">162</td><td align="left" rowspan="1" colspan="1">3.27</td><td align="left" rowspan="1" colspan="1">6</td></tr><tr><td align="left" rowspan="1" colspan="1">Legumain</td><td align="left" rowspan="1" colspan="1">C13</td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="4AW9">4AW9</ext-link></td><td align="left" rowspan="1" colspan="1">0.13</td><td align="left" rowspan="1" colspan="1">5.5</td><td align="left" rowspan="1" colspan="1">31</td><td align="left" rowspan="1" colspan="1">53</td><td align="left" rowspan="1" colspan="1">13</td><td align="left" rowspan="1" colspan="1">161</td><td align="left" rowspan="1" colspan="1">2.05</td><td align="left" rowspan="1" colspan="1">6</td></tr><tr><td align="left" rowspan="1" colspan="1">TcdB-CPD</td><td align="left" rowspan="1" colspan="1">C80</td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="3PEE">3PEE</ext-link></td><td align="left" rowspan="1" colspan="1">0.10</td><td align="left" rowspan="1" colspan="1">4.9</td><td align="left" rowspan="1" colspan="1">28</td><td align="left" rowspan="1" colspan="1">50</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">138</td><td align="left" rowspan="1" colspan="1">3.18</td><td align="left" rowspan="1" colspan="1">9</td></tr><tr><td align="left" rowspan="1" colspan="1">Gingipain</td><td align="left" rowspan="1" colspan="1">C25</td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="4TKX">4TKX</ext-link></td><td align="left" rowspan="1" colspan="1">0.07</td><td align="left" rowspan="1" colspan="1">5.4</td><td align="left" rowspan="1" colspan="1">28</td><td align="left" rowspan="1" colspan="1">27</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">153</td><td align="left" rowspan="1" colspan="1">2.97</td><td align="left" rowspan="1" colspan="1">10</td></tr></tbody></table> 26525 Enzyme Family PDB code QH Z-score %SSEPC-X %SSEX-PC % Seq. ID Nalign RMSD (Å) NStrands PmC11 C11 3UWS 1.00 33.4 100 100 100 352 0.00 9 Caspase-7 C14A 4HQ0 0.16 4.3 38 79 14 162 3.27 6 Legumain C13 4AW9 0.13 5.5 31 53 13 161 2.05 6 TcdB-CPD C80 3PEE 0.10 4.9 28 50 12 138 3.18 9 Gingipain C25 4TKX 0.07 5.4 28 27 12 153 2.97 10 zbc0191642560002.jpg F2 FIG fig_caption 26866 Biochemical and structural characterization of PmC11. A, ribbon representation of the overall structure of PmC11 illustrating the catalytic site, cleavage site displacement, and potential S1 binding site. The overall structure of PmC11 is shown in gray, looking down into the catalytic site with the catalytic dyad in red. The two ends of the autolytic cleavage site (Lys147 and Ala148, green) are displaced by 19.5 Å (thin black line) from one another and residues in the potential substrate binding pocket are highlighted in blue. B, size exclusion chromatography of PmC11. PmC11 migrates as a monomer with a molecular mass around 41 kDa calculated from protein standards of known molecular weights. Elution fractions across the major peak (1–6) were analyzed by SDS-PAGE on a 4–12% gel in MES buffer. C, the active form of PmC11 and two mutants, PmC11C179A (C) and PmC11K147A (K), were examined by SDS-PAGE (lane 1) and Western blot analysis using an anti-His antibody (lane 2) show that PmC11 autoprocesses, whereas mutants, PmC11C179A and PmC11K147A, do not show autoprocessing in vitro. D, cysteine peptidase activity of PmC11. Km and Vmax of PmC11 and K147A mutant were determined by monitoring change in the fluorescence corresponding to AMC release from Bz-R-AMC. Reactions were performed in triplicate and representative values ± S.D. are shown. E, intermolecular processing of PmC11C179A by PmC11. PmC11C179A (20 μg) was incubated overnight at 37 °C with increasing amounts of processed PmC11 and analyzed on a 10% SDS-PAGE gel. Inactive PmC11C179A was not processed to a major extent by active PmC11 until around a ratio of 1:4 (5 μg of active PmC11). A single lane of 20 μg of active PmC11 (labeled 20) is shown for comparison. F, activity of PmC11 against basic substrates. Specific activity is shown ± S.D. from three independent experiments. G, electrostatic surface potential of PmC11 shown in a similar orientation, where blue and red denote positively and negatively charged surface potential, respectively, contoured at ±5 kT/e. The position of the catalytic dyad, one potential key substrate binding residue Asp177, and the ends of the cleavage site Lys147 and Ala148 are indicated. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:32:47Z Biochemical and structural characterization protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 site SO: melaniev@ebi.ac.uk 2023-03-16T14:11:52Z catalytic site site SO: melaniev@ebi.ac.uk 2023-03-16T14:32:59Z S1 binding site evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:32:52Z structure protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 site SO: melaniev@ebi.ac.uk 2023-03-16T14:11:52Z catalytic site site SO: melaniev@ebi.ac.uk 2023-03-16T14:10:24Z catalytic dyad site SO: melaniev@ebi.ac.uk 2023-03-16T14:33:21Z autolytic cleavage site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:33:27Z Lys147 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:33:27Z Lys147 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:33:30Z Ala148 site SO: melaniev@ebi.ac.uk 2023-03-16T14:33:35Z substrate binding pocket experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:33:38Z size exclusion chromatography protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:33:43Z monomer experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:33:49Z SDS-PAGE protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:33:56Z active protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T14:34:03Z PmC11C179A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T14:34:09Z PmC11K147A experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:33:49Z SDS-PAGE experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:34:17Z Western blot protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 ptm MESH: melaniev@ebi.ac.uk 2023-03-16T14:34:33Z autoprocesses mutant MESH: melaniev@ebi.ac.uk 2023-03-16T14:34:03Z PmC11C179A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T14:34:09Z PmC11K147A ptm MESH: melaniev@ebi.ac.uk 2023-03-16T14:34:45Z autoprocessing protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:34:54Z Km evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:34:59Z Vmax protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T14:35:04Z K147A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T14:54:37Z Bz-R-AMC ptm MESH: melaniev@ebi.ac.uk 2023-03-16T14:35:16Z intermolecular processing mutant MESH: melaniev@ebi.ac.uk 2023-03-16T14:34:03Z PmC11C179A protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T14:34:03Z PmC11C179A protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:33:49Z SDS-PAGE protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:35:32Z Inactive mutant MESH: melaniev@ebi.ac.uk 2023-03-16T14:34:03Z PmC11C179A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:33:56Z active protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:33:57Z active protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:33:57Z active protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:35:38Z activity protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 site SO: melaniev@ebi.ac.uk 2023-03-16T14:10:24Z catalytic dyad site SO: melaniev@ebi.ac.uk 2023-03-16T14:35:48Z key substrate binding residue residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:11:08Z Asp177 site SO: melaniev@ebi.ac.uk 2023-03-16T13:57:19Z cleavage site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:33:27Z Lys147 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:33:30Z Ala148 RESULTS paragraph 29083 Five of the α-helices surrounding the β-sheet of PmC11 (α1, α2, α4, α6, and α7) are found in similar positions to the five structurally conserved helices in caspases and other members of clan CD, apart from family C80. Other than its more extended β-sheet, PmC11 differs most significantly from other clan CD members at its C terminus, where the CTD contains a further seven α-helices and four β-strands after β8. structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:38:43Z α-helices structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:38:52Z β-sheet protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:39:00Z α1 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:39:03Z α2 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:39:07Z α4 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:39:09Z α6 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:39:13Z α7 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:38:39Z structurally conserved structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:39:17Z helices protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:32:04Z caspases protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:39:59Z clan CD protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T14:39:22Z C80 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:39:25Z extended β-sheet protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:39:59Z clan CD structure_element SO: melaniev@ebi.ac.uk 2023-03-16T13:58:04Z CTD structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:40:08Z α-helices structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:40:43Z β-strands structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:22:18Z β8 RESULTS title_4 29537 Autoprocessing of PmC11 ptm MESH: melaniev@ebi.ac.uk 2023-03-16T14:41:11Z Autoprocessing protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 RESULTS paragraph 29561 Purification of recombinant PmC11 (molecular mass = 42.6 kDa) revealed partial processing into two cleavage products of 26.4 and 16.2 kDa, related to the observed cleavage at Lys147 in the crystal structure (Fig. 2A). Incubation of PmC11 at 37 °C for 16 h, resulted in a fully processed enzyme that remained as an intact monomer when applied to a size-exclusion column (Fig. 2B). The single cleavage site of PmC11 at Lys147 is found immediately after α3, in loop L5 within the central β-sheet (Figs. 1, A and B, and 2A). The two ends of the cleavage site are remarkably well ordered in the crystal structure and displaced from one another by 19.5 Å (Fig. 2A). Moreover, the C-terminal side of the cleavage site resides near the catalytic dyad with Ala148 being 4.5 and 5.7 Å from His133 and Cys179, respectively. Consequently, it appears feasible that the helix attached to Lys147 (α3) could be responsible for steric autoinhibition of PmC11 when Lys147 is covalently bonded to Ala148. Thus, the cleavage would be required for full activation of PmC11. To investigate this possibility, two mutant forms of the enzyme were created: PmC11C179A (a catalytically inactive mutant) and PmC11K147A (a cleavage-site mutant). Initial SDS-PAGE and Western blot analysis of both mutants revealed no discernible processing occurred as compared with active PmC11 (Fig. 2C). The PmC11K147A mutant enzyme had a markedly different reaction rate (Vmax) compared with WT, where the reaction velocity of PmC11 was 10 times greater than that of PmC11K147A (Fig. 2D). Taken together, these data reveal that PmC11 requires processing at Lys147 for optimum activity. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:44:17Z Purification protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 ptm MESH: melaniev@ebi.ac.uk 2023-03-16T13:34:11Z cleavage residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:33:28Z Lys147 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:28:56Z crystal structure experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:44:25Z Incubation protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:44:29Z fully processed protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:44:38Z intact oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:33:43Z monomer site SO: melaniev@ebi.ac.uk 2023-03-16T13:57:19Z cleavage site protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:33:28Z Lys147 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:03:18Z α3 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T15:37:56Z loop structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:03:12Z L5 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T15:38:00Z β-sheet site SO: melaniev@ebi.ac.uk 2023-03-16T13:57:19Z cleavage site evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:28:56Z crystal structure site SO: melaniev@ebi.ac.uk 2023-03-16T13:57:19Z cleavage site site SO: melaniev@ebi.ac.uk 2023-03-16T14:10:24Z catalytic dyad residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:33:30Z Ala148 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:28:08Z His133 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:28:13Z Cys179 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:45:34Z helix residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:33:28Z Lys147 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:03:18Z α3 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:33:28Z Lys147 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:33:30Z Ala148 ptm MESH: melaniev@ebi.ac.uk 2023-03-16T13:34:11Z cleavage protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:45:43Z full activation protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T14:34:03Z PmC11C179A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:46:11Z catalytically inactive mutant mutant MESH: melaniev@ebi.ac.uk 2023-03-16T14:34:09Z PmC11K147A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:46:17Z cleavage-site mutant experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:33:49Z SDS-PAGE experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:34:17Z Western blot protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:33:57Z active protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T14:34:09Z PmC11K147A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:46:21Z mutant evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:46:33Z reaction rate evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:34:59Z Vmax protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:46:27Z WT evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:46:31Z reaction velocity protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T14:34:09Z PmC11K147A protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:33:28Z Lys147 RESULTS paragraph 31216 To investigate whether processing is a result of intra- or intermolecular cleavage, the PmC11C179A mutant was incubated with increasing concentrations of processed and activated PmC11. These studies revealed that there was no apparent cleavage of PmC11C179A by the active enzyme at low concentrations of PmC11 and that only limited cleavage was observed when the ratio of active enzyme (PmC11:PmC11C179A) was increased to ∼1:10 and 1:4, with complete cleavage observed at a ratio of 1:1 (Fig. 2E). This suggests that cleavage of PmC11C179A was most likely an effect of the increasing concentration of PmC11 and intermolecular cleavage. Collectively, these data suggest that the pro-form of PmC11 is autoinhibited by a section of L5 blocking access to the active site, prior to intramolecular cleavage at Lys147. This cleavage subsequently allows movement of the region containing Lys147 and the active site to open up for substrate access. mutant MESH: melaniev@ebi.ac.uk 2023-03-16T14:34:03Z PmC11C179A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:49:25Z mutant experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:49:28Z incubated with increasing concentrations protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:49:31Z processed protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:49:35Z activated protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T14:34:03Z PmC11C179A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:33:57Z active experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:49:46Z at low concentrations protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:33:57Z active protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T14:34:03Z PmC11C179A experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:49:53Z increased to ∼1:10 and 1:4 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:49:56Z ratio of 1:1 ptm MESH: melaniev@ebi.ac.uk 2023-03-16T13:34:11Z cleavage mutant MESH: melaniev@ebi.ac.uk 2023-03-16T14:34:03Z PmC11C179A protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:50:12Z pro-form protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:50:16Z autoinhibited structure_element SO: melaniev@ebi.ac.uk 2023-03-16T14:03:12Z L5 site SO: melaniev@ebi.ac.uk 2023-03-16T13:34:26Z active site ptm MESH: melaniev@ebi.ac.uk 2023-03-16T14:50:36Z intramolecular cleavage residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:33:28Z Lys147 ptm MESH: melaniev@ebi.ac.uk 2023-06-15T08:23:33Z cleavage residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:33:28Z Lys147 site SO: melaniev@ebi.ac.uk 2023-03-16T13:34:26Z active site protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:51:01Z open RESULTS title_4 32158 Substrate Specificity of PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 RESULTS paragraph 32189 The autocatalytic cleavage of PmC11 at Lys147 (sequence KLK∧A) demonstrates that the enzyme accepts substrates with Lys in the P1 position. The substrate specificity of the enzyme was further tested using a variety of fluorogenic substrates. As expected, PmC11 showed no activity against substrates with Pro or Asp in P1 but was active toward substrates with a basic residue in P1 such as Bz-R-AMC, Z-GGR-AMC, and BOC-VLK-AMC. The rate of cleavage was ∼3-fold greater toward the single Arg substrate Bz-R-AMC than for the other two (Fig. 2F) and, unexpectedly, PmC11 showed no activity toward BOC-K-AMC. These results confirm that PmC11 accepts substrates containing Arg or Lys in P1 with a possible preference for Arg. ptm MESH: melaniev@ebi.ac.uk 2023-03-16T14:54:06Z autocatalytic cleavage protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:33:28Z Lys147 residue_name SO: melaniev@ebi.ac.uk 2023-03-16T14:55:14Z Lys residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:48:22Z P1 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 residue_name SO: melaniev@ebi.ac.uk 2023-03-16T14:54:23Z Pro residue_name SO: melaniev@ebi.ac.uk 2023-03-16T14:54:26Z Asp residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:48:22Z P1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:33:57Z active residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:48:22Z P1 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T14:54:37Z Bz-R-AMC chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T14:54:42Z Z-GGR-AMC chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T14:54:48Z BOC-VLK-AMC residue_name SO: melaniev@ebi.ac.uk 2023-03-16T14:54:55Z Arg chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T14:54:37Z Bz-R-AMC protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T14:55:07Z BOC-K-AMC protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 residue_name SO: melaniev@ebi.ac.uk 2023-03-16T14:54:55Z Arg residue_name SO: melaniev@ebi.ac.uk 2023-03-16T14:55:14Z Lys residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:48:22Z P1 residue_name SO: melaniev@ebi.ac.uk 2023-03-16T14:54:55Z Arg RESULTS paragraph 32913 The catalytic dyad of PmC11 sits near the bottom of an open pocket on the surface of the enzyme at a conserved location in the clan CD family. The PmC11 structure reveals that the catalytic dyad forms part of a large acidic pocket (Fig. 2G), consistent with a binding site for a basic substrate. This pocket is lined with the potential functional side chains of Asn50, Asp177, and Thr204 with Gly134, Asp207, and Met205 also contributing to the pocket (Fig. 2A). Interestingly, these residues are in regions that are structurally similar to those involved in the S1 binding pockets of other clan CD members (shown in Ref.). site SO: melaniev@ebi.ac.uk 2023-03-16T14:10:24Z catalytic dyad protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:56:36Z open site SO: melaniev@ebi.ac.uk 2023-03-16T14:56:39Z pocket protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:57:02Z conserved location protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T14:57:14Z CD family protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:57:19Z structure site SO: melaniev@ebi.ac.uk 2023-03-16T14:10:24Z catalytic dyad site SO: melaniev@ebi.ac.uk 2023-03-16T14:57:24Z acidic pocket site SO: melaniev@ebi.ac.uk 2023-03-16T14:57:27Z binding site site SO: melaniev@ebi.ac.uk 2023-03-16T14:57:30Z pocket residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:57:35Z Asn50 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:11:08Z Asp177 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:57:45Z Thr204 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:57:50Z Gly134 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:57:55Z Asp207 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:58:02Z Met205 site SO: melaniev@ebi.ac.uk 2023-03-16T14:58:05Z pocket protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:58:11Z structurally similar site SO: melaniev@ebi.ac.uk 2023-03-16T14:58:14Z S1 binding pockets protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T14:58:18Z clan CD members RESULTS paragraph 33537 Because PmC11 recognizes basic substrates, the tetrapeptide inhibitor Z-VRPR-FMK was tested as an enzyme inhibitor and was found to inhibit both the autoprocessing and activity of PmC11 (Fig. 3A). Z-VRPR-FMK was also shown to bind to the enzyme: a size-shift was observed, by SDS-PAGE analysis, in the larger processed product of PmC11 suggesting that the inhibitor bound to the active site (Fig. 3B). A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). Asp177 is located near the catalytic cysteine and is conserved throughout the C11 family, suggesting it is the primary S1 binding site residue. In the structure of PmC11, Asp207 resides on a flexible loop pointing away from the S1 binding pocket (Fig. 3C). However, this loop has been shown to be important for substrate binding in clan CD and this residue could easily rotate and be involved in substrate binding in PmC11. Thus, Asn50, Asp177, and Asp207 are most likely responsible for the substrate specificity of PmC11. Asp177 is highly conserved throughout the clan CD C11 peptidases and is thought to be primarily responsible for substrate specificity of the clan CD enzymes, as also illustrated from the proximity of these residues relative to the inhibitor Z-VRPR-FMK when PmC11 is overlaid on the MALT1-P structure (Fig. 3C). protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:02:52Z Z-VRPR-FMK protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:02:33Z inhibit ptm MESH: melaniev@ebi.ac.uk 2023-03-16T15:02:44Z autoprocessing protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:02:52Z Z-VRPR-FMK evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:03:03Z size-shift experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:33:49Z SDS-PAGE protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:03:26Z inhibitor bound site SO: melaniev@ebi.ac.uk 2023-03-16T13:34:26Z active site experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T15:03:33Z structure overlay protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T15:03:45Z MALT1-paracacaspase protein PR: melaniev@ebi.ac.uk 2023-03-16T15:04:29Z MALT1-P protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:04:01Z complex chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:02:52Z Z-VRPR-FMK protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 site SO: melaniev@ebi.ac.uk 2023-03-16T15:38:12Z dyad protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:33:57Z active protein PR: melaniev@ebi.ac.uk 2023-03-16T15:04:29Z MALT1-P residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:57:35Z Asn50 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:11:08Z Asp177 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:57:55Z Asp207 protein PR: melaniev@ebi.ac.uk 2023-03-16T15:04:29Z MALT1-P site SO: melaniev@ebi.ac.uk 2023-03-16T15:05:32Z inhibitor binding residues residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:05:39Z Asp365 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:05:44Z Asp462 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:05:49Z Glu500 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:05:57Z VRPR-FMK protein PR: melaniev@ebi.ac.uk 2023-03-16T15:04:29Z MALT1-P protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T15:06:02Z structural overlay residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:11:08Z Asp177 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:06:05Z catalytic residue_name SO: melaniev@ebi.ac.uk 2023-03-16T15:06:08Z cysteine protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:06:11Z conserved throughout protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:06:13Z C11 family site SO: melaniev@ebi.ac.uk 2023-03-16T15:06:20Z S1 binding site residue evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:06:23Z structure protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:57:55Z Asp207 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T15:38:04Z loop site SO: melaniev@ebi.ac.uk 2023-03-16T14:10:59Z S1 binding pocket structure_element SO: melaniev@ebi.ac.uk 2023-03-16T15:38:07Z loop protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:39:59Z clan CD protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:57:35Z Asn50 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:11:08Z Asp177 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:57:55Z Asp207 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:11:08Z Asp177 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:48:11Z highly conserved protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:06:36Z clan CD C11 peptidases protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:06:45Z clan CD enzymes chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:02:52Z Z-VRPR-FMK protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T15:06:50Z overlaid protein PR: melaniev@ebi.ac.uk 2023-03-16T15:04:29Z MALT1-P evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:06:52Z structure zbc0191642560003.jpg F3 FIG fig_caption 35240 PmC11 binds and is inhibited by Z-VRPR-FMK and does not require Ca2+ for activity. A, PmC11 activity is inhibited by Z-VRPR-FMK. Cleavage of Bz-R-AMC by PmC11 was measured in a fluorometric activity assay with (+, purple) and without (−, red) Z-VRPR-FMK. The relative fluorescence units of AMC released are plotted against time (min) (n = 3; ±S.D.). B, gel-shift assay reveals that Z-VRPR-FMK binds to PmC11. PmC11 was incubated with (+) or without (−) Z-VRPR-FMK and the samples analyzed on a 10% SDS-PAGE gel. A size shift can be observed in the larger processed product of PmC11 (26.1 kDa). C, PmC11 with the Z-VRPR-FMK from the MALT1-paracacaspase (MALT1-P) superimposed. A three-dimensional structural overlay of Z-VRPR-FMK from the MALT1-P complex onto PmC11. The position and orientation of Z-VRPR-FMK was taken from superposition of the PmC11 and MALTI_P structures and indicates the presumed active site of PmC11. Residues surrounding the inhibitor are labeled and represent potentially important binding site residues, labeled in black and shown in an atomic representation. Carbon atoms are shown in gray, nitrogen in blue, and oxygen in red. C, divalent cations do not increase the activity of PmC11. The cleavage of Bz-R-AMC by PmC11 was measured in the presence of the cations Ca2+, Mn2+, Zn2+, Co2+, Cu2+, Mg2+, and Fe3+ with EGTA as a negative control, and relative fluorescence measured against time (min). The addition of cations produced no improvement in activity of PmC11 when compared in the presence of EGTA, suggesting that PmC11 does not require metal ions for proteolytic activity. Furthermore, Cu2+, Fe2+, and Zn2+ appear to inhibit PmC11. protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:02:52Z Z-VRPR-FMK chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:10:14Z Ca2+ chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:02:52Z Z-VRPR-FMK chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T14:54:37Z Bz-R-AMC protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T15:10:42Z fluorometric activity assay chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:02:52Z Z-VRPR-FMK experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T15:10:46Z gel-shift assay chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:02:52Z Z-VRPR-FMK protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T15:10:49Z incubated chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:02:52Z Z-VRPR-FMK experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T14:33:49Z SDS-PAGE evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:10:54Z size shift protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:02:52Z Z-VRPR-FMK protein PR: melaniev@ebi.ac.uk 2023-03-16T15:10:59Z MALT1-paracacaspase protein PR: melaniev@ebi.ac.uk 2023-03-16T15:04:29Z MALT1-P experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T15:11:02Z superimposed experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T15:11:05Z three-dimensional structural overlay chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:02:52Z Z-VRPR-FMK protein PR: melaniev@ebi.ac.uk 2023-03-16T15:04:29Z MALT1-P protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:02:52Z Z-VRPR-FMK experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T15:11:08Z superposition protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T15:37:07Z MALTI_P evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:11:11Z structures site SO: melaniev@ebi.ac.uk 2023-03-16T13:34:26Z active site protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 site SO: melaniev@ebi.ac.uk 2023-03-16T15:11:18Z binding site residues protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:58Z PmC11 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T14:54:37Z Bz-R-AMC protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:11:41Z Ca2+ chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:11:46Z Mn2+ chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:11:49Z Zn2+ chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:11:52Z Co2+ chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:11:56Z Cu2+ chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:11:59Z Mg2+ chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:12:01Z Fe3+ chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:12:04Z EGTA experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T15:12:11Z relative fluorescence measured against time experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T15:12:14Z addition of cations protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:12:17Z EGTA protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:12:23Z Cu2+ chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:12:25Z Fe2+ chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:12:28Z Zn2+ protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:13:11Z inhibit protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 RESULTS title_4 36912 Comparison with Clostripain protein PR: melaniev@ebi.ac.uk 2023-03-16T13:50:12Z Clostripain RESULTS paragraph 36940 Clostripain from C. histolyticum is the founding member of the C11 family of peptidases and contains an additional 149 residues compared with PmC11. A multiple sequence alignment revealed that most of the secondary structural elements are conserved between the two enzymes, although they are only ∼23% identical (Fig. 1A). Nevertheless, PmC11 may be a good model for the core structure of clostripain. protein PR: melaniev@ebi.ac.uk 2023-03-16T13:50:12Z Clostripain species MESH: melaniev@ebi.ac.uk 2023-03-16T14:10:15Z C. histolyticum protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:14:10Z C11 family protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:14:14Z peptidases residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:14:20Z 149 residues protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T15:14:23Z multiple sequence alignment structure_element SO: melaniev@ebi.ac.uk 2023-03-16T15:14:35Z secondary structural elements protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:14:38Z conserved protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:50:12Z clostripain RESULTS paragraph 37344 The primary structural alignment also shows that the catalytic dyad in PmC11 is structurally conserved in clostripain (Fig. 1A). Unlike PmC11, clostripain has two cleavage sites (Arg181 and Arg190), which results in the removal of a nonapeptide, and is required for full activation of the enzyme (highlighted in Fig. 1A). Interestingly, Arg190 was found to align with Lys147 in PmC11. In addition, the predicted primary S1-binding residue in PmC11 Asp177 also overlays with the residue predicted to be the P1 specificity determining residue in clostripain (Asp229, Fig. 1A). experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T15:16:18Z primary structural alignment site SO: melaniev@ebi.ac.uk 2023-03-16T14:10:24Z catalytic dyad protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:16:21Z structurally conserved protein PR: melaniev@ebi.ac.uk 2023-03-16T13:50:13Z clostripain protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:50:13Z clostripain site SO: melaniev@ebi.ac.uk 2023-03-16T15:16:37Z cleavage sites residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:17:03Z Arg181 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:17:09Z Arg190 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T15:17:16Z nonapeptide protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:17:19Z full activation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:17:09Z Arg190 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:33:28Z Lys147 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 site SO: melaniev@ebi.ac.uk 2023-03-16T15:17:37Z S1-binding residue protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T14:11:08Z Asp177 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T15:17:41Z overlays site SO: melaniev@ebi.ac.uk 2023-03-16T15:17:47Z P1 specificity determining residue protein PR: melaniev@ebi.ac.uk 2023-03-16T13:50:13Z clostripain residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:17:51Z Asp229 RESULTS paragraph 37919 As studies on clostripain revealed addition of Ca2+ ions are required for full activation, the Ca2+ dependence of PmC11 was examined. Surprisingly, Ca2+ did not enhance PmC11 activity and, furthermore, other divalent cations, Mg2+, Mn2+, Co2+, Fe2+, Zn2+, and Cu2+, were not necessary for PmC11 activity (Fig. 3D). In support of these findings, EGTA did not inhibit PmC11 suggesting that, unlike clostripain, PmC11 does not require Ca2+ or other divalent cations, for activity. protein PR: melaniev@ebi.ac.uk 2023-03-16T13:50:13Z clostripain chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:19:08Z Ca2+ protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:19:12Z full activation chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:19:15Z Ca2+ protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:19:18Z Ca2+ protein PR: melaniev@ebi.ac.uk 2023-03-22T09:56:52Z PmC11 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:19:22Z Mg2+ chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:19:25Z Mn2+ chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:19:28Z Co2+ chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:19:31Z Fe2+ chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:19:34Z Zn2+ chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:19:38Z Cu2+ protein PR: melaniev@ebi.ac.uk 2023-03-22T09:57:02Z PmC11 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:19:42Z EGTA protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:50:13Z clostripain protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:19:44Z Ca2+ DISCUSS title_1 38397 Discussion DISCUSS paragraph 38408 The crystal structure of PmC11 now provides three-dimensional information for a member of the clostripain C11 family of cysteine peptidases. The enzyme exhibits all of the key structural elements of clan CD members, but is unusual in that it has a nine-stranded central β-sheet with a novel C-terminal domain. The structural similarity of PmC11 with its nearest structural neighbors in the PDB is decidedly low, overlaying better with six-stranded caspase-7 than any of the other larger members of the clan (Table 2). The substrate specificity of PmC11 is Arg/Lys and the crystal structure revealed an acidic pocket for specific binding of such basic substrates. In addition, the structure suggested a mechanism of self-inhibition in both PmC11 and clostripain and an activation mechanism that requires autoprocessing. PmC11 differs from clostripain in that is does not appear to require divalent cations for activation. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:28:56Z crystal structure protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:50:13Z clostripain protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:26:34Z C11 family protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:39:31Z cysteine peptidases protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:26:43Z clan CD members structure_element SO: melaniev@ebi.ac.uk 2023-03-16T15:26:50Z β-sheet structure_element SO: melaniev@ebi.ac.uk 2023-03-16T13:33:06Z C-terminal domain protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T15:26:56Z caspase-7 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 residue_name SO: melaniev@ebi.ac.uk 2023-03-16T14:54:55Z Arg residue_name SO: melaniev@ebi.ac.uk 2023-03-16T14:55:14Z Lys evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T13:28:57Z crystal structure site SO: melaniev@ebi.ac.uk 2023-03-16T15:27:08Z acidic pocket evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:27:11Z structure protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:50:13Z clostripain ptm MESH: melaniev@ebi.ac.uk 2023-03-16T15:27:18Z autoprocessing protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:50:13Z clostripain DISCUSS paragraph 39332 Several other members of clan CD require processing for full activation including legumain, gingipain-R, MARTX-CPD, and the effector caspases, e.g. caspase-7. To date, the effector caspases are the only group of enzymes that require cleavage of a loop within the central β-sheet. This is also the case in PmC11, although the cleavage loop is structurally different to that found in the caspases and follows the catalytic His (Fig. 1A), as opposed to the Cys in the caspases. protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:39:59Z clan CD ptm MESH: melaniev@ebi.ac.uk 2023-03-16T15:38:44Z processing protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:28:29Z full activation protein PR: melaniev@ebi.ac.uk 2023-03-16T13:44:14Z legumain protein PR: melaniev@ebi.ac.uk 2023-03-16T15:37:13Z gingipain-R protein PR: melaniev@ebi.ac.uk 2023-03-16T15:28:33Z MARTX-CPD protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:28:36Z effector caspases protein PR: melaniev@ebi.ac.uk 2023-03-16T15:37:18Z caspase-7 protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:28:41Z effector caspases ptm MESH: melaniev@ebi.ac.uk 2023-03-16T13:34:11Z cleavage structure_element SO: melaniev@ebi.ac.uk 2023-03-16T15:28:45Z loop structure_element SO: melaniev@ebi.ac.uk 2023-03-16T15:28:49Z β-sheet protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 ptm MESH: melaniev@ebi.ac.uk 2023-03-16T15:29:29Z cleavage structure_element SO: melaniev@ebi.ac.uk 2023-03-16T15:29:39Z loop protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:32:04Z caspases protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:29:57Z catalytic residue_name SO: melaniev@ebi.ac.uk 2023-03-16T15:29:51Z His residue_name SO: melaniev@ebi.ac.uk 2023-03-16T15:29:47Z Cys protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:32:04Z caspases DISCUSS paragraph 39810 All other clan CD members requiring cleavage for full activation do so at sites external to their central sheets. The caspases and gingipain-R both undergo intermolecular (trans) cleavage and legumain and MARTX-CPD are reported to perform intramolecular (cis) cleavage. In addition, several members of clan CD exhibit self-inhibition, whereby regions of the enzyme block access to the active site. Like PmC11, these structures show preformed catalytic machinery and, for a substrate to gain access, movement and/or cleavage of the blocking region is required. protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:31:05Z clan CD members ptm MESH: melaniev@ebi.ac.uk 2023-03-16T13:34:11Z cleavage protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:31:08Z full activation site SO: melaniev@ebi.ac.uk 2023-03-16T15:38:18Z sites structure_element SO: melaniev@ebi.ac.uk 2023-03-16T15:31:27Z sheets protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:32:04Z caspases protein PR: melaniev@ebi.ac.uk 2023-03-16T15:37:21Z gingipain-R ptm MESH: melaniev@ebi.ac.uk 2023-03-16T15:32:28Z intermolecular (trans) cleavage protein PR: melaniev@ebi.ac.uk 2023-03-16T13:44:14Z legumain protein PR: melaniev@ebi.ac.uk 2023-03-16T15:37:25Z MARTX-CPD ptm MESH: melaniev@ebi.ac.uk 2023-03-16T15:32:39Z intramolecular (cis) cleavage protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T13:40:00Z clan CD structure_element SO: melaniev@ebi.ac.uk 2023-03-16T15:32:54Z regions site SO: melaniev@ebi.ac.uk 2023-03-16T13:34:26Z active site protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 ptm MESH: melaniev@ebi.ac.uk 2023-03-16T13:34:11Z cleavage structure_element SO: melaniev@ebi.ac.uk 2023-03-16T15:32:57Z blocking region DISCUSS paragraph 40370 The structure of PmC11 gives the first insight into this class of relatively unexplored family of proteins and should allow important catalytic and substrate binding residues to be identified in a variety of orthologues. Indeed, insights gained from an analysis of the PmC11 structure revealed the identity of the Trypanosoma brucei PNT1 protein as a C11 cysteine peptidase with an essential role in organelle replication. The PmC11 structure should provide a good basis for structural modeling and, given the importance of other clan CD enzymes, this work should also advance the exploration of these peptidases and potentially identify new biologically important substrates. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:33:43Z structure protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:33:45Z structure species MESH: melaniev@ebi.ac.uk 2023-03-16T15:33:36Z Trypanosoma brucei protein PR: melaniev@ebi.ac.uk 2023-03-16T15:33:40Z PNT1 protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:33:58Z C11 cysteine peptidase protein PR: melaniev@ebi.ac.uk 2023-03-16T13:32:59Z PmC11 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:33:47Z structure experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T15:34:20Z structural modeling protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:34:03Z clan CD enzymes protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:33:55Z peptidases AUTH_CONT title_1 41047 Author Contributions AUTH_CONT paragraph 41068 K. M., J. S. G., D. D., I. A. W., and J. C. M. designed the research; K. M., J. S. G., and D. D. performed the research; K. M., J. S. G., D. D., G. H. C., A. S., M. A. E., and J. C. M. analyzed the data; A. G., S. A. L., A. M. D., M. A. E., and I. A. W. supervised various components of the JCSG structural genomics pipeline; M. K. G., A. G., S. A. L., A. M. D., and M. A. E. contributed reagents, materials, and analysis tools; and K. M., J. S. G., G. H. C., M. A. E., I. A. W., and J. C. M. wrote the paper. AUTH_CONT footnote 41578 This work was supported by the Medical Research Council Grant MR/K019384, Wellcome Trust Grants 091790 and 104111, and National Institutes of Health Grant U54 GM094586 (JCSG). The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and National Institutes of Health (NIH), National Center for Research Resources, Biomedical Technology Program Grant P41RR001209, and the NIMGS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIGMS or NIH. The authors declare that they have no conflicts of interest with the contents of this article. AUTH_CONT footnote 42243 The atomic coordinates and structure factors (code 3UWS) have been deposited in the Protein Data Bank (http://wwpdb.org/). AUTH_CONT footnote 42366 JCSG AUTH_CONT footnote 42371 Joint Center for Structural Genomics AUTH_CONT footnote 42408 PIPE AUTH_CONT footnote 42413 polymerase incomplete primer extension AUTH_CONT footnote 42452 TCEP AUTH_CONT footnote 42457 Tris(2-carboxyethyl)phosphine AUTH_CONT footnote 42487 AMC AUTH_CONT footnote 42491 7-amino-4-methylcoumarin AUTH_CONT footnote 42516 PDB AUTH_CONT footnote 42520 Protein Data Bank AUTH_CONT footnote 42538 BisTris AUTH_CONT footnote 42546 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol AUTH_CONT footnote 42609 Z AUTH_CONT footnote 42611 benzyloxycarbonyl AUTH_CONT footnote 42629 FMK AUTH_CONT footnote 42633 fluoromethyl ketone AUTH_CONT footnote 42653 CTD AUTH_CONT footnote 42657 C-terminal domain AUTH_CONT footnote 42675 Bz-R-AMC AUTH_CONT footnote 42684 benzoyl-l-Arg-4-methylcoumaryl-7-amide AUTH_CONT footnote 42723 Z-GGR-AMC AUTH_CONT footnote 42733 benzyloxycarbonyl-Gly-Gly-Arg-AMC AUTH_CONT footnote 42767 BOC-VLK-AMC AUTH_CONT footnote 42779 t-butyloxycarbonyl-Val-Leu-Lys. 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