NIH Public Access Author Manuscript Structure. Author manuscript; available in PMC 2010 February 13.
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Published in final edited form as: Structure. 2009 February 13; 17(2): 303–313. doi:10.1016/j.str.2008.12.008.
Structural Basis of Murein Peptide Specificity of a γ-D-glutamyl-Ldiamino Acid Endopeptidase
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Qingping Xu1,2, Sebastian Sudek1,3, Daniel McMullan1,4, Mitchell D. Miller1,2, Bernhard Geierstanger4, David H. Jones4, S. Sri Krishna1,5,6, Glen Spraggon1,4, Badry Bursalay4, Polat Abdubek1,4, Claire Acosta1,4, Eileen Ambing1,4, Tamara Astakhova1,5, Herbert L. Axelrod1,2, Dennis Carlton1,3, Jonathan Caruthers1,2, Hsiu-Ju Chiu1,2, Thomas Clayton1,3, Marc C. Deller1,3, Lian Duan1,5, Ylva Elias1,3, Marc-Andre Elsliger1,3, Julie Feuerhelm1,4, Slawomir K. Grzechnik1,5, Joanna Hale1,4, Gye Won Han1,3, Justin Haugen1,4, Lukasz Jaroszewski1,5,6, Kevin K. Jin1,2, Heath E. Klock1,4, Mark W. Knuth1,4, Piotr Kozbial1,6, Abhinav Kumar1,2, David Marciano1,3, Andrew T. Morse1,5, Edward Nigoghossian1,4, Linda Okach1,4, Silvya Oommachen1,2, Jessica Paulsen1,4, Ron Reyes1,2, Christopher L. Rife1,2, Christina V. Trout1,3, Henry van den Bedem1,2, Dana Weekes1,6, Aprilfawn White1,4, Guenter Wolf1,2, Chloe Zubieta1,2, Keith O. Hodgson1,2, John Wooley1,5, Ashley M. Deacon1,2, Adam Godzik1,5,6, Scott A. Lesley1,3,4, and Ian A. Wilson1,3,* 1Joint Center for Structural Genomics, http://www.jcsg.org 2Stanford Synchrotron Radiation Lightsource (SSRL), Stanford University, Menlo Park, California 3The Scripps Research Institute, La Jolla, California 4Genomics Institute of the Novartis Research Foundation, San Diego, California 5Center for Research in Biological Systems, University of California, San Diego, La Jolla, California 6Burnham Institute for Medical Research, La Jolla, California
Abstract
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Crystal structures of two homologous peptidases from cyanobacteria Anabaena variabilis and Nostoc punctiforme at 1.05 Å and 1.60 Å resolution represent the first structures of a large class of cell-wall, cysteine peptidases that contain an N-terminal bacterial SH3-like domain (SH3b) and a C-terminal NlpC/P60 cysteine peptidase domain. The NlpC/P60 domain is a primitive, papain-like peptidase in the CA clan of cysteine peptidases with a Cys126/His176/His188 catalytic triad and a conserved catalytic core. We deduced from structure and sequence analysis, and then experimentally, that that these two proteins act as γ-D-glutamyl-L-diamino acid endopeptidases (EC 3.4.22.-). The active site is located near the interface between the SH3b and NlpC/P60 domains, where the SH3b domain may help define substrate specificity, instead of functioning as a targeting domain, so that only muropeptides with an N-terminal L-alanine can bind to the active site.
Correspondence to: Dr. Ian Wilson, JCSG, The Scripps Research Institute, BCC206, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail:
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Keywords
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Peptidoglycan hydrolase; SH3b; NlpC/P60 domain; CHAP domain; Cysteine protease; γ-Dglutamyl-diaminopimelate DL-endopeptidases
INTRODUCTION The peptidoglycan (murein) of bacteria consists of glycan chains that are crosslinked via short peptides (Martin, 1966; Schleifer and Kandler, 1972; Shockman and Barrett, 1983; Strominger and Ghuysen, 1967; Vollmer et al., 2008a). This strong and protective, yet highly dynamic structure, must reshape, reorganize, and disassemble during cell growth, cell division, and cell lysis. Many enzymes are involved in degradation of peptidoglycan. The D-Ala, D-Glu, and meso-diaminopimelic acid (DAP) amino acids, that are present in the stem peptides, are not usually present in natural proteins and may protect the peptidoglycan against most peptidases. As a result, cell wall-specific peptidases are required to catalyze the hydrolysis of peptide bonds in the crossbridges (Firczuk and Bochtler, 2007; Vollmer et al., 2008b). An amidase generally cleaves the linkage between MurNAc and the stem peptide, whereas an endopeptidase generally targets a specific peptide linkage (Figure 1A).
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Recently, a ubiquitous Cysteine, Histidine-dependent Amidohydrolases/Peptidase (CHAP) superfamily was discovered that is involved in cell wall hydrolysis (Anantharaman and Aravind, 2003; Bateman and Rawlings, 2003; Rigden et al., 2003). This superfamily primarily consists of proteins from two Pfam families (Bateman et al., 2004), the NlpC/P60 family (PF00877) and the CHAP family (PF05257). Despite low sequence similarity, these two families are both predicted to have papain-like folds (Anantharaman and Aravind, 2003). As a result, the nomenclature NlpC/P60 and CHAP domain are sometimes used interchangeably. Both domains consist of ~110-140 residues with a strictly conserved cysteine and histidine residues. Proteins that contain these domains are highly modular, and the multiple components are often fused to form a multifunctional protein. An NlpC/P60 or CHAP domain is frequently fused with an N-terminal signal peptide, a MurNAc amidase, one or multiple targeting domains, such as the LysM domain, the choline-binding domain (CBD), and the bacterial SH3b domain. The NlpC/P60 or CHAP domains are widespread in bacteria; members of this superfamily have also been detected in bacteriophages, viruses, archaea, and eukaryotes (Anantharaman and Aravind, 2003).
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Characterized NlpC/P60 members include Escherichia coli membrane-associated lipoprotein NlpC, Listeria monocytogenes secreted autolysin P60 (Kuhn and Goebel, 1989), Bacillus sphaericus dipeptidyl-peptidase VI (DPP VI), and Bacillus subtilis autolysins LytF, LytE, and CwlS (YojL) (Smith et al., 2000) (Figure 1B). LytF, LytE, and CwlS, each with a multiple tandem repeat of the LysM and NlpC/P60 domains, are localized at the cell-separation sites during vegetative growth (Fukushima et al., 2006; Margot et al., 1999; Yamamoto et al., 2003). All known members of the NlpC/P60 family are γ-glutamyl DL-endopeptidases. LytF breaks the linkage of γ-D-glutamyl-DAP in a murein peptide (Margot et al., 1999; Ohnishi et al., 1999), while CwlO of Bacillus subtilis (yvcE), a protein of unknown function, contains a C-terminal CHAP domain that was characterized as a cell-wall DL-endopeptidase (Yamaguchi et al., 2004). Another Bacillus subtilis enzyme, YwtD, specifically cleaves the γ-glutamyl bond between D-glutamic acid and L-glutamic acid of γ-polyglutamic acid (Suzuki and Tahara, 2003). DPP VI is involved in cell sporulation and hydrolyzes γ-D-Glu-DAP(Lys) linkages in peptides that have a free N-terminal L-alanine (Bourgogne et al., 1992). Many members of the CHAP family are MurNAc amidases that cleave the N-acetylmuramyl-L-alanine bond, but other linkage preferences are also observed. CHAP-containing N-acetylmuramyl-L-alanine amidases include Skl from Gram-positive Streptococcus mitis (Llull et al., 2006), Sle1 from Structure. Author manuscript; available in PMC 2010 February 13.
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Staphylococcus aureus (Kajimura et al., 2005), and the CA subunit of PlyC, a multimeric lysin from bacteriophage (Nelson et al., 2006). Streptococcus agalactiae bacteriophage B30 endolysin cleaves bonds (in crosslinks) between D-Ala and L-Ala (Donovan et al., 2006; Pritchard et al., 2004). Cysteine peptidases are widely distributed in all kingdoms of life and play important roles under normal and pathological conditions. Development of cysteine peptidase inhibitors is of considerable medical interest (Chapman et al., 1997; McGrath, 1999; Vicik et al., 2006). Steadily increasing interest has arisen in studying bacterial CHAP superfamily cysteine peptidases due to their potential as antibacterial drug targets. The unique ability of endolysins to cleave rapidly peptidoglycan in a species-specific manner, for example, renders them promising antibacterial agents (Borysowski et al., 2006; Nelson et al., 2001).
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Although cell-wall cysteine peptidases have been biochemically characterized for decades, little structural information is available. In order to explore and interrogate the structure and function of enzymes from these families, two cyanobacterial peptidoglycan cysteine endopeptidase (PCP) orthologs were selected from the NlpC/P60 family for structural studies: AvPCP (GenBank: YP_323898.1; gi|75909602) from Anabaena variabilis ATCC 29413 (molecular weight 25,313 Da, residues 1-234, and calculated isoelectric point 5.33) and NpPCP (GenBank: ZP_00105875.2; gi|53686717) from Nostoc punctiforme PCC 73102 (MW 25,921 Da, residues 1-234, calculated PI 4.71) (Figure 1B). Here, we report their crystal structures, which were determined using the semiautomated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG). AvPCP and NpPCP are novel cysteine peptidases with an Nterminal bacterial SH3-like (SH3b) domain and a C-terminal NlpC/P60 catalytic domain. The bacterial SH3b domains feature a common β-hairpin structural motif in their RT loops. The NlpC/P60 domain is a novel and primitive papain-like cysteine peptidase that retains the conserved active site, as well as the core structure, of the papain superfamily. AvPCP is characterized as a γ-D-glutamyl-L-diamino acid endopeptidase. We also show that AvPCP might use a unique mechanism for substrate recognition and specificity, which involves a possible novel role for the SH3b domain.
RESULTS Overall Description
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The crystal structure of AvPCP was determined in the orthorhombic space group P21212 at 1.05 Å resolution (PDB 2hbw) with one molecule per asymmetric unit (ASU) using the MAD method. Two crystal structures of NpPCP were also determined by the MAD method: a tetragonal P4122 crystal form at 1.60 Å resolution with one monomer per ASU (PDB 2evr) and a P41212 crystal form at 1.79 Å resolution with two monomers per ASU (PDB 2fg0). The monomers in 2fg0 and 2evr are essentially identical (Root-Mean-Square Deviation (RMSD) of 0.21~0.35 Å for 222 Cα atoms). Analytical size exclusion chromatography in combination with static light scattering indicated that AvPCP and NpPCP both exist as monomers in solution, consistent with the paucity of contacts in the monomer-monomer packing in the crystals of both proteins. All models have good geometry, and the Ramachandran plots produced by MolProbity (Davis et al., 2004) show that all residues are in allowed regions, with 99.3% (2hbw), 98.6% (2evr), and 97.7% (2fg0) in favored regions. The final model of AvPCP contains residues 14 to 234, an unknown ligand (UNL) that appears to mimic L-alanine and a second disordered moiety, and 345 water molecules. The final models of NpPCP contain residues 13 to 234, but no substrates or other ligands in the active site. Residues 1-13 (2hbw) and 1-12 (2evr, 2fg0) were omitted due to the lack of interpretable electron density. Data collection, model, and refinement statistics are summarized in Table I.
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NpPCP and AvPCP are of equal length and are highly homologous (80% sequence identity). The crystal structures, including the active sites, are highly conserved, suggesting they are functionally identical. Since the RMSD between apo NpPCP and the AvPCP complex is only ~0.6 Å for 220 Cα atoms, no significant structural changes appear to result from “substrate” binding. We will refer to the higher resolution AvPCP structure in all analyses and discussions, except where significant differences are observed between the two structures. AvPCP is composed of 13 β-strands (β1-β13) and six helices (H1-H6), including two 310helices (H1 and H5) (Figure 2A). The structure can be divided into two domains (14-81 and 82-234), with the C-terminal residues (230-234) extending towards the N-terminal domain. The N-terminal domain adopts an SH3-like fold (hereafter referred to as the SH3b domain), which is an all-β fold with seven strands that form a β-barrel. A 310 helix is located between β6 and β7. The C-terminal catalytic domain (i.e., the NlpC/P60 domain or AvPCP-C) can be classified as a α+β-fold with segregated α and β regions. Structural similarity searches using DALI (Holm and Sander, 1995) found no matches to the full-length structure of AvPCP.
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Homologous proteins to AvPCP, with the same two-domain architecture, are found in many cyanobacteria (Figure S1). Since the similarity is observed across the full length of the sequence, these proteins are likely to bind the same or similar substrates with a conserved catalytic mechanism. Hereafter, we will refer to sequence conservation within the context of these close homologs, unless otherwise stated. The N-terminal SH3b Domain
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The presence of SH3b domains in prokaryotes was predicted previously (Ponting et al., 1999; Whisstock and Lesk, 1999). SH3b domains, like the LysM (Bateman and Bycroft, 2000) and choline-binding (Fernandez-Tornero et al., 2001) domains, are commonly thought of as targeting domains, which are involved in cell wall recognition and binding. The SH3b domain of AvPCP is structurally similar to many eukaryotic SH3 domains, where 75% of the Cα atoms can be aligned with RMSDs between 2.3 and 2.5 Å (e.g., PDB 1jo8, 1bbz). Conventional eukaryotic SH3 domains consist of five β-strands, which are termed βA through βE (Mayer and Saksella, 2005). The SH3b domain of AvPCP contains seven β-strands, with the following correspondence to eukaryotic SH3 domains: β1→ βA, β4→βB, β5→βC, β6→βD, and β7→βE (Figure 3). One significant structural variation is that the long RT loop in AvPCP (residues 20-42) forms a two-stranded β-hairpin (β2 and β3), but follows a similar path to the shorter RT loops found in conventional SH3 domains. This motif (β2 and β3) packs against strand β6 in almost perpendicular orientation. The overall connection between β1 and β4 is around six residues longer than the corresponding RT loop of a eukaryotic SH3 domain, such as Abl SH3 (Pisabarro et al., 1998). The AvPCP equivalent of the N-Src loop between βB and βC (i.e., β4 and β5) contains only two residues (52-53). Another notable structural difference between the AvPCP SH3b domain and eukaryotic SH3 domains is that the RT loop and the N-Src loop fold towards each other, effectively closing the gap between them, in contrast with typical eukaryotic SH3 domains, which have a much wider opening (Figure 3). The structural similarities between the SH3b domain of AvPCP, the GW domains of Internalin B (Marino et al., 2002), and the SH3b domain of ALE-1 (Lu et al., 2006) are particularly interesting, since both the ALE-1 and the GW domains bind cell-wall components (Figure 3). Despite very low sequence identity (5%), 58 Cα positions can be aligned structurally with an RMSD of 2.6 Å between the AvPCP SH3b domain and that of ALE-1. Similarly, the AvPCP SH3b domain can be aligned to the GW domains (GW1, GW2, and GW3) of InlB, with an RMSD of 2.5 Å for 58 Cα atoms of GW3 551-628, for example. The InlB GW domains and the AvPCP SH3b domain have the same secondary structural elements. Unlike the AvPCP SH3b and InlB GW domains, the SH3b domain of ALE-1 lacks the characteristic 310-helix between the last two β-strands, where it is replaced instead with an extended loop. Surprisingly, Structure. Author manuscript; available in PMC 2010 February 13.
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all three domains possess a β-hairpin (β2 and β3 of AvPCP) in their RT-loop regions that is unique to prokaryotic SH3b domains and, interestingly, also essential in the choline-binding domain (CBD) of Streptococcus pneumoniae LytA (Fernandez-Tornero et al., 2001). Large differences are found between the N-Src loops and the distal loops in these domains, but the gap between the RT and N-Src loops is consistently closed for all of these SH3b domains, resulting in a significant loss of binding surface compared with eukaryotic SH3 domains (Marino et al., 2002). AvPCP Catalytic (NlpC/P60) Domain Is a Primitive Cysteine Peptidase
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The NlpC/P60 domain represents a new family of papain-like cysteine peptidases according to SCOP (Murzin et al., 1995). The NlpC/P60 domain adopts a much simpler topology, consisting of a six-stranded, central β-sheet and five α-helices, compared to the classical eightstranded, central β-sheet and seven α-helices of papain (Kamphuis et al., 1984). However, the core of the papain-like cysteine proteases is retained in the NlpC/P60 domain (Figure 4A) and consists of one α-helix (H3) and a five-stranded, antiparallel β-sheet (β8, β13, β9, β10, and β11). The RMSD between AvPCP and papain is 3.3 Å for 85 aligned Cα atoms (Table S1). The β-strands are positioned such that the first strand is followed immediately by the last strand and then by the three sequentially incremental strands (β-strand ordering is 16234 in the NlpC/ P60 domain of AvPCP, while the equivalent in papain is 28356). The more complex topology of the papain-like proteases can be derived from the NlpC/P60 domain via several significant insertions between the conserved secondary structural elements. In NlpC/P60, a single α-helix is inserted between the conserved α-helix, H3, and the first β-strand, β8. In comparison, three α-helices are present at the corresponding location in papain. This insertion also varies in other papain-like cysteine proteases. For example, the plant cysteine protease Ervatamin C (PDB 1o0e) contains two α-helices, while human Cathepsin S (PDB 2fg9) has four α-helices and two strands. Papain has one other insertion that is not present in the NlpC/P60 domain: an α-helix and a strand between the third (β9) and fourth (β10) β-strands of the core. Thus, the structure of the NlpC/P60 domain suggests that it corresponds to a minimal functional unit of the papainlike cysteine protease fold and may represent an early branch in the divergent evolution of cysteine proteases (Anantharaman and Aravind, 2003; Barrett and Rawlings, 2001). Comparisons of NlpC/P60 Domains and the CHAP Domains
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Since the release of our AvPCP/NpPCP coordinates in the PDB, a few new structures containing either a CHAP or a related domain have become available, that enable more extensive structural comparisons (Figure 4B, Table S1), including glutathionylsperidine synthetase (GspS) from E. coli (PDB 2ioa) (Pai et al., 2006), trypanothione synthetase-amidase (PDB 2vps) (Fyfe et al., 2008) and several uncharacterized structural genomics targets, Spr (PDB 2k1g), 2p1g (PDB 2p1g), 2im9 (PDB 2im9) and Syr11 (PDB 2k3a). AvPCP-C is also similar to phytochelatin synthase from Nostoc sp. (NsPCS) (Vivares et al., 2005). The catalytic core, the helix carrying the catalytic cysteine and the 5 central β-stands, are all conserved. Spr is essentially identical to AvPCP-C, but without the SH3b domain. 2p1g and 2im9, representing the DUF1460 family, are surprisingly similar to AvPCP-C with RMSD’s of 2.6 Å (111 aligned Cα ’s) and 2.8 Å (123 aligned Cα’s), respectively. This similarity and complete conservation of catalytic residues strongly suggest that these uncharacterized proteins are cysteine peptidases (Table S1). Beyond the catalytic core, these structures are highly diverged, through deletions and insertions of structural elements. The CHAP domain of GspS, consisting of mostly βstrands, is the most distant from AvPCP-C. Each active pocket displays its own unique features due to these structural variations (Figure 4C). For example, the S site of 2p1g is significantly larger than that of AvPCP-C, the active site groove of the GspS-CHAP domain is extended and wide, whereas NsPCS has a highly restrictive binding site. These observations suggest the NlpC/P60 and CHAP families should be considered as one superfamily with the same basic
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fold, but with distinct structural sub-features that account for their diverse substrate preferences (Anantharaman and Aravind, 2003).
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SH3b-NlpC/P60 Domain Interface The SH3b domain is located on the same side of the subdomain formed by the first three αhelices of the NlpC/P60 domain (H2-H4) and lies between the H2-H3 loop and the H3-H4 loop (Figure 2A). The SH3b domain interacts with the NlpC/P60 domain through its distal loop (i.e., β5-β6 loop, residues 61-65) and β3 (residues 34-39 and 42). The distal loop interacts with the H2-H3 loop (residues 115-120) and β3 interacts with the H3-H4 loop (residues 131, 140-147). The domain interface between SH3b and the catalytic domain is highly conserved. The interaction between the two domains buries 1510 Å2 of surface area with seven hydrogen bonds and three water-mediated interactions. Additional contacts between the SH3b domain and the NlpC/P60 domain are maintained through the linker (81-87) and C-terminal region (231-234).
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The conserved residues of the NlpC/P60 domain are clustered near the domain interface, with exception of Gly163/Asp164 (G/D), that are located near the N-terminus of β9, and Gly223/ Arg224 near the C-terminus of β13. Asp164 is buried and forms a salt bridge with Arg224. Interestingly, this G/D sequence conservation is observed in other NlpC/P60 domains (Figure 2C), even although these residues are located far from the active site. Most of the highly conserved residues in sequences homologous to the AvPCP SH3b domain are also located at or near the SH3b and NlpC/P60 domain interface, except for a few that are buried inside the SH3b domain (Leu44, Val46, Val57, Trp67, and Leu68). All conserved residues near the domain interface are important in forming the active site or maintaining domain interactions (Figure 5A-B). The conserved β2-β3 hairpin in SH3b contributes to the domain interface. AvPCP SH3b has conserved residues (Tyr26 and Pro29) that were thought to be unique to the GWB subtype of InlB GW domains (Marino et al., 2002). The corresponding region of the GW domain is also near the domain interface, and mutations at this site in ALE-1 (Arg 296) significantly affect its structure (Lu et al., 2006). Therefore, it seems likely that this conserved structural motif could be employed more generally to form protein-protein interfaces due to the structural conservation of these prokaryotic RT loops. The Active Site of AvPCP
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The active site of AvPCP is identified by its well-defined shape and strictly conserved residues (Figure 5C). A V-shaped long groove curves around the perimeter of Trp116. The bulk of the groove is located on the surface of the catalytic domain near the domain interface. The entrance to the groove is formed by Ala35, Tyr64, Gly117, Asp125, Arg143, and Asp144. Asp125 is neutralized by a salt bridge to the nearby Arg143 that also interacts with the strictly conserved Thr36 located on the SH3b domain. The middle section of the groove consists of Trp116, Ala145, Cys126, Ser127, and Thr175. Additional active-site residues (Tyr114, Trp116, Lys173, Thr175, His176, Ser190, Gly191, Lys192, Ala193, Gln194, and Tyr215) are located on the other side of Cys126 and between the β11-β12 and H2-H3 loops. Residues lining the bottom of the groove are the most conserved in the curved AvPCP active site that differs from papain, which contains a straight channel that runs along the interface between its two subdomains. AvPCP-C contains a variant of the catalytic triad found in other cysteine proteases (Cys126/ His176/His188), in which the third residue is a histidine instead of the more commonly observed asparagine or glutamine. This triad is located near the domain interface, but resides exclusively on the NlpC/P60 domain. Cys126 is located on the N-terminus of the helix H3.
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The spatial configuration of the catalytic triad is strictly conserved with papain (Figure 5D). The catalytic dyad of AvPCP (Cys126/His176) superimposes well with that of papain (Cys25/ His159, RMSD 0.32 Å for all side-chain atoms). The distance between His188Nδ1, which is hydrogen-bonded to His176Nε2 distance 2.68 Å), and its papain equivalent, Asn175Oδ1, is only 0.22 Å upon superimposition of the Cys/His dyads. His188 is further stabilized by a hydrogen-bond network with the strictly conserved Asp202 and Arg196. Another important residue in papain, Gln19, which helps form the “oxyanion hole” (McGrath, 1999), has a counterpart, Tyr114, in AvPCP. This tyrosine is also conserved in Spr (Tyr56), 2p1g (Tyr38), 2im9 (Tyr92). Thus, the mechanism of proteolysis for AvPCP is expected to be similar to that of papain or serine proteases (Cstorer et al., 1994; Otto and Schirmeister, 1997). The thiol group of Cys126 is polarized by the imidazole group of His176 and deprotonates to form a nucleophilic thiolate/imidazolium ion pair. The thiolate anion attacks the carbonyl carbon of the scissile bond of the substrate to form a tetrahedral intermediate. The transition state could be stabilized by the hydroxyl group of Tyr114. The reaction is completed by the hydrolysis of the acyl-thioester through an activated water.
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The Cys/His/His triads are also present in Spr and 2p1g (Table S1). However, the hydrogen bond network to the Nε2 of the third histidine differs. The third histidine in 2p1g is exposed to solvent and a water interacts with Nε2 (Figure 4C), whereas the corresponding histidine in Spr is buried as in AvPCP, with a serine forming a hydrogen bond with Nε2. The human cytomegalovirus serine protease (HCMV) contains a Ser/His/His triad, where the third histidine makes less contribution compared to the classic triad, due to a weaker hydrogen bond between the two histidines (Khayat et al., 2001). However, the side chains of the triads in AvPCP and HCMV do not superimpose very well (RMSD 1.6 Å for three Cα atoms). The His/ His hydrogen bond involves different atom pairs (Nδ2 and Nε2 of the second and third histidine in HCMV, Nε2-Nδ2 in AvPCP). This hydrogen bond in AvPCP appears stronger (shorter distance) compared to HCMV. Although the active sites of AvPCP and NpPCP are strictly conserved, no ligand density is observed in either crystal form of NpPCP. The main structural difference among the three structures concerns the side-chain rotamer of Trp116. Trp116 is critical for the formation and integrity of the binding pocket, as it likely contributes to multiple subsites by residing directly above Cys126, and is strictly conserved in AvPCP, lytE, lytF, and CwlS. Interestingly, the Trp116 indole adopts two conformations in the solved crystal structures (rotamer 1: χ1 = -67.4°; rotamer 2: χ1 = -177.3°). Only rotamer 2 (the main conformation in AvPCP) is active, since rotamer 1 changes the shape of the binding pocket and blocks access to Cys126 by the substrate. In conformation 1, the S pocket becomes more solvent-exposed. Thus, the flexibility and reorientation of Trp116 may play a role in product release.
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Functional Characterization of AvPCP Several characterized γ-D-Glu-DAP DL-endopeptidases, such as Bacillus subtilis LytE, LytF, and CwlO and Bacillus sphaericus DPP VI, contain a NlpC/P60 domain that is homologous to the AvPCP-C, with sequence identities ranging from 22 to 27%. The active-site residues of these enzymes are highly conserved (Figure 2C). These data suggest that AvPCP-C is likely a DL-endopeptidase. We have now shown that AvPCP can, indeed, hydrolyze Ala-γ-D-GluDAP to Ala-γ-D-Glu and DAP, confirming it as a γ-D-Glu-DAP DL-endopeptidase (Supplemental Data).
DISCUSSION The modular architecture of AvPCP suggests that it is likely evolved from gene fusion of two independent domains (Pasek et al., 2006). The papain-like superfamily (PFAM CL0125, clan CA), one of the largest peptidase families, have extremely divergent sequences. It is widely Structure. Author manuscript; available in PMC 2010 February 13.
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accepted that members of the papain-like superfamily have evolved from one general purpose ancestral peptidase to acquire multiple and more specific activities through repeated gene duplications, lateral gene transfers and gene fusions (Berti and Storer, 1995). The evolutionary relationship between the NlpC/P60 proteins and papain-like peptidases can be established at the sequence level through highly sensitive sequence profile analysis (Anantharaman and Aravind, 2003). This relationship is confirmed by the crystal structure of AvPCP. The SH3b domains in the NlpC/P60 family also diverge rapidly. Although it is general accepted that the SH3b domains bind cell-wall components, the structural determinants of SH3b for cellwall targeting are currently not well understood. No readily identifiable sites can be discerned on the SH3b surface of AvPCP that may be involved in cell-wall recognition. There have been some indications that SH3b domains may use different binding interfaces compared to their eukaryotic SH3 counterparts (Lu et al., 2006; Marino et al., 2002; Ponting et al., 1999). The SH3b groove on AvPCP, that is equivalent to a typical eukaryotic groove, is narrow and not conserved in sequence (Figure 5B). A different groove formed by the N-terminus of the ALE-1 SH3b-targeting domain is important for binding polyglycine (Lu et al., 2006). The AvPCP SH3b domain, however, does not have a corresponding groove (near Asn49; Figure 5B). If the SH3b of AvPCP could indeed interact with the cell wall, one possible site is near the domain interface where the β2-β3 hairpin of the SH3b domain interacts with AvPCP-C. From this site, a shallow, but wide, groove extends directly to Cys126 (Figure 5B).
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AvPCP is not inhibited by 10 μM E-64, a known cysteine protease inhibitor, suggesting that the active site of AvPCP is distinct from other non-cell-wall cysteine peptidases, despite sharing a similar proposed catalytic mechanism. Interestingly, residual electron density was present in the active site of AvPCP, although it was difficult to conclusively identify the ligand based solely on the electron density maps due to disorder. This density was, therefore, modeled as an unknown ligand (UNL) that may represent an L-alanine and a second disordered moiety (Figure 7A).
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Although AvPCP and NpPCP were crystallized at different conditions and crystal packing environments, no significant interdomain movements between SH3b and NlpC/P60 domains were observed. Together with the highly conserved nature of the domain interface, these data indicate that any inter-domain motion in AvPCP/NpPCP is small. Since the active site of AvPCP is well defined and spatially restricted, we utilized molecular docking to better understand the substrate specificity using the assumption of that the full length AvPCP is a rigid structure. Docking of the murein tripeptide (L-Ala-γ-D-Glu-Xaa, Xaa=DAP or Lys) or tetrapeptide (L-Ala-γ-D-Glu-Xaa-D-Ala) to the active site of AvPCP clearly favors a model that places the L-Ala at the sites where an L-Ala-like moiety is clearly observed in the density map (Figure 7B). The docked ligand is geometrically feasible and is engaged in extensive hydrogen bond interaction network with the protein. Further docking studies with large ligands which contain the murein tripeptide showed that the active site of AvPCP was unable to accommodate any moiety larger than a methyl group attached to L-Ala. Therefore, we propose that AvPCP has a strict specificity for muropeptides with an N-terminal L-alanine, similar to DPP VI of Bacillus sphaericus. The catalytic domains of these two enzymes contain identical S2/S1 sites and a very similar S1’ site (Figure 2C). Although the SH3b domain of AvPCP does not affect the shape of the binding site significantly, it does participate in formation of the S2 pocket by contributing two residues, Ala35 and Tyr64 (Figure 7C), that create a very restricted S2 binding pocket. Furthermore, the SH3b domain blocks an extension of the binding site on the NlpC/P60 domain. Thus, we speculate that the SH3b domain is crucial for substrate specificity of AvPCP, representing a novel role for SH3b. The SH3b domain is also required for the amidase domain activity of B30 lysin (Donovan et al., 2006).
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The biological function of AvPCP and NpPCP and their physiological implications are currently unknown. As no N-terminal signal peptide is associated with AvPCP (or NpPCP), it is likely that AvPCP is located in the cytoplasm where possible substrates, such as L-Ala-γD-Glu-DAP tripeptides, are produced during cell-wall recycling (Park, 1995; Uehara et al., 2005). A. variabilis contains homologs of the E. coli cell-wall, recycling enzymes, except for MpaA and PepD, indicating that it may lack or utilize a different mechanism for murein peptide metabolism compared to E. coli. Interestingly, the metallocarboxypeptidase MpaA also has strict substrate specificity for murein peptides with a free L-Ala (Uehara and Park, 2003), suggesting the intriguing possibility that AvPCP may play a similar role to MpaA. If AvPCP is located in the cytoplasm, strict substrate specificity is plausible since it prevents AvPCP from hydrolyzing intermediates of peptidoglycans and interfering with cell-wall biosynthesis.
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World-wide structural genomics projects have produced a significant number of new structures that sample vastly diverse sequence space. Most of these structures have confirmed the notion that structure and fold space is more limited compared to sequence and functional space. Nature tends to adapt conserved structural modules for different functions, so that knowing the structure of a previously uncharacterized protein does not necessarily lead to a specific functional annotation. Thus, deriving function from a structure can be a significant challenge, and an urgent task given the prodigious output of structural genomics projects. In this study, we combined structural, experimental, and computational approaches to explore and ascertain the function of a ‘hypothetical’ protein. Our efforts also led to better annotation of other related structures produced in a structural genomics context. Such new information provides a more solid foundation for the further study of the structure and function of cell-wall peptidases.
EXPERIMENTAL PROCEDURES Protein Production and Crystallization The selenomethionine derivatives of the full length AvPCP and NpPCP were cloned and expressed in E. coli with an N-terminal TEV cleavable His-tag, purified by metal affinity chromatography, and crystallized using the high-throughput structural genomics pipeline of JCSG (Lesley et al., 2002). Details can be found in Supplemental Data. Data Collection, Structure Solution, and Refinement Multi-wavelength anomalous diffraction (MAD) data were collected at SSRL beamline 11-1 (AvPCP) and the Advanced Light Source (ALS, Berkeley, CA) beamline 8.2.1 (NpPCP). Data processing and structure solution were carried out using an automatic structure solution pipeline developed at the JCSG. Details of the data processing, structure solution and refinement can be found in Supplemental Data.
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Molecular Docking Fully flexible ligand docking was performed with Glide 5.0 (Schrödinger LLC, Portland, OR) for models of L-Ala-D-Glu-DAP and L-Ala-L-Glu-DAP. The initial conformations of the ligands were generated by ChemBioOffice (CambridgeSoft Corporation, Cambridge, MA) in random orientations. The protein was prepared for grid generation and subsequent docking using the Protein Preparation Wizard tool from the Schrödinger’s FirstDiscovery suite. Optimal grid dimensions for the docking were determined with SiteMap (Halgren, 2007). The default settings for grid calculations and docking were used.
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Accession Codes
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Atomic coordinates and experimental structure factors for AvPCP at 1.05 Å resolution and NpPCP at 1.60 Å and 1.79 Å have been deposited in the Worldwide Protein Data Bank (PDB) under access codes 2hbw, 2evr, and 2fg0, respectively.
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgements The project is sponsored by NIGMS Protein Structure Initiative (U54 GM074898). Portions of this research were carried out at the SSRL and the Advanced Light Source (ALS). The SSRL is a national user facility operated by Stanford University on behalf of the U.S. DOE, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by NIH. The ALS is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division, of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory. Genomic DNA from Anabaena variabilis ATCC 29413 was a gift from Dr. Teresa Thiel (
[email protected]), Univ. Missouri-St. Louis. Genomic DNA from Nostoc punctiforme PCC 73102 (ATCC 29133) was a gift from Jack Meeks Ph.D. (
[email protected]), Univ. California-Davis. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIGMS. We greatly appreciate valuable comments on the manuscript from Professor William Hunter.
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References
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Schematic representation of type A1γ peptidoglycan and cell-wall amidases/peptidases containing NlpC/P60 or CHAP domain. (A). Peptidoglycans of type A1γ in Gram-negative bacteria (including cyanobacteria) consist of glycan chains that comprise alternating GlcNAc and MurNAc residues, stem peptides (usually tetrapeptides, such as L-Ala-γ-D-Glu-DAP-DAla), and crossbridges between DAP of one stem peptide and D-Ala of a nearby stem peptide. The enzymes involved in peptidoglycan hydrolysis and their potential sites of action are shown. (B). Schematic representation of the domain organization of AvPCP from Anabaena variabilis and some biochemically characterized NlpC/P60 or CHAP domain-containing proteins: DPP VI, dipeptidyl-peptidase VI, Bacillus sphaericus; LytE/LytF/CwlS (only LytE is shown) and CwlO (ycvE), Bacillus subtilis; Skl, Streptococcus mitis SK137; B30 Endolysin, Streptococcus agalactiae; and P60, Listeria monocytogenes. LytE, LytF, and CwlS contain three, five, and four N-terminal LysM domains, respectively. The caption is as follows: NlpC/ P60/CHAP domain, blue box; amidase domain (AMI), magenta box; choline-binding domain (CBD), cyan circle; LysM domain, green circle; SH3b domain, red circle; and signal peptide, green arrow. The domain of unknown function (DUF) is shown as a gray box.
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Figure 2.
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Crystal structure of AvPCP. (A). Crystal structure of peptidoglycan cysteine peptidase AvPCP from Anabaena variabilis. Stereo ribbon diagram of the AvPCP monomer color-coded from N-terminus (blue) to C-terminus (red). Helices H1–H6 and β-strands β1–β13 are labeled. (B). Sequence alignment of the SH3b domains of AvPCP, NpPCP, and some of their protein homologs. The primary sequence number (every 10 residues) and the secondary structure of AvPCP (2hbw) are both shown on the top of the alignment; the equivalent eukaryotic SH3 domain secondary structural elements, βA-βE, are shown in parentheses. (C). Sequence alignment of the catalytic domains of LytF, LytE, CwlS, and YwtD from Bacillus subtilis, AvPCP, NpPCP, the putative lipoprotein Spr from E. coli (PDB 2k1g), and bacterial dipeptidylpeptidase VI (DPP VI) from Bacillus sphaericus. The secondary structure on the top corresponds to that of AvPCP, while that on the bottom corresponds to Spr. The tentatively assigned S2, S1, S1’, and S2’ binding sites of AvPCP are shown in green, cyan, magenta, and blue stars, respectively.
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Figure 3.
The bacterial SH3b domains. Structural comparison between the SH3b domain of AvPCP, Abl SH3 domain (PDB 1bbz), ALE-1 targeting domain (PDB 1r77), and GW3 domain (PDB 1m9s; residue range 551-629) of invasion protein InlB. The four structures are shown in the same superimposed orientation. Residues of the ALE-1 targeting domain, the GW3 domain, and the Abl SH3 domain that can be superimposed with AvPCP SH3b are colored red.
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Structural comparisons of the catalytic domain of AvPCP. (A). Structural superimposition of the catalytic domains of AvPCP (green) and papain (PDB 9pap, orange) in stereo. The catalytic triad of each protein is shown in sticks (green). (B). Comparison of AvPCP with representative papain-like proteins: papain, Spr, the CHAP domain of GspS (Gsps-N), 2p1g (PDB 2p1g), and NsPCS (PDB 2bu3). The structures are shown in the same orientation of their catalytic domains. (C). Four representatives active site pockets. The cysteine in the catalytic triad is colored in red, the histidine in blue, and the third polar residue in cyan (may not be seen if buried). The S sites (labeled S) are tentatively assigned based on papain.
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Figure 5.
Domain interface and the active site of AvPCP. (A). Interaction between SH3b and NlpC/P60 domain. The NlpC/P60 domain is shown in surface and colored according to sequence conservation of the full-length homologs shown in Figure S1. (B). Domain interface and surface features of the SH3b domain. The eukaryotic SH3 peptide-binding groove and the ALE-1 glycine-binding site are mapped onto the SH3b domain of AvPCP. The active site Cys126 is colored blue. (C). The active site of AvPCP is identified by highly conserved residues. (D). Comparison of the catalytic triad of papain (orange) with that of AvPCP (green). The functionally important Gln19 of papain and the AvPCP equivalent, Tyr114, is also shown.
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Figure 6.
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Proposed models for binding murein peptides (L-Ala-γ-D-Glu-DAP and L-Ala-γ-D-Glu-DAPD-Ala) by AvPCP. (A). Stereo view of the active site residues of the refined model superposed with the experimental map (obtained by density modification of initial MAD phases, contoured at 1.5σ). The non-protein/solvent residual density (blue) was modeled as an UNL (unknown ligand consisting of free oxygen atoms, gray spheres) with an occupancy of 0.7. The distribution of atoms in the UNL matches an L-Ala (yellow sticks) and an additional disordered moiety. An acetate ion is labeled as ACT. Waters are shown in red spheres. (B). Modeled interaction between AvPCP (surface) and a murein tripeptide L-Ala-γ-D-Glu-DAP (sticks) and tetrapeptide L-Ala-γ-D-Glu-DAP-D-Ala (thin lines). The binding sites of AvPCP (S2, S1, S1’, and S2’) are colored as green, cyan, magenta, and blue, respectively. The S2 site consists of residues: W116, G117, A35, Y64, D125, R143, and D144; S1 site: W116, C126, S127, A145, and T175; S1’ site: Y114, W116, H176, G191, and Y215; and S2’ site: A193, Q194, and Y215. Some residues (W116 and Y215) help form more than one site and are thus shown in italics at the site where they make their main contribution. (C). Interaction between L-Ala (shown in sticks and dots) and the domain interface between SH3b and NlpC/P60 (spheres).
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NIH-PA Author Manuscript P 41 2 2
51,615
10.5 (75.2)
Rsym on I (%)a
Structure. Author manuscript; available in PMC 2010 February 13. 290 / 7
222 / 1826
Protein residues / atoms
Water molecules / ligands
20.0 0.060
ESU based on Rfree (Å)
1.51
Bond angle (°)
Average isotropic B-value (Å )
0.016
Bond length (Å)
Restraints (RMS observed)
Ramachandran plot (%)b 98.6 (0.0)
0.176
Rfree
Stereochemical parameters
99.4 0.159
Rcryst
2,624
No. of reflections (test)
Completeness (% total)
51,594
No. of reflections (total)
2
28.7 - 1.60
Resolution range(Å) |F|>0
2evr-remo
Data set used in refinement
Model and refinement statistics
9.1 (2.0)
Mean I/σ (I)a
99.4 (96.9)
Number of unique reflections
Completeness(%)a
430,704
Number of observations
28.7 - 1.60
1.0163
Wavelength (Å)
Resolution range (Å)
2evr-remo
Data collection
11.4 (73.3)
8.8 (2.0)
99.6 (98.1)
51,699
439,024
28.7 - 1.60
0.9797
2evr-infl
a=b=90.47 Å c=93.81 Å
Space group
Unit cell parameters
ALS 8.2.1
NpPCP - 2evr
Beamline
Structure - PDB ID
Cutoff criteria
Table I
428 / 3
444 / 3531
0.076
22.2
1.51
0.016
97.7 (0.0)
0.172
0.154
99.6
3,659
72,429
|F|>0
29.0 - 1.79
2fg0-remo
13.9 (115.2)
12.3 (1.9)
99.6 (96.9)
72,488
1,051,466
29.5 - 1.79
1.0163
2fg0-remo
14.1 (115.4)
11.6 (1.9)
99.6 (97.0)
72,520
1,050,603
28.9 - 1.79
0.9798
2fg0-infl
a=b=125.08 Å c=97.67 Å
P 41 21 2
ALS 8.2.1
NpPCP - 2fg0
345 / 3
221 / 1926
0.023
9.3
1.94
0.020
99.3 (0.0)
0.149
0.125
100.0
5,839
116,513
|F|>0
35.0-1.05
2hbw-peak
7.6 (75.6)
10.5 (2.0)
100.0 (100.0)
116,587
634,927
35.0 - 1.05
0.9790
2hbw-peak
8.4 (72.2)
9.7 (2.1)
99.9 (100.)
101,490
477,760
35.0 - 1.10
0.9184
2hbw-remo
8.8 (74.1)
9.7 (2.0)
99.9 (100.)
96,283
450,687
35.0 - 1.12
0.9793
2hbw-infl
a=76.48 Å b=86.89 Å c=37.29 Å
P 21 21 2
SSRL BL 11-1
AvPCP - 2hbw
NIH-PA Author Manuscript
Summary of crystal parameters, data collection, and refinement statistics Xu et al. Page 20
NIH-PA Author Manuscript
Rfree = as for Rcryst, but for 5% of the total reflections chosen at random and omitted from refinement.
Rcryst = Σ| |Fobs|-|Fcalc| | / Σ|Fobs|, where Fcalc and Fobs are the calculated and observed structure factor amplitudes, respectively.
Rsym = Σ|Ii-| / Σ|Ii|, where Ii is the scaled intensity of the ith measurement, and is the mean intensity for that reflection.
ESU = Estimated overall coordinate error.
The percent of residues in the favored region of the Ramachandran plot generated by MolProbity (outliers in parenthesis).
Statistics for the highest resolution shell in parentheses.
NIH-PA Author Manuscript
b
NIH-PA Author Manuscript
a
Xu et al. Page 21
Structure. Author manuscript; available in PMC 2010 February 13.
