Deciphering how Cpl-7 cell wall-binding repeats recognize the bacterial peptidoglycan (original) (raw)
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Journal of Biological Chemistry, 2007
Pneumococcal bacteriophage-encoded lysins are modular proteins that have been shown to act as enzymatic antimicrobial agents (enzybiotics) in treatment of streptococcal infections. The first x-ray crystal structures of the Cpl-1 lysin, encoded by the pneumococcal phage Cp-1, in complex with three bacterial cell wall peptidoglycan (PG) analogues are reported herein. The Cpl-1 structure is folded in two well defined modules, one responsible for anchoring to the pneumococcal cell wall and the other, a catalytic module, that hydrolyzes the PG. Conformational rearrangement of Tyr-127 is a critical event in molecular recognition of a stretch of five saccharide rings of the polymeric peptidoglycan (cell wall). The PG is bound at a stretch of the surface that is defined as the peptidoglycan-binding sites 1 and 2, the juncture of which catalysis takes place. The peptidoglycan-binding site 1 binds to a stretch of three saccharides of the peptidoglycan in a conformation essentially identical to that of the peptidoglycan in solution. In contrast, binding of two peptidoglycan saccharides at the peptidoglycan-binding site 2 introduces a kink into the solution structure of the peptidoglycan, en route to catalytic turnover. These findings provide the first structural evidence on recognition of the peptidoglycan and shed light on the discrete events of cell wall degradation by Cpl-1.
Cpl-7, a Lysozyme Encoded by a Pneumococcal Bacteriophage with a Novel Cell Wall-binding Motif*
Journal of Biological Chemistry, 2010
Bacteriophage endolysins include a group of new antibacterials reluctant to development of resistance. We present here the first structural study of the Cpl-7 endolysin, encoded by pneumococcal bacteriophage Cp-7. It contains an N-terminal catalytic module (CM) belonging to the GH25 family of glycosyl hydrolases and a C-terminal region encompassing three identical repeats of 42 amino acids (CW_7 repeats). These repeats are unrelated to choline-targeting motifs present in other cell wall hydrolases produced by Streptococcus pneumoniae and its bacteriophages, and are responsible for the protein attachment to the cell wall. By combining different biophysical techniques and molecular modeling, a three-dimensional model of the overall protein structure is proposed, consistent with circular dichroism and sequence-based secondary structure prediction, small angle x-ray scattering data, and Cpl-7 hydrodynamic behavior. Cpl-7 is an ϳ115-Å long molecule with two well differentiated regions, corresponding to the CM and the cell wall binding region (CWBR), arranged in a lateral disposition. The CM displays the (␣) 5  3 barrel topology characteristic of the GH25 family, and the impact of sequence differences with the CM of the Cpl-1 lysozyme in substrate binding is discussed. The CWBR is organized in three tandemly assembled three-helical bundles whose dispositions remind us of a super-helical structure. Its approximate dimensions are 60 ؋ 20 ؋ 20 Å and presents a concave face that might constitute the functional region involved in bacterial surface recognition. The distribution of CW_7 repeats in the sequences deposited in the Entrez Database have been examined, and the results drastically expanded the antimicrobial potential of the Cpl-7 endolysin.
2020
Endolysins are bacteriophage-encoded peptidoglycan hydrolases targeting the cell wall of host bacteria via their cell wall-binding domains (CBDs). The molecular basis for selective recognition of surface carbohydrate ligands by CBDs remains elusive. Here, we describe, in atomic detail, the interaction between the Listeria phage endolysin domain CBD500 and its cell wall teichoic acid (WTA) ligands. We show that 3’ O-acetylated GlcNAc residues integrated into the WTA polymer chain are the key epitope recognized by a CBD binding cavity located at the interface of tandem copies of beta-barrel, pseudo-symmetric SH3b-like repeats. This cavity consists of multiple aromatic residues making extensive interactions with two GlcNAc acetyl groups via hydrogen bonds and van der Waals contacts, while permitting the docking of the diastereomorphic ligands. The multidisciplinary approach described here delineates a previously unknown recognition mechanism by which a phage endolysin specifically reco...
Journal of Biological Chemistry, 2013
Background: Peptidoglycan hydrolases, including bacterial autolysins and bacteriophage endolysins, contain generally a cell wall-binding domain (CWBD), responsible for their high affinity and specificity toward target cell walls. Results: Two Lactobacillus casei endolysins lyse only bacterial cells with a D-Asn cross-bridge in their peptidoglycan. Conclusion: The CWBD of these two endolysins recognizes specifically peptidoglycan with a D-Asn cross-bridge. Significance: This CWBD is a novel type of peptidoglycan-binding domain. Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-L-alanine amidase, whereas Lc-Lys-2 is a ␥-D-glutamyl-L-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with D-Ala 4 3D-Asx-L-Lys 3 in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting D-Ala 4 3L-Ala-(L-Ala/L-Ser)-L-Lys 3 ; moreover, they do not lyse the L. lactis mutant containing only the nonamidated D-Asp cross-bridge, i.e. D-Ala 4 3 D-Asp-L-Lys 3. In contrast, Lc-Lys could lyse the ampicillinresistant E. faecium mutant with 333 L-Lys 3-D-Asn-L-Lys 3 bridges replacing the wild-type 433 D-Ala 4-D-Asn-L-Lys 3 bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly D-Asn but not PG with only the nonamidated D-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the D-Asn interpeptide bridge of PG. Peptidoglycan hydrolases (PGHs) 3 synthesized by Grampositive bacteria and their bacteriophages are able to degrade the protective cell wall peptidoglycan (PG) that surrounds the bacterial cytoplasmic membrane. PG is a macromolecule consisting of glycan chains made of alternating -1,4-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) cross-linked by peptide chains, the composition of which varies between bacterial species. Bacterial PGHs or autolysins are required for cell wall remodeling during bacterial cell growth and division (1, 2). Bacteriophage PGHs, named endolysins, are synthesized in phage-infected cells at the end of the multiplication cycle leading to bacterial lysis and release of phage progeny (3, 4). Most PGHs have a modular structure with multiple domains, including a catalytic domain with PG hydrolyzing activity and a cell wall-binding domain (CWBD) that targets the enzyme specifically to the cell wall. The ligands of CWBDs may be structural motifs present either in PG or in secondary cell wall polymers, including polysaccharides or teichoic acids that decorate PG (3). Endolysins usually lack a signal peptide for their export and therefore rely on the synthesis of holins, which are able to insert into the cytoplasmic membrane and make pores (5). Most often, their catalytic domain is located at the N terminus and their CWBD at the C terminus (6). Generally, the catalytic domains found in endolysins belong to the same families as those encountered in bacterial PGHs (2). In contrast, certain endolysin CWBDs do not display any sequence similarity with well characterized CWBDs found in bacterial PGHs such as LysM or SH3b domains. CWBDs are considered to confer to endolysins high affinity and high specificity for their target bacteria (7). Endolysins from bacteriophages infecting Gram-positive bacteria can lyse bacteria from the outside and thus have been proposed as alternatives for preservatives and antibiotics used to destroy pathogens in food and medical applications (3, 4, 6, 8). Because of their high affinity for the cell wall, endolysin * This work was supported in part by INRA and Ré gion Ile de France and was an associated project from the Marie Curie FP7 Initial Training Network Cross Talk (Grant Agreement 21553-2). 1 Recipient of a fellowship from INRA.
Journal of Molecular Biology, 2008
The mammalian peptidoglycan recognition protein-S (PGRP-S) binds to peptidoglycans (PGNs), which are essential components of the cell wall of bacteria. The protein was isolated from the samples of milk obtained from camels with mastitis and purified to homogeneity and crystallized. The crystals belong to orthorhombic space group I222 with a = 87.0 Å, b = 101.7 Å and c = 162.3 Å having four crystallographically independent molecules in the asymmetric unit. The structure has been determined using X-ray crystallographic data and refined to 1.8 Å resolution. Overall, the structures of all the four crystallographically independent molecules are identical. The folding of PGRP-S consists of a central β-sheet with five β-strands, four parallel and one antiparallel, and three α-helices. This protein fold provides two functional sites. The first of these is the PGN-binding site, located on the groove that opens on the surface in the direction opposite to the location of the N terminus. The second site is implicated to be involved in the binding of non-PGN molecules, it also includes putative N-terminal segment residues (1-31) and helix α2 in the extended binding. The structure reveals a novel arrangement of PGRP-S molecules in which two pairs of molecules associate to form two independent dimers. The first dimer is formed by two molecules with N-terminal segments at the interface in which non-PGN binding sites are buried completely, whereas the PGN-binding sites of two participating molecules are fully exposed at the opposite ends of the dimer. In the second dimer, PGN-binding sites are buried at the interface while non-PGN binding sites are fully exposed at the opposite ends of the dimer. This form of dimeric arrangement is unique and seems to be aimed at enhancing the capability of the protein against specific invading bacteria. This mode of functional dimerization enhances efficiency and specificity, and is observed for the first time in the family of PGRP molecules.
Journal of Biological Chemistry, 2006
ALE-1, a homologue of lysostaphin, is a peptidoglycan hydrolase that specifically lyses Staphylococcus aureus cell walls by cleaving the pentaglycine linkage between the peptidoglycan chains. Binding of ALE-1 to S. aureus cells through its C-terminal 92 residues, known as the targeting domain, is functionally important for staphylolytic activity. The ALE-1-targeting domain belongs to the SH3b domain family, the prokaryotic counterpart of the eukaryotic SH3 domains. The 1.75 Å crystal structure of the targeting domain shows an all- fold similar to typical SH3s but with unique features. The structure reveals patches of conserved residues among orthologous targeting domains, forming surface regions that can potentially interact with some common features of the Gram-positive cell wall. ALE-1-targeting domain binding studies employing various bacterial peptidoglycans demonstrate that the length of the interpeptide bridge, as well as the amino acid composition of the peptide, confers the maximum binding of the targeting domain to the staphylococcal peptidoglycan. Truncation of the highly conserved first 9 N-terminal residues results in loss of specificity to S. aureus cell wall-targeting, suggesting that these residues confer specificity to S. aureus cell wall.
Three-dimensional structure of the bacterial cell wall peptidoglycan
Proceedings of the National Academy of Sciences, 2006
The 3D structure of the bacterial peptidoglycan, the major constituent of the cell wall, is one of the most important, yet still unsolved, structural problems in biochemistry. The peptidoglycan comprises alternating N-acetylglucosamine (NAG) and N-acetylmuramic disaccharide (NAM) saccharides, the latter of which has a peptide stem. Adjacent peptide stems are cross-linked by the transpeptidase enzymes of cell wall biosynthesis to provide the cell wall polymer with the structural integrity required by the bacterium. The cell wall and its biosynthetic enzymes are targets of antibiotics. The 3D structure of the cell wall has been elusive because of its complexity and the lack of pure samples. Herein we report the 3D solution structure as determined by NMR of the 2-kDa NAG-NAM(pentapeptide)-NAG-NAM(pentapeptide) synthetic fragment of the cell wall. The glycan backbone of this peptidoglycan forms a right-handed helix with a periodicity of three for the NAG-NAM repeat (per turn of the helix). The first two amino acids of the pentapeptide adopt a limited number of conformations. Based on this structure a model for the bacterial cell wall is proposed. murein sacculus ͉ bacterial envelope T he peptidoglycan scaffold of the bacterial cell wall is a repeating N-acetylglucosamine (NAG)-N-acetylmuramic disaccharide (NAM) [NAG-(-1,4)-NAM] having a pentapeptide attached to the D-lactyl moiety of each NAM. This pentapeptide stem participates in an interglycan cross-linking reaction, thus creating the cell wall polymer. In contrast to the two other -1,4-linked glycan biopolymers, cellulose (repeating glucose) (1-4) and chitin (repeating NAG) (5-7) for which the 3D structure is solved, the structure of the bacterial cell wall has remained elusive because of its complexity and the lack of pure and discrete segments for structural study . Herein we describe the 3D structure, determined in aqueous solution by NMR, of a 2-kDa synthetic NAG-NAM(pentapeptide)-NAG-NAM(pentapeptide) tetrasaccharide cell wall segment. The defining aspect of this structure is an ordered, right-handed helical saccharide conformation corresponding to three NAG-NAM pairs per turn of the helix. The structure of this peptidoglycan segment is the basis for a proposal for the structure of the bacterial cell wall polymer.
Frontiers in Microbiology
The cell wall (CW) of bacteria is an intricate arrangement of macromolecules, at least constituted of peptidoglycan (PG) but also of (lipo)teichoic acids, various polysaccharides, polyglutamate and/or proteins. During bacterial growth and division, there is a constant balance between CW degradation and biosynthesis. The CW is remodeled by bacterial hydrolases, whose activities are carefully regulated to maintain cell integrity or lead to bacterial death. Each cell wall hydrolase (CWH) has a specific role regarding the PG: (i) cell wall amidase (CWA) cleaves the amide bond between N-acetylmuramic acid and L-alanine residue at the N-terminal of the stem peptide, (ii) cell wall glycosidase (CWG) catalyses the hydrolysis of the glycosidic linkages, whereas (iii) cell wall peptidase (CWP) cleaves amide bonds between amino acids within the PG chain. After an exhaustive overview of all known conserved catalytic domains responsible for CWA, CWG, and CWP activities, this review stresses that the CWHs frequently display a modular architecture combining multiple and/or different catalytic domains, including some lytic transglycosylases as well as CW binding domains. From there, direct physiological and collateral roles of CWHs in bacterial cells are further discussed.
Molecular basis for bacterial peptidoglycan recognition by LysM domains
Nature Communications, 2014
Carbohydrate recognition is essential for growth, cell adhesion and signalling in all living organisms. A highly conserved carbohydrate binding module, LysM, is found in proteins from viruses, bacteria, fungi, plants and mammals. LysM modules recognize polysaccharides containing N-acetylglucosamine (GlcNAc) residues including peptidoglycan, an essential component of the bacterial cell wall. However, the molecular mechanism underpinning LysM-peptidoglycan interactions remains unclear. Here we describe the molecular basis for peptidoglycan recognition by a multimodular LysM domain from AtlA, an autolysin involved in cell division in the opportunistic bacterial pathogen Enterococcus faecalis. We explore the contribution of individual modules to the binding, identify the peptidoglycan motif recognized, determine the structures of free and bound modules and reveal the residues involved in binding. Our results suggest that peptide stems modulate LysM binding to peptidoglycan. Using these results, we reveal how the LysM module recognizes the GlcNAc-X-GlcNAc motif present in polysaccharides across kingdoms.