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An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase.

Wong JE, Midtgaard SR, Gysel K, Thygesen MB, Sørensen KK, Jensen KJ, Stougaard J, Thirup S, Blaise M - Acta Crystallogr. D Biol. Crystallogr. (2015)

Bottom Line: The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer.The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers.By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus, Denmark.

ABSTRACT
LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of the Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers. By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed.

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Proposed model of PGN recognition by P60_tth. (a) PGN recognition model depicting how the LysM domain interacts with a PGN fragment. Each MurNAc is linked to a peptide [l-Ala-γ-d-Gln-l-Lys-(d-Asn)]; the MurNAc-peptide molecule was extracted from the l,d-carboxypeptidase crystal structure (PDB entry 4oxd; Hoyland et al., 2014 ▶). (b) Distances observed between cross-linked PGN strands and the length of the peptide stem in the three-dimensional model of S. aureus PGN proposed by Meroueh et al. (2006 ▶). The distance between the two LysM binding sites in the P60_tth full-length crystal structure and between the entrance of the two active sites (red) of the P60_tth catalytic domains are also indicated. (c) Scheme explaining how the P60_tth homodimer could anchor the protein onto PGN. The PGN GlcNAc-MurNAc strands are represented by hexagons and the cross-linked peptide-stem composition of T. thermophilus is indicated by three-letter amino-acid codes; the amino-acid composition has been described previously (Quintela et al., 1995 ▶). ‘Cys’ represents the catalytic cysteines and the red arrows indicate the putative cleavage sites in the peptide stem.
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fig9: Proposed model of PGN recognition by P60_tth. (a) PGN recognition model depicting how the LysM domain interacts with a PGN fragment. Each MurNAc is linked to a peptide [l-Ala-γ-d-Gln-l-Lys-(d-Asn)]; the MurNAc-peptide molecule was extracted from the l,d-carboxypeptidase crystal structure (PDB entry 4oxd; Hoyland et al., 2014 ▶). (b) Distances observed between cross-linked PGN strands and the length of the peptide stem in the three-dimensional model of S. aureus PGN proposed by Meroueh et al. (2006 ▶). The distance between the two LysM binding sites in the P60_tth full-length crystal structure and between the entrance of the two active sites (red) of the P60_tth catalytic domains are also indicated. (c) Scheme explaining how the P60_tth homodimer could anchor the protein onto PGN. The PGN GlcNAc-MurNAc strands are represented by hexagons and the cross-linked peptide-stem composition of T. thermophilus is indicated by three-letter amino-acid codes; the amino-acid composition has been described previously (Quintela et al., 1995 ▶). ‘Cys’ represents the catalytic cysteines and the red arrows indicate the putative cleavage sites in the peptide stem.

Mentions: First of all, we superposed a MurNAc peptide (Hoyland et al., 2014 ▶) onto GlcNAc 6, GlcNAc 4 and GlcNAc 2 of the chitohexaose from our P60_2LysM–chito­hexaose structure. With minimal additional modelling (rotating only the bond between l-Ala and MurNAc), we could fit the peptide stem without inducing any steric hindrance with the residues from the LysM binding site (Fig. 9 ▶a). This suggests that a MurNAc-GlcNAc oligosaccharide might interact in a similar way to that observed with a GlcNAc oligosaccharide and that the peptide portion of PGN might not be recognized at all by the residues in the LysM domains. However, we do not exclude the possibility that the peptide portion of PGN might trigger steric hindrance upon binding in the LysM groove. This hypothesis was demonstrated in a recent study by Mesnage and coworkers, who proposed that the peptide portion of PGN reduces the affinity of the Enterococcus faecalis AtlA single LysM domain for PGN (Mesnage et al., 2014 ▶).


An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase.

Wong JE, Midtgaard SR, Gysel K, Thygesen MB, Sørensen KK, Jensen KJ, Stougaard J, Thirup S, Blaise M - Acta Crystallogr. D Biol. Crystallogr. (2015)

Proposed model of PGN recognition by P60_tth. (a) PGN recognition model depicting how the LysM domain interacts with a PGN fragment. Each MurNAc is linked to a peptide [l-Ala-γ-d-Gln-l-Lys-(d-Asn)]; the MurNAc-peptide molecule was extracted from the l,d-carboxypeptidase crystal structure (PDB entry 4oxd; Hoyland et al., 2014 ▶). (b) Distances observed between cross-linked PGN strands and the length of the peptide stem in the three-dimensional model of S. aureus PGN proposed by Meroueh et al. (2006 ▶). The distance between the two LysM binding sites in the P60_tth full-length crystal structure and between the entrance of the two active sites (red) of the P60_tth catalytic domains are also indicated. (c) Scheme explaining how the P60_tth homodimer could anchor the protein onto PGN. The PGN GlcNAc-MurNAc strands are represented by hexagons and the cross-linked peptide-stem composition of T. thermophilus is indicated by three-letter amino-acid codes; the amino-acid composition has been described previously (Quintela et al., 1995 ▶). ‘Cys’ represents the catalytic cysteines and the red arrows indicate the putative cleavage sites in the peptide stem.
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Related In: Results  -  Collection

License
Show All Figures
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fig9: Proposed model of PGN recognition by P60_tth. (a) PGN recognition model depicting how the LysM domain interacts with a PGN fragment. Each MurNAc is linked to a peptide [l-Ala-γ-d-Gln-l-Lys-(d-Asn)]; the MurNAc-peptide molecule was extracted from the l,d-carboxypeptidase crystal structure (PDB entry 4oxd; Hoyland et al., 2014 ▶). (b) Distances observed between cross-linked PGN strands and the length of the peptide stem in the three-dimensional model of S. aureus PGN proposed by Meroueh et al. (2006 ▶). The distance between the two LysM binding sites in the P60_tth full-length crystal structure and between the entrance of the two active sites (red) of the P60_tth catalytic domains are also indicated. (c) Scheme explaining how the P60_tth homodimer could anchor the protein onto PGN. The PGN GlcNAc-MurNAc strands are represented by hexagons and the cross-linked peptide-stem composition of T. thermophilus is indicated by three-letter amino-acid codes; the amino-acid composition has been described previously (Quintela et al., 1995 ▶). ‘Cys’ represents the catalytic cysteines and the red arrows indicate the putative cleavage sites in the peptide stem.
Mentions: First of all, we superposed a MurNAc peptide (Hoyland et al., 2014 ▶) onto GlcNAc 6, GlcNAc 4 and GlcNAc 2 of the chitohexaose from our P60_2LysM–chito­hexaose structure. With minimal additional modelling (rotating only the bond between l-Ala and MurNAc), we could fit the peptide stem without inducing any steric hindrance with the residues from the LysM binding site (Fig. 9 ▶a). This suggests that a MurNAc-GlcNAc oligosaccharide might interact in a similar way to that observed with a GlcNAc oligosaccharide and that the peptide portion of PGN might not be recognized at all by the residues in the LysM domains. However, we do not exclude the possibility that the peptide portion of PGN might trigger steric hindrance upon binding in the LysM groove. This hypothesis was demonstrated in a recent study by Mesnage and coworkers, who proposed that the peptide portion of PGN reduces the affinity of the Enterococcus faecalis AtlA single LysM domain for PGN (Mesnage et al., 2014 ▶).

Bottom Line: The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer.The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers.By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus, Denmark.

ABSTRACT
LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of the Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers. By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed.

Show MeSH