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Structural determinants for activity and specificity of the bacterial toxin LlpA.

Ghequire MG, Garcia-Pino A, Lebbe EK, Spaepen S, Loris R, De Mot R - PLoS Pathog. (2013)

Bottom Line: The N-terminal MMBL domain (N-domain) adopts the same fold but is structurally more divergent and lacks a functional mannose-binding site.Differential activity of engineered N/C-domain chimers derived from two LlpA homologues with different killing spectra, disclosed that the N-domain determines target specificity.Apparently this bacteriocin is assembled from two structurally similar domains that evolved separately towards dedicated functions in target recognition and bacteriotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Centre of Microbial and Plant Genetics, University of Leuven, Heverlee-Leuven, Belgium.

ABSTRACT
Lectin-like bacteriotoxic proteins, identified in several plant-associated bacteria, are able to selectively kill closely related species, including several phytopathogens, such as Pseudomonas syringae and Xanthomonas species, but so far their mode of action remains unrevealed. The crystal structure of LlpABW, the prototype lectin-like bacteriocin from Pseudomonas putida, reveals an architecture of two monocot mannose-binding lectin (MMBL) domains and a C-terminal β-hairpin extension. The C-terminal MMBL domain (C-domain) adopts a fold very similar to MMBL domains from plant lectins and contains a binding site for mannose and oligomannosides. Mutational analysis indicates that an intact sugar-binding pocket in this domain is crucial for bactericidal activity. The N-terminal MMBL domain (N-domain) adopts the same fold but is structurally more divergent and lacks a functional mannose-binding site. Differential activity of engineered N/C-domain chimers derived from two LlpA homologues with different killing spectra, disclosed that the N-domain determines target specificity. Apparently this bacteriocin is assembled from two structurally similar domains that evolved separately towards dedicated functions in target recognition and bacteriotoxicity.

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Carbohydrate binding in site IIIC of LlpABW.(A) Stereoview of methyl-α-D-mannopyranoside bound to subdomain IIIC. Methyl-α-D-mannopyranoside is shown in blue and indicated by M. Residues belonging to the QxDxNxVxY motif and hydrogen bonding to the sugar as well as Asn188 are labeled. Water molecules bridging protein and carbohydrate are shown in cyan (B) Similar view of the pentasaccharide GlcNAcβ(1–2)Manα(1–3)[GlcNAcβ(1–2)Manα(1–6)]Man. The mannose residue occupying the primary binding site is shown in blue and labeled M. The additional two mannoses (labeled +1 and −1) and two N-acetyl glucosamine residues (labeled +2 and −2) are shown in green. Other colors are as in panel A. (C) Binding of the disaccharide Manα(1–2)Man. The non-reducing mannose residue occupying the primary binding site is shown in blue and labeled M. The second, reducing mannose is shown in green. Other colors are as in panel A.
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ppat-1003199-g003: Carbohydrate binding in site IIIC of LlpABW.(A) Stereoview of methyl-α-D-mannopyranoside bound to subdomain IIIC. Methyl-α-D-mannopyranoside is shown in blue and indicated by M. Residues belonging to the QxDxNxVxY motif and hydrogen bonding to the sugar as well as Asn188 are labeled. Water molecules bridging protein and carbohydrate are shown in cyan (B) Similar view of the pentasaccharide GlcNAcβ(1–2)Manα(1–3)[GlcNAcβ(1–2)Manα(1–6)]Man. The mannose residue occupying the primary binding site is shown in blue and labeled M. The additional two mannoses (labeled +1 and −1) and two N-acetyl glucosamine residues (labeled +2 and −2) are shown in green. Other colors are as in panel A. (C) Binding of the disaccharide Manα(1–2)Man. The non-reducing mannose residue occupying the primary binding site is shown in blue and labeled M. The second, reducing mannose is shown in green. Other colors are as in panel A.

Mentions: Subdomains IIC and IIICof LlpABW contain the typical sugar-binding signature (QxDxNxVxY) of an active MMBL mannose-binding site (Figure S1 and S4). Soaking crystals of LlpABW with 200 mM methyl-α-D-mannopyranoside (Me-Man) led to clear electron density of a single Me-Man in site IIIC of each of the two LlpABW monomers in the asymmetric unit (Figure S6A). This site comprises the side chains from Gln171, Asp173, Asn175 and Tyr179, which contribute to hydrogen bond interactions and the side chains of residues Val177, Asn188, Gln192 and Ala185, which contribute to van der Waals contacts with the carbohydrate ligand (Figure 3A, Figure S7A,C). This architecture is very similar to what is observed for mannose bound to other MMBL-type lectins such as snowdrop and garlic lectin (Figure S7B).


Structural determinants for activity and specificity of the bacterial toxin LlpA.

Ghequire MG, Garcia-Pino A, Lebbe EK, Spaepen S, Loris R, De Mot R - PLoS Pathog. (2013)

Carbohydrate binding in site IIIC of LlpABW.(A) Stereoview of methyl-α-D-mannopyranoside bound to subdomain IIIC. Methyl-α-D-mannopyranoside is shown in blue and indicated by M. Residues belonging to the QxDxNxVxY motif and hydrogen bonding to the sugar as well as Asn188 are labeled. Water molecules bridging protein and carbohydrate are shown in cyan (B) Similar view of the pentasaccharide GlcNAcβ(1–2)Manα(1–3)[GlcNAcβ(1–2)Manα(1–6)]Man. The mannose residue occupying the primary binding site is shown in blue and labeled M. The additional two mannoses (labeled +1 and −1) and two N-acetyl glucosamine residues (labeled +2 and −2) are shown in green. Other colors are as in panel A. (C) Binding of the disaccharide Manα(1–2)Man. The non-reducing mannose residue occupying the primary binding site is shown in blue and labeled M. The second, reducing mannose is shown in green. Other colors are as in panel A.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585409&req=5

ppat-1003199-g003: Carbohydrate binding in site IIIC of LlpABW.(A) Stereoview of methyl-α-D-mannopyranoside bound to subdomain IIIC. Methyl-α-D-mannopyranoside is shown in blue and indicated by M. Residues belonging to the QxDxNxVxY motif and hydrogen bonding to the sugar as well as Asn188 are labeled. Water molecules bridging protein and carbohydrate are shown in cyan (B) Similar view of the pentasaccharide GlcNAcβ(1–2)Manα(1–3)[GlcNAcβ(1–2)Manα(1–6)]Man. The mannose residue occupying the primary binding site is shown in blue and labeled M. The additional two mannoses (labeled +1 and −1) and two N-acetyl glucosamine residues (labeled +2 and −2) are shown in green. Other colors are as in panel A. (C) Binding of the disaccharide Manα(1–2)Man. The non-reducing mannose residue occupying the primary binding site is shown in blue and labeled M. The second, reducing mannose is shown in green. Other colors are as in panel A.
Mentions: Subdomains IIC and IIICof LlpABW contain the typical sugar-binding signature (QxDxNxVxY) of an active MMBL mannose-binding site (Figure S1 and S4). Soaking crystals of LlpABW with 200 mM methyl-α-D-mannopyranoside (Me-Man) led to clear electron density of a single Me-Man in site IIIC of each of the two LlpABW monomers in the asymmetric unit (Figure S6A). This site comprises the side chains from Gln171, Asp173, Asn175 and Tyr179, which contribute to hydrogen bond interactions and the side chains of residues Val177, Asn188, Gln192 and Ala185, which contribute to van der Waals contacts with the carbohydrate ligand (Figure 3A, Figure S7A,C). This architecture is very similar to what is observed for mannose bound to other MMBL-type lectins such as snowdrop and garlic lectin (Figure S7B).

Bottom Line: The N-terminal MMBL domain (N-domain) adopts the same fold but is structurally more divergent and lacks a functional mannose-binding site.Differential activity of engineered N/C-domain chimers derived from two LlpA homologues with different killing spectra, disclosed that the N-domain determines target specificity.Apparently this bacteriocin is assembled from two structurally similar domains that evolved separately towards dedicated functions in target recognition and bacteriotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Centre of Microbial and Plant Genetics, University of Leuven, Heverlee-Leuven, Belgium.

ABSTRACT
Lectin-like bacteriotoxic proteins, identified in several plant-associated bacteria, are able to selectively kill closely related species, including several phytopathogens, such as Pseudomonas syringae and Xanthomonas species, but so far their mode of action remains unrevealed. The crystal structure of LlpABW, the prototype lectin-like bacteriocin from Pseudomonas putida, reveals an architecture of two monocot mannose-binding lectin (MMBL) domains and a C-terminal β-hairpin extension. The C-terminal MMBL domain (C-domain) adopts a fold very similar to MMBL domains from plant lectins and contains a binding site for mannose and oligomannosides. Mutational analysis indicates that an intact sugar-binding pocket in this domain is crucial for bactericidal activity. The N-terminal MMBL domain (N-domain) adopts the same fold but is structurally more divergent and lacks a functional mannose-binding site. Differential activity of engineered N/C-domain chimers derived from two LlpA homologues with different killing spectra, disclosed that the N-domain determines target specificity. Apparently this bacteriocin is assembled from two structurally similar domains that evolved separately towards dedicated functions in target recognition and bacteriotoxicity.

Show MeSH
Related in: MedlinePlus