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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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ITC analysis of carbohydrate binding to LlpABW and mutants.(A) Binding of LlpABW to the pentasaccharide GlcNAcβ(1–2)Manα(1–3)[GlcNAcβ(1–2)Manα(1–6)]Man. (B) Binding of LlpABW (blue circles, wild type) and the mutants LlpAV177Y (green circles, site IIIC knockout), LlpAV208Y (red circles, site IIC knockout) and LlpAV177Y-V208Y (black circles, site IIC and IIIC knockout) to α-methyl mannoside. There is no heat exchanged in the titration of the double mutant or the site IIIC knockout LlpAV177Y, whereas the site IIC knockout LlpAV208Y, binds the monosaccharide in a “wildtype”-like fashion, showing that only site IIIC is involved in sugar binding.
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ppat-1003199-g005: ITC analysis of carbohydrate binding to LlpABW and mutants.(A) Binding of LlpABW to the pentasaccharide GlcNAcβ(1–2)Manα(1–3)[GlcNAcβ(1–2)Manα(1–6)]Man. (B) Binding of LlpABW (blue circles, wild type) and the mutants LlpAV177Y (green circles, site IIIC knockout), LlpAV208Y (red circles, site IIC knockout) and LlpAV177Y-V208Y (black circles, site IIC and IIIC knockout) to α-methyl mannoside. There is no heat exchanged in the titration of the double mutant or the site IIIC knockout LlpAV177Y, whereas the site IIC knockout LlpAV208Y, binds the monosaccharide in a “wildtype”-like fashion, showing that only site IIIC is involved in sugar binding.

Mentions: Purified proteins were prepared to further quantify these effects. Far UV CD spectra of these mutant forms are identical to that of native protein LlpABW, indicating that the mutations do not affect the overall structure of the protein. Isothermal titration calorimetry (ITC) showed that LlpABW has an affinity of 2.1 mM for the pentasaccharide GlcNAcβ(1–2)Manα(1–3)[GlcNAcβ(1–2)Manα(1–6)]Man, the highest among all the tested oligo-mannosides (See Figure 5 and Table 1 for a summary of the experimentally validated LlpABW-carbohydrate interactions). This is in agreement with the crystal structures of the different complexes since this sugar is the one with the largest binding interface (Figure 3). Titrations of LlpABW, of the mutants LlpAV177Y (a site IIIC knockout), LlpAV208Y (a site IIC knockout) and of the double mutant LlpAV177Y-V208Y with α-methyl mannoside clearly pinpoint site IIIC as the only responsible for the sugar binding activity. Point mutations in both sites or IIIC (V177Y) alone, completely abrogate sugar binding. However knocking out site IIC (V208Y) has little effect in binding and the affinities of LlpAV208Y for α-methyl mannoside and Manα(1–3)Man are very close to the ones measured for the wild-type protein (See Table 1 and Figure 5B).


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)

ITC analysis of carbohydrate binding to LlpABW and mutants.(A) Binding of LlpABW to the pentasaccharide GlcNAcβ(1–2)Manα(1–3)[GlcNAcβ(1–2)Manα(1–6)]Man. (B) Binding of LlpABW (blue circles, wild type) and the mutants LlpAV177Y (green circles, site IIIC knockout), LlpAV208Y (red circles, site IIC knockout) and LlpAV177Y-V208Y (black circles, site IIC and IIIC knockout) to α-methyl mannoside. There is no heat exchanged in the titration of the double mutant or the site IIIC knockout LlpAV177Y, whereas the site IIC knockout LlpAV208Y, binds the monosaccharide in a “wildtype”-like fashion, showing that only site IIIC is involved in sugar binding.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003199-g005: ITC analysis of carbohydrate binding to LlpABW and mutants.(A) Binding of LlpABW to the pentasaccharide GlcNAcβ(1–2)Manα(1–3)[GlcNAcβ(1–2)Manα(1–6)]Man. (B) Binding of LlpABW (blue circles, wild type) and the mutants LlpAV177Y (green circles, site IIIC knockout), LlpAV208Y (red circles, site IIC knockout) and LlpAV177Y-V208Y (black circles, site IIC and IIIC knockout) to α-methyl mannoside. There is no heat exchanged in the titration of the double mutant or the site IIIC knockout LlpAV177Y, whereas the site IIC knockout LlpAV208Y, binds the monosaccharide in a “wildtype”-like fashion, showing that only site IIIC is involved in sugar binding.
Mentions: Purified proteins were prepared to further quantify these effects. Far UV CD spectra of these mutant forms are identical to that of native protein LlpABW, indicating that the mutations do not affect the overall structure of the protein. Isothermal titration calorimetry (ITC) showed that LlpABW has an affinity of 2.1 mM for the pentasaccharide GlcNAcβ(1–2)Manα(1–3)[GlcNAcβ(1–2)Manα(1–6)]Man, the highest among all the tested oligo-mannosides (See Figure 5 and Table 1 for a summary of the experimentally validated LlpABW-carbohydrate interactions). This is in agreement with the crystal structures of the different complexes since this sugar is the one with the largest binding interface (Figure 3). Titrations of LlpABW, of the mutants LlpAV177Y (a site IIIC knockout), LlpAV208Y (a site IIC knockout) and of the double mutant LlpAV177Y-V208Y with α-methyl mannoside clearly pinpoint site IIIC as the only responsible for the sugar binding activity. Point mutations in both sites or IIIC (V177Y) alone, completely abrogate sugar binding. However knocking out site IIC (V208Y) has little effect in binding and the affinities of LlpAV208Y for α-methyl mannoside and Manα(1–3)Man are very close to the ones measured for the wild-type protein (See Table 1 and Figure 5B).

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