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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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Inhibitory activity of wild-type LlpABW and selected mutants with modified (potential) mannose-binding sites.The domain structure (N-domain in red, C-domain in blue and C-terminal extension in green) and the position of the MMBL motifs (potentially active binding sites in orange, inactive ones in grey) are shown. The positions of conserved valine residues converted to tyrosine residues by site-directed mutagenesis are indicated with a black bar. Inhibitory activity of E. coli strains expressing mutant LlpABW forms was assayed against P. syringae GR12-2R3 and semi-quantified according to the size (inner zone radius) of the growth inhibition halo relative to LlpABW (+++, native LlpABW; ++, halo size reduced; + halo size strongly reduced; −, no halo; NT, not tested). For wild-type LlpABW and three purified His-tagged mutant forms (LlpAV177Y, LlpAV208Y and LlpAV177Y-V208Y) the MIC values were determined with indicator P. syringae GR12-2R3. Molar minimal inhibitory concentrations of recombinant proteins (with standard deviations): LlpA, 2.08 nM (±0.58 nM); LlpAV177Y, 10.9 nM (±0.66 nM); LlpAV208Y, 1.98 nM (±0.066 nM); 65.72 nM (±2.80 nM).
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ppat-1003199-g004: Inhibitory activity of wild-type LlpABW and selected mutants with modified (potential) mannose-binding sites.The domain structure (N-domain in red, C-domain in blue and C-terminal extension in green) and the position of the MMBL motifs (potentially active binding sites in orange, inactive ones in grey) are shown. The positions of conserved valine residues converted to tyrosine residues by site-directed mutagenesis are indicated with a black bar. Inhibitory activity of E. coli strains expressing mutant LlpABW forms was assayed against P. syringae GR12-2R3 and semi-quantified according to the size (inner zone radius) of the growth inhibition halo relative to LlpABW (+++, native LlpABW; ++, halo size reduced; + halo size strongly reduced; −, no halo; NT, not tested). For wild-type LlpABW and three purified His-tagged mutant forms (LlpAV177Y, LlpAV208Y and LlpAV177Y-V208Y) the MIC values were determined with indicator P. syringae GR12-2R3. Molar minimal inhibitory concentrations of recombinant proteins (with standard deviations): LlpA, 2.08 nM (±0.58 nM); LlpAV177Y, 10.9 nM (±0.66 nM); LlpAV208Y, 1.98 nM (±0.066 nM); 65.72 nM (±2.80 nM).

Mentions: To assess whether the mannose-recognizing QxDxNxVxY motifs in LlpABW are nevertheless relevant for bactericidal activity, the conserved valine residue was mutated to tyrosine in subdomains IIIN, IIIC, and IIC. These mutations sterically preclude mannose or any other ligand to enter the binding sites (Figure S7C). Semi-quantitative activity assays with permeabilized E. coli cells expressing the LlpA variants in motifs IIIN, IIIC and IIC were used to assess the relationship between carbohydrate binding and bactericidal activity. Modification of the IIIN site, for which no mannose binding was observed, does not affect the antibacterial activity against P. syringae GR12-2R3 (Figure 4). In contrast, the altered IIIC pocket strongly diminishes activity, either alone or in pairwise combination with the other mutated sites (IIIN or IIC). A minor negative effect of the IIC mutation is only apparent in a double mutant, when combined with a modified IIIN motif.


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)

Inhibitory activity of wild-type LlpABW and selected mutants with modified (potential) mannose-binding sites.The domain structure (N-domain in red, C-domain in blue and C-terminal extension in green) and the position of the MMBL motifs (potentially active binding sites in orange, inactive ones in grey) are shown. The positions of conserved valine residues converted to tyrosine residues by site-directed mutagenesis are indicated with a black bar. Inhibitory activity of E. coli strains expressing mutant LlpABW forms was assayed against P. syringae GR12-2R3 and semi-quantified according to the size (inner zone radius) of the growth inhibition halo relative to LlpABW (+++, native LlpABW; ++, halo size reduced; + halo size strongly reduced; −, no halo; NT, not tested). For wild-type LlpABW and three purified His-tagged mutant forms (LlpAV177Y, LlpAV208Y and LlpAV177Y-V208Y) the MIC values were determined with indicator P. syringae GR12-2R3. Molar minimal inhibitory concentrations of recombinant proteins (with standard deviations): LlpA, 2.08 nM (±0.58 nM); LlpAV177Y, 10.9 nM (±0.66 nM); LlpAV208Y, 1.98 nM (±0.066 nM); 65.72 nM (±2.80 nM).
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Related In: Results  -  Collection

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ppat-1003199-g004: Inhibitory activity of wild-type LlpABW and selected mutants with modified (potential) mannose-binding sites.The domain structure (N-domain in red, C-domain in blue and C-terminal extension in green) and the position of the MMBL motifs (potentially active binding sites in orange, inactive ones in grey) are shown. The positions of conserved valine residues converted to tyrosine residues by site-directed mutagenesis are indicated with a black bar. Inhibitory activity of E. coli strains expressing mutant LlpABW forms was assayed against P. syringae GR12-2R3 and semi-quantified according to the size (inner zone radius) of the growth inhibition halo relative to LlpABW (+++, native LlpABW; ++, halo size reduced; + halo size strongly reduced; −, no halo; NT, not tested). For wild-type LlpABW and three purified His-tagged mutant forms (LlpAV177Y, LlpAV208Y and LlpAV177Y-V208Y) the MIC values were determined with indicator P. syringae GR12-2R3. Molar minimal inhibitory concentrations of recombinant proteins (with standard deviations): LlpA, 2.08 nM (±0.58 nM); LlpAV177Y, 10.9 nM (±0.66 nM); LlpAV208Y, 1.98 nM (±0.066 nM); 65.72 nM (±2.80 nM).
Mentions: To assess whether the mannose-recognizing QxDxNxVxY motifs in LlpABW are nevertheless relevant for bactericidal activity, the conserved valine residue was mutated to tyrosine in subdomains IIIN, IIIC, and IIC. These mutations sterically preclude mannose or any other ligand to enter the binding sites (Figure S7C). Semi-quantitative activity assays with permeabilized E. coli cells expressing the LlpA variants in motifs IIIN, IIIC and IIC were used to assess the relationship between carbohydrate binding and bactericidal activity. Modification of the IIIN site, for which no mannose binding was observed, does not affect the antibacterial activity against P. syringae GR12-2R3 (Figure 4). In contrast, the altered IIIC pocket strongly diminishes activity, either alone or in pairwise combination with the other mutated sites (IIIN or IIC). A minor negative effect of the IIC mutation is only apparent in a double mutant, when combined with a modified IIIN motif.

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