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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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Related in: MedlinePlus

Differential inhibitory activity of wild-type LlpABW and LlpA/LlpA1 domain chimers.The domain structures of LlpABW (as in Figure 4) and of LlpA1 (inferred by pairwise alignment; N-domain in orange, C-domain in purple and C-terminal extension in grey) are depicted, along with those of chimeric forms (in dashed box). The LlpA variant lacking the terminal phenylalanine residue is marked with a yellow hexagon. Inhibitory activity of the respective E. coli recombinants was tested with diagnostic indicators for LlpABW (P. syringae GR12-2R3) and LlpA1 (P. fluorescens LMG 1794). Halo sizes are semi-quantified according to size of the growth inhibition halo (+++, native halo size of LlpABW and LlpA1; ++, halo size reduced; C, local clearing confined to producer colony spot; −, no halo or clearing; NT, not tested). Additional chimeric and domain deletion constructs not conferring bacteriocin activity against one of the indicator strains are specified in Figure S11.
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ppat-1003199-g007: Differential inhibitory activity of wild-type LlpABW and LlpA/LlpA1 domain chimers.The domain structures of LlpABW (as in Figure 4) and of LlpA1 (inferred by pairwise alignment; N-domain in orange, C-domain in purple and C-terminal extension in grey) are depicted, along with those of chimeric forms (in dashed box). The LlpA variant lacking the terminal phenylalanine residue is marked with a yellow hexagon. Inhibitory activity of the respective E. coli recombinants was tested with diagnostic indicators for LlpABW (P. syringae GR12-2R3) and LlpA1 (P. fluorescens LMG 1794). Halo sizes are semi-quantified according to size of the growth inhibition halo (+++, native halo size of LlpABW and LlpA1; ++, halo size reduced; C, local clearing confined to producer colony spot; −, no halo or clearing; NT, not tested). Additional chimeric and domain deletion constructs not conferring bacteriocin activity against one of the indicator strains are specified in Figure S11.

Mentions: None of these deletion constructs resulted in the production of an active protein, indicating that none of the domains are dispensable. Removal of the terminal phenylalanine residue still allows expression of a functional bacteriocin in E. coli (Figure 7), but a further C-terminal truncation (deletion of Trp-His-Phe tail) resulted in a negative bacteriocin assay (data not shown). From these data we conclude that both MMBL domains as well as the C-terminal hairpin extension are required for activity of LlpA. Whether the role of the C-terminal hairpin is any other than simply stabilization of the C-domain cannot be concluded.


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)

Differential inhibitory activity of wild-type LlpABW and LlpA/LlpA1 domain chimers.The domain structures of LlpABW (as in Figure 4) and of LlpA1 (inferred by pairwise alignment; N-domain in orange, C-domain in purple and C-terminal extension in grey) are depicted, along with those of chimeric forms (in dashed box). The LlpA variant lacking the terminal phenylalanine residue is marked with a yellow hexagon. Inhibitory activity of the respective E. coli recombinants was tested with diagnostic indicators for LlpABW (P. syringae GR12-2R3) and LlpA1 (P. fluorescens LMG 1794). Halo sizes are semi-quantified according to size of the growth inhibition halo (+++, native halo size of LlpABW and LlpA1; ++, halo size reduced; C, local clearing confined to producer colony spot; −, no halo or clearing; NT, not tested). Additional chimeric and domain deletion constructs not conferring bacteriocin activity against one of the indicator strains are specified in Figure S11.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003199-g007: Differential inhibitory activity of wild-type LlpABW and LlpA/LlpA1 domain chimers.The domain structures of LlpABW (as in Figure 4) and of LlpA1 (inferred by pairwise alignment; N-domain in orange, C-domain in purple and C-terminal extension in grey) are depicted, along with those of chimeric forms (in dashed box). The LlpA variant lacking the terminal phenylalanine residue is marked with a yellow hexagon. Inhibitory activity of the respective E. coli recombinants was tested with diagnostic indicators for LlpABW (P. syringae GR12-2R3) and LlpA1 (P. fluorescens LMG 1794). Halo sizes are semi-quantified according to size of the growth inhibition halo (+++, native halo size of LlpABW and LlpA1; ++, halo size reduced; C, local clearing confined to producer colony spot; −, no halo or clearing; NT, not tested). Additional chimeric and domain deletion constructs not conferring bacteriocin activity against one of the indicator strains are specified in Figure S11.
Mentions: None of these deletion constructs resulted in the production of an active protein, indicating that none of the domains are dispensable. Removal of the terminal phenylalanine residue still allows expression of a functional bacteriocin in E. coli (Figure 7), but a further C-terminal truncation (deletion of Trp-His-Phe tail) resulted in a negative bacteriocin assay (data not shown). From these data we conclude that both MMBL domains as well as the C-terminal hairpin extension are required for activity of LlpA. Whether the role of the C-terminal hairpin is any other than simply stabilization of the C-domain cannot be concluded.

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