Limits...
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.

Show MeSH

Related in: MedlinePlus

Killing activity of LlpABW and mutant proteins.Percentages of dead cells after live/dead staining as quantified by flow cytometry analysis (Figure S10). P. syringae GR12-2R3 was used as indicator strain and treated at a final concentration of 50 µg/ml for 1 h. Average values (with standard deviations; indicated by error bars): LlpA, 10.1 (±1.04); LlpAV177Y, 6.1 (±0.44); LlpAV208Y, 9.7 (±1.39); LlpAV177Y-V208Y, 3.7 (±0.90); buffer (control), 1.0 (±0.11).Values are significantly different for (a) and (b), (b) and (c) (p<0.01).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585409&req=5

ppat-1003199-g006: Killing activity of LlpABW and mutant proteins.Percentages of dead cells after live/dead staining as quantified by flow cytometry analysis (Figure S10). P. syringae GR12-2R3 was used as indicator strain and treated at a final concentration of 50 µg/ml for 1 h. Average values (with standard deviations; indicated by error bars): LlpA, 10.1 (±1.04); LlpAV177Y, 6.1 (±0.44); LlpAV208Y, 9.7 (±1.39); LlpAV177Y-V208Y, 3.7 (±0.90); buffer (control), 1.0 (±0.11).Values are significantly different for (a) and (b), (b) and (c) (p<0.01).

Mentions: While the V208Y mutation in the IIC site has no observable effect on the MIC value for P. syringae GR12-2R3, the altered IIIC motif engenders a 5.2-fold increase in MIC (Figure 4). The mutant protein LlpAV177Y-V208Y suffers a further reduction in activity, yielding a 31.6-fold increased MIC compared to native LlpABW. The biological activities of LlpA and its mutants were further assessed by live/dead staining and subsequent flow cytometry analysis (Figure 6, Figure S10). Proportions of dead cells after 1 hour of exposure to LlpA or LlpAV208Y were comparable (10.1% and 9.7%, respectively). For LlpAV177Y this value was reduced to 6.1%, significantly lower than for LlpA. Killing activity was even further reduced for LlpAV177Y-V208Y (3.7%). These results are consistent with the MIC determination and ITC data, indicating that an active site IIIC is required to generate a fully active LlpA bacteriocin. The difference in bacteriotoxicity between LlpAV177Y and LlpAV177Y-V208Y suggests that site IIC has a supporting role in the LlpABW bacteriotoxicity.


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)

Killing activity of LlpABW and mutant proteins.Percentages of dead cells after live/dead staining as quantified by flow cytometry analysis (Figure S10). P. syringae GR12-2R3 was used as indicator strain and treated at a final concentration of 50 µg/ml for 1 h. Average values (with standard deviations; indicated by error bars): LlpA, 10.1 (±1.04); LlpAV177Y, 6.1 (±0.44); LlpAV208Y, 9.7 (±1.39); LlpAV177Y-V208Y, 3.7 (±0.90); buffer (control), 1.0 (±0.11).Values are significantly different for (a) and (b), (b) and (c) (p<0.01).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003199-g006: Killing activity of LlpABW and mutant proteins.Percentages of dead cells after live/dead staining as quantified by flow cytometry analysis (Figure S10). P. syringae GR12-2R3 was used as indicator strain and treated at a final concentration of 50 µg/ml for 1 h. Average values (with standard deviations; indicated by error bars): LlpA, 10.1 (±1.04); LlpAV177Y, 6.1 (±0.44); LlpAV208Y, 9.7 (±1.39); LlpAV177Y-V208Y, 3.7 (±0.90); buffer (control), 1.0 (±0.11).Values are significantly different for (a) and (b), (b) and (c) (p<0.01).
Mentions: While the V208Y mutation in the IIC site has no observable effect on the MIC value for P. syringae GR12-2R3, the altered IIIC motif engenders a 5.2-fold increase in MIC (Figure 4). The mutant protein LlpAV177Y-V208Y suffers a further reduction in activity, yielding a 31.6-fold increased MIC compared to native LlpABW. The biological activities of LlpA and its mutants were further assessed by live/dead staining and subsequent flow cytometry analysis (Figure 6, Figure S10). Proportions of dead cells after 1 hour of exposure to LlpA or LlpAV208Y were comparable (10.1% and 9.7%, respectively). For LlpAV177Y this value was reduced to 6.1%, significantly lower than for LlpA. Killing activity was even further reduced for LlpAV177Y-V208Y (3.7%). These results are consistent with the MIC determination and ITC data, indicating that an active site IIIC is required to generate a fully active LlpA bacteriocin. The difference in bacteriotoxicity between LlpAV177Y and LlpAV177Y-V208Y suggests that site IIC has a supporting role in the LlpABW bacteriotoxicity.

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