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Allosteric signalling in the outer membrane translocation domain of PapC usher.

Farabella I, Pham T, Henderson NS, Geibel S, Phan G, Thanassi DG, Delcour AH, Waksman G, Topf M - Elife (2014)

Bottom Line: Their translocation domain is a large β-barrel occluded by a plug domain, which is displaced to allow the translocation of pilus subunits across the membrane.To investigate the role of these elements in allosteric signal communication, we developed a method combining evolutionary and molecular dynamics studies of the native translocation domain and mutants lacking the β-hairpin and/or the α-helix.This study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability.

View Article: PubMed Central - PubMed

Affiliation: Institute of Structural and Molecular Biology, Birkbeck College and University College London, London, United Kingdom.

ABSTRACT
PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili in uropathogenic E. coli. Their translocation domain is a large β-barrel occluded by a plug domain, which is displaced to allow the translocation of pilus subunits across the membrane. Previous studies suggested that this gating mechanism is controlled by a β-hairpin and an α-helix. To investigate the role of these elements in allosteric signal communication, we developed a method combining evolutionary and molecular dynamics studies of the native translocation domain and mutants lacking the β-hairpin and/or the α-helix. Analysis of a hybrid residue interaction network suggests distinct regions (residue 'communities') within the translocation domain (especially around β12-β14) linking these elements, thereby modulating PapC gating. Antibiotic sensitivity and electrophysiology experiments on a set of alanine-substitution mutants confirmed functional roles for four of these communities. This study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability.

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Combined RIN of the difference in non-covalent interaction score (ΔNCI score).RINs showing the difference in non-covalent interaction score (ΔNCI score) between the native TD system and the hairpin mutant (A), helix mutant (B), and helix-hairpin mutant (C). The combined RIN (D) was created by merging the three RINs. The nodes (representing residues) are coloured as in Figure 2A. Edges (connecting two residues) are shown in blue, with edge width proportional to its corresponding ΔNCI score (from lower to higher values).DOI:http://dx.doi.org/10.7554/eLife.03532.008
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fig2s2: Combined RIN of the difference in non-covalent interaction score (ΔNCI score).RINs showing the difference in non-covalent interaction score (ΔNCI score) between the native TD system and the hairpin mutant (A), helix mutant (B), and helix-hairpin mutant (C). The combined RIN (D) was created by merging the three RINs. The nodes (representing residues) are coloured as in Figure 2A. Edges (connecting two residues) are shown in blue, with edge width proportional to its corresponding ΔNCI score (from lower to higher values).DOI:http://dx.doi.org/10.7554/eLife.03532.008

Mentions: Comparative analysis between the RINs of native and mutants systems revealed slight changes, suggesting a rearrangement in the interaction network. To better understand the mutation-induced changes in network components, we calculated the difference in non-covalent interaction score (ΔNCI score) between the native TD system and each of the mutant systems (the weakened interactions are shown in Figure 2—figure supplement 2A–C). This information was then added as a weighted undirected edge to the pre-existing native non-covalent RIN (the ΔNCI edges are shown in Figure 2—figure supplement 2D). Interestingly, 24% of the strong interactions in the native RIN were weakened relative to the RIN of the mutant lacking the β-hairpin, 22.6% relative to the mutant lacking the α-helix, and 23.3% relative to the mutant lacking both, suggesting that interactions between nodes that are not part of the deleted secondary structure elements were consistently weakened in the absence of these elements.


Allosteric signalling in the outer membrane translocation domain of PapC usher.

Farabella I, Pham T, Henderson NS, Geibel S, Phan G, Thanassi DG, Delcour AH, Waksman G, Topf M - Elife (2014)

Combined RIN of the difference in non-covalent interaction score (ΔNCI score).RINs showing the difference in non-covalent interaction score (ΔNCI score) between the native TD system and the hairpin mutant (A), helix mutant (B), and helix-hairpin mutant (C). The combined RIN (D) was created by merging the three RINs. The nodes (representing residues) are coloured as in Figure 2A. Edges (connecting two residues) are shown in blue, with edge width proportional to its corresponding ΔNCI score (from lower to higher values).DOI:http://dx.doi.org/10.7554/eLife.03532.008
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356140&req=5

fig2s2: Combined RIN of the difference in non-covalent interaction score (ΔNCI score).RINs showing the difference in non-covalent interaction score (ΔNCI score) between the native TD system and the hairpin mutant (A), helix mutant (B), and helix-hairpin mutant (C). The combined RIN (D) was created by merging the three RINs. The nodes (representing residues) are coloured as in Figure 2A. Edges (connecting two residues) are shown in blue, with edge width proportional to its corresponding ΔNCI score (from lower to higher values).DOI:http://dx.doi.org/10.7554/eLife.03532.008
Mentions: Comparative analysis between the RINs of native and mutants systems revealed slight changes, suggesting a rearrangement in the interaction network. To better understand the mutation-induced changes in network components, we calculated the difference in non-covalent interaction score (ΔNCI score) between the native TD system and each of the mutant systems (the weakened interactions are shown in Figure 2—figure supplement 2A–C). This information was then added as a weighted undirected edge to the pre-existing native non-covalent RIN (the ΔNCI edges are shown in Figure 2—figure supplement 2D). Interestingly, 24% of the strong interactions in the native RIN were weakened relative to the RIN of the mutant lacking the β-hairpin, 22.6% relative to the mutant lacking the α-helix, and 23.3% relative to the mutant lacking both, suggesting that interactions between nodes that are not part of the deleted secondary structure elements were consistently weakened in the absence of these elements.

Bottom Line: Their translocation domain is a large β-barrel occluded by a plug domain, which is displaced to allow the translocation of pilus subunits across the membrane.To investigate the role of these elements in allosteric signal communication, we developed a method combining evolutionary and molecular dynamics studies of the native translocation domain and mutants lacking the β-hairpin and/or the α-helix.This study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability.

View Article: PubMed Central - PubMed

Affiliation: Institute of Structural and Molecular Biology, Birkbeck College and University College London, London, United Kingdom.

ABSTRACT
PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili in uropathogenic E. coli. Their translocation domain is a large β-barrel occluded by a plug domain, which is displaced to allow the translocation of pilus subunits across the membrane. Previous studies suggested that this gating mechanism is controlled by a β-hairpin and an α-helix. To investigate the role of these elements in allosteric signal communication, we developed a method combining evolutionary and molecular dynamics studies of the native translocation domain and mutants lacking the β-hairpin and/or the α-helix. Analysis of a hybrid residue interaction network suggests distinct regions (residue 'communities') within the translocation domain (especially around β12-β14) linking these elements, thereby modulating PapC gating. Antibiotic sensitivity and electrophysiology experiments on a set of alanine-substitution mutants confirmed functional roles for four of these communities. This study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability.

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