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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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MD simulations of the native PapC TD and its mutants.(A) Cutaway view across the membrane plane of the native PapC TD starting model in a POPE/POPG lipid bilayer (sim1, t = 0). Molecular surface of PapC TD is coloured as in Figure 1, the lipids are shown in grey with the lipid head group coloured by element, the water is coloured by element, and the ions (the Na+ in blue and the CL− in yellow) are represented as sphere. The Cα-RMSD values for each system from the starting structure (t = 0) for the native TD (B), the hairpin mutant (C), helix mutant (D), and helix-hairpin mutant (E) are plotted as a function of time.DOI:http://dx.doi.org/10.7554/eLife.03532.004
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fig1s1: MD simulations of the native PapC TD and its mutants.(A) Cutaway view across the membrane plane of the native PapC TD starting model in a POPE/POPG lipid bilayer (sim1, t = 0). Molecular surface of PapC TD is coloured as in Figure 1, the lipids are shown in grey with the lipid head group coloured by element, the water is coloured by element, and the ions (the Na+ in blue and the CL− in yellow) are represented as sphere. The Cα-RMSD values for each system from the starting structure (t = 0) for the native TD (B), the hairpin mutant (C), helix mutant (D), and helix-hairpin mutant (E) are plotted as a function of time.DOI:http://dx.doi.org/10.7554/eLife.03532.004

Mentions: To investigate if the β-hairpin or α-helix (or both) of the TD (residues 146–637 in the full length PapC) have a role in the allosteric communication leading to the displacement of the PD (residues 264–324), we performed four independent MD simulations, corresponding to the PapC TD model (sim1, Table 1) and three mutants embedded in a mixed lipid bilayer (Table 1): (i) where the region corresponding to the hairpin between β5 and β6 (residues 233–240) is deleted (sim2); (ii) where the α-helix between β13 and β14 (residues 447–460) is removed; and where both the regions were removed (sim4). The last 50 ns of simulation were considered for analysis, where the averaged root-mean-square deviation of Cα atoms (Cα-RMSD) from the averaged structures stabilized around 2.00 ± 0.09 Å, 1.80 ± 0.09 Å, 1.86 ± 0.11 Å, and 2.03 ± 0.10 Å, for the native (sim1), hairpin mutant (sim2), helix mutant (sim3), and helix-hairpin mutant (sim4), respectively (Figure 1—figure supplement 1). This timescale, although limited for a full exploration of the structural changes induced by the mutations, was informative in revealing how local structural perturbations may affect allosteric changes leading to the plug displacement in PapC TD.10.7554/eLife.03532.005Table 1.


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)

MD simulations of the native PapC TD and its mutants.(A) Cutaway view across the membrane plane of the native PapC TD starting model in a POPE/POPG lipid bilayer (sim1, t = 0). Molecular surface of PapC TD is coloured as in Figure 1, the lipids are shown in grey with the lipid head group coloured by element, the water is coloured by element, and the ions (the Na+ in blue and the CL− in yellow) are represented as sphere. The Cα-RMSD values for each system from the starting structure (t = 0) for the native TD (B), the hairpin mutant (C), helix mutant (D), and helix-hairpin mutant (E) are plotted as a function of time.DOI:http://dx.doi.org/10.7554/eLife.03532.004
© Copyright Policy
Related In: Results  -  Collection

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

fig1s1: MD simulations of the native PapC TD and its mutants.(A) Cutaway view across the membrane plane of the native PapC TD starting model in a POPE/POPG lipid bilayer (sim1, t = 0). Molecular surface of PapC TD is coloured as in Figure 1, the lipids are shown in grey with the lipid head group coloured by element, the water is coloured by element, and the ions (the Na+ in blue and the CL− in yellow) are represented as sphere. The Cα-RMSD values for each system from the starting structure (t = 0) for the native TD (B), the hairpin mutant (C), helix mutant (D), and helix-hairpin mutant (E) are plotted as a function of time.DOI:http://dx.doi.org/10.7554/eLife.03532.004
Mentions: To investigate if the β-hairpin or α-helix (or both) of the TD (residues 146–637 in the full length PapC) have a role in the allosteric communication leading to the displacement of the PD (residues 264–324), we performed four independent MD simulations, corresponding to the PapC TD model (sim1, Table 1) and three mutants embedded in a mixed lipid bilayer (Table 1): (i) where the region corresponding to the hairpin between β5 and β6 (residues 233–240) is deleted (sim2); (ii) where the α-helix between β13 and β14 (residues 447–460) is removed; and where both the regions were removed (sim4). The last 50 ns of simulation were considered for analysis, where the averaged root-mean-square deviation of Cα atoms (Cα-RMSD) from the averaged structures stabilized around 2.00 ± 0.09 Å, 1.80 ± 0.09 Å, 1.86 ± 0.11 Å, and 2.03 ± 0.10 Å, for the native (sim1), hairpin mutant (sim2), helix mutant (sim3), and helix-hairpin mutant (sim4), respectively (Figure 1—figure supplement 1). This timescale, although limited for a full exploration of the structural changes induced by the mutations, was informative in revealing how local structural perturbations may affect allosteric changes leading to the plug displacement in PapC TD.10.7554/eLife.03532.005Table 1.

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.

Show MeSH