Structural and energetic basis of folded-protein transport by the FimD usher - PubMed (original) (raw)

Structural and energetic basis of folded-protein transport by the FimD usher

Sebastian Geibel et al. Nature. 2013.

Abstract

Type 1 pili, produced by uropathogenic Escherichia coli, are multisubunit fibres crucial in recognition of and adhesion to host tissues. During pilus biogenesis, subunits are recruited to an outer membrane assembly platform, the FimD usher, which catalyses their polymerization and mediates pilus secretion. The recent determination of the crystal structure of an initiation complex provided insight into the initiation step of pilus biogenesis resulting in pore activation, but very little is known about the elongation steps that follow. Here, to address this question, we determine the structure of an elongation complex in which the tip complex assembly composed of FimC, FimF, FimG and FimH passes through FimD. This structure demonstrates the conformational changes required to prevent backsliding of the nascent pilus through the FimD pore and also reveals unexpected properties of the usher pore. We show that the circular binding interface between the pore lumen and the folded substrate participates in transport by defining a low-energy pathway along which the nascent pilus polymer is guided during secretion.

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Figures

Figure 1

Figure 1. Structure of the FimD:FimC:FimF:FimG:FimH complex

A) Crystal structure FimD:FimC:FimF:FimG:FimH. All proteins are in ribbon representation; FimD NTD, β-barrel, plug domain, CTD1 and CTD2 in blue, slate, magenta, cyan and purple, respectively; FimC, FimF, FimG and FimH in yellow, red, orange, and green, respectively. B) Recruitment of the next chaperone:subunit FimC:FimA complex in assembly by the FimD:FimC:FimF:FimG:FimH complex. FimD is in cartoon representation, coloured as in A. Chaperone FimC (yellow), pilus subunits FimF (red), FimG (orange) and adhesin FimH (green) are in sphere representation. Recruited chaperone:subunit complex FimC’:FimA (PDB ID 4DWH; pale yellow cartoon and purple spheres, respectively) is modelled at the NTD based on the crystal structure of the isolated FimD NTD domain bound to FimC:FimF (PDB ID 3BWU; see Supplementary Methods for details).

Figure 2

Figure 2. Comparison of the structure of FimH before and after translocation

A) Superposition of the structure of FimH from the FimD:FimC:FimH initiation complex (in dark green) with that of FimH from the elongation FimD:FimC:FimF:FimG:FimH complex (same color coding as in Figure 1, panel A). FimHp was used for the superposition. B) Superposition of the structure of FimHL from the initiation complex (dark green) with that of the same domain from the elongation complex (light green).

Figure 3

Figure 3. Steep energy funnels and opposing binding surfaces position the translocating substrate at the centre of the pore

A) The heat map is equivalent to looking down the pore axis, with the energy increase plotted as FimG is laterally translated (i.e. perpendicular to the pore axis) along a finely-spaced grid within the usher lumen. FimG located at any position on the grid will experience a force dependent on the slope of the potential energy well, returning FimG to its central ground state. REU, Rosetta Energy Units. B) As in (A), with FimHL now inside the usher lumen. C) Subunits/domains occupying the pore were randomly rotated within ± 8° about their geometric centres and translated up to 6 Å parallel to the pore axis (for the plug domain, displacement was only in the direction of the periplasm to mimic plug extrusion during activation). Calculated energies are plotted against the rmsd from the respective minimized crystal structures. 8000 perturbations were made for each subunit, with the outline of the lowest energy conformations shown. FimG, orange; FimHL, green; plug, magenta. D) Plot of binding energy as a 60°-sector window emanating from the pore axis is rotated around the respective structures. FimG, orange. FimHL, green.

Figure 4

Figure 4. A low energy pathway through the pore lumen facilitates translocation of subunits and their transfer from NTD to CTDs

A) Starting from the native crystal structure with FimG and its complementing strand from FimF inside the FimD pore (subunits FimH, FimF and FimC were not considered), FimG was translated by 1 Å steps up to 40 Å out of the pore, and up to 20 Å back towards the periplasm, along the pore axis. At each translational step, FimG is rotated around the pore axis by 2° increments. Each sampled FimG rotation-translation conformation was minimized after rotamer repacking to resolve small clashes, and the calculated potential energy is plotted against the initial perturbation. Lower, favourable energies are hotter colours. B) The energy landscape calculated in panel A is modified by addition of a torsional spring potential (E = ½.k.Δθ2; represented by the coil with spring constant k twisted around the pore axis in the overlaid schematic at top-left) to derive a connected trajectory, with Δθ the angle from the lowest energy FimG conformation prior to each 1 Å step, and starting with FimG positioned at 0 Å, 0°.

References

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