Crystal structure of the FimD usher bound to its cognate FimC–FimH substrate

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Abstract

Type 1 pili are the archetypal representative of a widespread class of adhesive multisubunit fibres in Gram-negative bacteria. During pilus assembly, subunits dock as chaperone-bound complexes to an usher, which catalyses their polymerization and mediates pilus translocation across the outer membrane. Here we report the crystal structure of the full-length FimD usher bound to the FimC–FimH chaperone–adhesin complex and that of the unbound form of the FimD translocation domain. The FimD–FimC–FimH structure shows FimH inserted inside the FimD 24-stranded β-barrel translocation channel. FimC–FimH is held in place through interactions with the two carboxy-terminal periplasmic domains of FimD, a binding mode confirmed in solution by electron paramagnetic resonance spectroscopy. To accommodate FimH, the usher plug domain is displaced from the barrel lumen to the periplasm, concomitant with a marked conformational change in the β-barrel. The amino-terminal domain of FimD is observed in an ideal position to catalyse incorporation of a newly recruited chaperone–subunit complex. The FimD–FimC–FimH structure provides unique insights into the pilus subunit incorporation cycle, and captures the first view of a protein transporter in the act of secreting its cognate substrate.

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Figure 1: Structure of the FimD–FimC–FimH complex.
Figure 2: Channel conformations in apo and activated (FimC–FimH-engaged) FimD usher.
Figure 3: FimC–FimH interactions with FimD in the FimD–FimC–FimH complex.
Figure 4: Chaperone–subunit incorporation cycle at the FimD usher.

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Acknowledgements

This work was funded by Medical Research Council grant 85602 to G.W., NIH grant GM62987 to D.G.T., NIH grants 49950, 29549 and 48689 to S.J.H., and NIH grant GM74985 and BNL LDRD grant 10-16 to H.L.; H.R. is supported by a VIB Young PI project grant and the Odysseus program of the FWO-Vlaanderen. K.F.P. is supported by a Schrödinger Fellowship from the Austrian Science Fund, project J 2959-N17. We thank the staff of beamlines X25 and X29 at NSLS, the staff of beamline ID23-1 at ESRF, N. Cronin for technical assistance during data collection, and H. Saibil and E. Orlova for comments on the manuscript.

Author information

Author contribution G.P. produced the FimD–FimC–FimH complex, grew the crystals of this complex, collected X-ray crystallographic data, and initiated the determination of the structure by molecular replacement, and participated in the building and refinement of the structure. H.R. produced the FimD–FimC–FimH complex, trained G.P., supervised the work, analysed the structures and wrote the paper. T.W. grew crystals of the FimD translocation domain, collected X-ray crystallographic data, and determined the structure. W.J.A. set up the DSE assay and prepared the samples for EPR. K.F.P. carried out the EPR experiments, which were analysed by K.F.P., M.B.A.K. and C.W.M.K.; A.L. completed the structure determination of the FimD–FimC–FimH complex, built and refined the structure. N.S.H., E.V., J.S.P. and B.F. cloned and purified the translocation domain of FimD, and cloned and analysed the FimD CTD mutants. S.G. participated in the building and refinement of the FimD–FimC–FimH structure and analysed the structure. J.Y. carried out the native mass spectrometry experiments on the FimD–FimC–FimH complex. C.W.M.K supervised the EPR work. H.L., S.J.H. and D.G.T. supervised the work on apo-FimD, analysed the structures, and wrote the paper. G.W. supervised the work on FimD–FimC–FimH, analysed the structures, and wrote the paper.

Correspondence to David G. Thanassi or Gabriel Waksman.

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Additional information

Structure factors and coordinates have been deposited in the Protein Data Bank (entry codes 3RFZ and 3OHN for coordinates and structure factors of the FimD–FimC–FimHcomplex and the translocation domain of FimD, respectively).

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The file contains Supplementary Tables 1-3, Supplementary Figures 1-10 with legends and additional references. (PDF 24878 kb)

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Phan, G., Remaut, H., Wang, T. et al. Crystal structure of the FimD usher bound to its cognate FimC–FimH substrate. Nature 474, 49–53 (2011) doi:10.1038/nature10109

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