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The pilus usher controls protein interactions via domain masking and is functional as an oligomer

Nature Structural & Molecular Biology volume 22, pages 540–546 (2015)Cite this article

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Abstract

The chaperone-usher (CU) pathway assembles organelles termed pili or fimbriae in Gram-negative bacteria. Type 1 pili expressed by uropathogenic Escherichia coli are prototypical structures assembled by the CU pathway. Biogenesis of pili by the CU pathway requires a periplasmic chaperone and an outer-membrane protein termed the usher (FimD). We show that the FimD C-terminal domains provide the high-affinity substrate-binding site but that these domains are masked in the resting usher. Domain masking requires the FimD plug domain, which serves as a switch controlling usher activation. We demonstrate that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer. These results reveal mechanisms by which molecular machines such as the usher regulate and harness protein-protein interactions and suggest that ushers may interact in a cooperative manner during pilus assembly in bacteria.

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Figure 1: Models for type 1–pilus biogenesis and usher domain architecture.
Figure 2: In vivo detection of FimC–FimH binding to the FimD usher.
Figure 3: Binding affinities of FimC–FimH chaperone–adhesin complexes for WT and domain-deleted FimD ushers.
Figure 4: FimC–FimH binds to the N, C1 and C2 domains of the FimDΔplug usher in vivo.
Figure 5: Assembly of type 1–pilus tip fibers by the WT and FimDΔplug ushers.
Figure 6: Coexpression of FimDΔNΔplug and FimDΔC1ΔC2 ushers results in pilus assembly on the bacterial surface.

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Acknowledgements

We thank the Schultz laboratory (Scripps Research Institute) for providing plasmid pEVOL-pBpF. We thank S. Van Horn of the Stony Brook University Central Microscopy Imaging Center and V. Sampath (Stony Brook University) for assistance with EM. We thank J. Haley, D. Martin and R. Rieger of the Stony Brook Proteomics Center for performing the mass spectrometry analysis and for helpful discussions. We thank S. Scarlata (Stony Brook University), A.W. Karzai (Stony Brook University), K.W. Dodson (Washington University) and A.H. Delcour (University of Houston) for helpful discussions and critical reading of the manuscript. This study was supported by US National Institutes of Health (NIH) grants R01GM062987 (to D.G.T. and H.L.) and R01AI029549 (to S.J.H.). G.T.W. was supported by Medical Scientist Training Program award T32GM008444 and National Research Service Award F30AI112252 from the NIH. The Stony Brook Proteomics Center receives support from NIH award S10RR023680.

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Authors and Affiliations

  1. Center for Infectious Diseases, Stony Brook University, Stony Brook, New York, USA

    Glenn T Werneburg, Nadine S Henderson, Erica B Portnoy & David G Thanassi

  2. Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, USA

    Glenn T Werneburg, Nadine S Henderson, Erica B Portnoy & David G Thanassi

  3. Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA

    Samema Sarowar & Huilin Li

  4. Biosciences Department, Brookhaven National Laboratory, Upton, New York, USA

    Samema Sarowar & Huilin Li

  5. Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, Missouri, USA

    Scott J Hultgren

  6. Center for Women's Infectious Disease Research, Washington University School of Medicine, Saint Louis, Missouri, USA

    Scott J Hultgren

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Contributions

G.T.W., N.S.H., S.J.H., H.L. and D.G.T. designed the experiments. G.T.W., N.S.H., E.B.P. and S.S. performed the experiments. All authors were involved in data interpretation and discussion. G.T.W. and D.G.T. wrote the manuscript with contributions from all other authors.

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Correspondence to David G Thanassi.

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Integrated supplementary information

Supplementary Figure 1 Structures depicting chaperone-subunit and subunit-subunit interactions, and the different states of the FimD usher.

(a) Structure of a FimC-FimH complex (PDB ID: 1QUN) showing donor strand complementation (DSC) between the chaperone and subunit. The FimC chaperone is in yellow and the FimH adhesin in green, with the FimH adhesin and pilin domains indicated. The chaperone donates its G1 ß-strand (in blue) to complete the Ig fold of the FimH pilin domain. (b) Structure of a FimG-FimH complex (taken from PDB ID: 4J3O) showing donor strand exchange (DSE) between the subunits. FimH is depicted as in a and FimG is in orange. The N-terminal extension (Nte) of FimG completes the Ig fold of the FimH pilin domain. (c) Structure of the apo-FimD usher (PDB ID: 3OHN). The ß-barrel channel domain is in light blue and the plug domain is in pink. The plug domain is positioned laterally within the ß-barrel domain, closing the usher channel. The N, C1, and C2 domains are not present in this structure. (d) Structure of the activated FimD usher (taken from PDB ID: 3RFZ). The channel and plug domains are depicted as in c, the N domain is in dark blue, the C1 domain is in cyan, and the C2 domain is in purple. In the activated usher, the plug domain resides in the periplasm, adjacent to the N domain.

Supplementary Figure 2 In vivo detection of FimC–FimH binding to the FimD usher.

(a,b,c) Immunoblot analysis of in vivo, site-directed photocrosslinking. (a) Samples are purified His-tagged WT FimD or FimD Phe4, Phe8, Gln109, Asn670, Tyr704, Thr717, Asn718, Leu754, or Tyr788 amber mutants and associated crosslinked products, analyzed with anti-FimC-FimH antibody. The position of the FimD monomer is indicated on the right. The asterisks (*) mark FimD crosslinked products. (b) Same as in a, except showing only FimD Phe4, Tyr704, Thr717, and Tyr788 amber mutants, and samples were analyzed with anti-His-tag antibody. The anti-His-tag antibody reacts with FimD only, confirming the presence of the usher in the crosslinked products (*). (c) Samples are as in b, except FimD contained a Strep-tag instead of a His-tag, and OM fractions were probed with anti-FimC-FimH antibody. The position of the FimD monomer is indicated on the right. The anti-FimC-FimH antibody does not cross-react with the strep-tagged FimD usher, confirming the presence of the chaperone or adhesin in the crosslinked products (*).

Supplementary Figure 3 Positions of the FimD amber mutations used for site-directed photo-cross-linking and predicted interactions with FimC and FimH.

Structures of the FimD N domain bound to a FimC-FimH pilin domain complex (a), and the FimD-FimC-FimH complex (b). The structures and colors are as in Figure 2. The FimD amber substitution sites Phe4 (N domain), Tyr704 (C1 domain), Thr717 (C1 domain), and Tyr788 (C2 domain) are depicted in red in stick representation. On the right are magnifications of the boxed areas, showing details of the interactions of the FimD residues with FimC or FimH.

Supplementary Figure 4 Positions of the FimC cysteine substitutions used for fluorescence labeling and predicted interactions with the FimD N and C domains.

Crystal structures of the FimD N domain bound to a FimC-FimH pilin domain complex (a), and the FimD-FimC-FimH complex (b). The structures and colors are as in Figure 3. The FimC cysteine substitution sites Gln19, Thr51, and Asn86 are shown in red in stick representation. On the right are magnifications of the boxed areas, showing details of the interface between the FimC residues and the FimD N domain (a) or C domains (b).

Supplementary Figure 5 Binding affinities of FimC–FimH for WT and domain-deleted FimD ushers.

(a–g) The graphs represent normalized changes in total fluorescence emission intensity plotted as a function of FimD concentration. The data points represent means ± SEM of at least three independent experiments, with three replicates per experiment. Apparent Kd values are indicated for each graph. (a,b) FimC-FimH was fluorescently labeled at FimC positions T51C (a) or N86C (b) with coumarin maleimide, and fluorescence spectra were recorded as WT FimD was titrated into the cuvettes. The total change in fluorescence intensity was 28% for both a and b. (c–g) FimC-FimH was fluorescently labeled at FimC position Q19C with coumarin maleimide and fluorescence spectra were recorded as FimD∆C2 (c), FimD∆C1∆C2 (d), FimD∆plug (e), FimD∆N (f), or FimD∆N∆plug (g) was titrated into the cuvettes. The total changes in fluorescence intensity for c-e and g ranged from 17 to 78%. The total change in fluorescence intensity for f was 427%.

Supplementary Figure 6 Proposed model for usher-chaperone-subunit interactions during pilus biogenesis at an oligomeric FimD usher.

FimD is depicted as a dimeric complex, with the usher domains colored as in Figure 1. In the resting state (a), the plug domain resides laterally within the usher channel. The plug closes the usher channel and also functions to mask the C domains, thus keeping the C domains inaccessible to off-target chaperone-subunit interactions. The N domains of the resting usher oligomer are accessible and able to sample the periplasm for chaperone-subunit complexes. Binding of a FimC-FimH chaperone-adhesin complex to the N domain of one FimD protomer activates that usher by triggering opening of the plug domain and unmasking of the C domains (b). FimC-FimH then undergoes affinity-driven handoff from the N to the C domains, concomitant with insertion of the FimH adhesin domain into the usher channel (c). The channel of the non-translocating FimD protomer remains closed, but the N domains of both ushers function to recruit additional chaperone-subunit complexes from the periplasm (FimC-FimG and FimC-FimF). As pictured, the FimC-FimG complex bound at N domain of the translocating usher is perfectly positioned to undergo DSE with FimC-FimH bound at the C domains, forming the first link in the pilus fiber and displacing FimC from FimH (d). FimC-FimG is handed off from the N to the C domains, concomitant with movement of the nascent pilus fiber through the usher channel toward the cell surface. Chaperone-subunit complexes bound at the non-translocating protomer could directly interact with the growing pilus fiber at the C domains of the translocating usher, or could first be transferred to the N domain of the translocating usher (e). Repeated cycles of chaperone-subunit (FimC-FimA) recruitment and DSE then result in assembly and secretion of a complete pilus organelle (f).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1–3 (PDF 781 kb)

Supplementary Data Set 1

Mass spectrometry analysis of FimD cross-linked products (XLSX 51 kb)

Supplementary Data Set 2

Uncropped gel images (PDF 5279 kb)

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Werneburg, G., Henderson, N., Portnoy, E. et al. The pilus usher controls protein interactions via domain masking and is functional as an oligomer. Nat Struct Mol Biol 22, 540–546 (2015). https://doi.org/10.1038/nsmb.3044

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