Handover mechanism of the growing pilus by the bacterial outer-membrane usher FimD

Abstract

Pathogenic bacteria such as Escherichia coli assemble surface structures termed pili, or fimbriae, to mediate binding to host-cell receptors¹. Type 1 pili are assembled via the conserved chaperone-usher pathway^2-5. The outer-membrane usher FimD recruits pilus subunits bound by the chaperone FimC via the periplasmic N-terminal domain of the usher. Subunit translocation through the β-barrel channel of the usher occurs at the two C-terminal domains (which we label CTD1 and CTD2) of this protein. How the chaperone-subunit complex bound to the N-terminal domain is handed over to the C-terminal domains, as well as the timing of subunit polymerization into the growing pilus, have previously been unclear. Here we use cryo-electron microscopy to capture a pilus assembly intermediate (FimD-FimC-FimF-FimG-FimH) in a conformation in which FimD is in the process of handing over the chaperone-bound end of the growing pilus to the C-terminal domains. In this structure, FimF has already polymerized with FimG, and the N-terminal domain of FimD swings over to bind CTD2; the N-terminal domain maintains contact with FimC-FimF, while at the same time permitting access to the C-terminal domains. FimD has an intrinsically disordered N-terminal tail that precedes the N-terminal domain. This N-terminal tail folds into a helical motif upon recruiting the FimC-subunit complex, but reorganizes into a loop to bind CTD2 during handover. Because both the N-terminal and C-terminal domains of FimD are bound to the end of the growing pilus, the structure further suggests a mechanism for stabilizing the assembly intermediate to prevent the pilus fibre diffusing away during the incorporation of thousands of subunits.

Extended Data Figure 1.. Pilus assembly occurs via donor-strand complementation (DSC) and donor-strand exchange (DSE).

This sketch is based on the crystal structure of FimDCFGH (PDB ID 4J3O). DSC: Pilus subunits (FimF in this case) have an immunoglobulin (Ig)-like structure, but with the C-terminal G strand missing. In the periplasm, the chaperone FimC donates its G1 strand to complete the subunit fold, but in a non-canonical parallel orientation with the subunit F strand. DSE: The donor strand of the FimC chaperone in the previous subunit (FimG in this case) is replaced by the N-terminal extension (NTE) of the incoming subunit, FimF, completing the FimG subunit Ig fold in a canonical, anti-parallel orientation.

Extended Data Figure 2.. Cryo-EM of FimD-tip complex.

(a) The gel filtration profile of FimDCFGH complex from a Superdex 200 10/300GL column. (b) Coomassie blue SDS–PAGE gel of the peak fraction showing the presence of all subunits of the purified FimDCFGH complex. Similar sample preparation by gel filtration and SDS-PAGE examination were carried out more than 3 times. (c) A raw cryo-EM micrograph of the purified FimD-tip complexes embedded in vitreous ice. A total of 12,000 such micrographs were recorded. (d) Selected 2D class averages showing the presence of many different views and well resolved structural features. Over 750,000 raw particles contributed to final 2D class averages. (e) 3D classification scheme. Over 1 million raw particles were selected from drift-corrected electron micrographs. 2D and 3D classification resulted in two 3D maps that were of the expected shape and the structure appeared complete, and the other three maps were either partial structures or distorted. Refinement with ~250,370 particles led to the 4.0-Å resolution 3D map of Conformer 1, and ~166,913 particles led to the 5.1-Å resolution 3D map of Conformer 2.

Extended Data Figure 3.. Resolution estimation and selected regions of the 3D EM maps.

(a) Local resolution estimation and the Gold-standard Fourier shell correlation estimation at the 0.143 correlation threshold of the FimD-tip in Conformer 1. (b) The same for Conformer 2. (c) Model fitting in the FimDCFGH density map. Selected densities for FimH-G, FimG-F, FimF, FimD, FimC of Conformer I are shown. Amino acids with clear side-chain densities are indicated.

Extended Data Figure 4.. Comparison between Conformer 1 and Conformer 2 of the FimD-tip complex.

(a) Overlap of FimD-Tip Conformer 1 in color cartoon with that of the FimD-Tip Conformer 2 in gray cartoon. The NTD movement is labeled in the blue box, and the CTD and FimF movement is labeled in the orange box. (b) Comparison of FimF and CTD2 of Comformer 1 with Conformer 2. FimF and CTD2 shift 10 Å and 5 Å, respectively. (c) Comparison of NTD of Comformer 1 with Conformer 2. The NTD shifts laterally by ~30 Å and rotated by ~45°.

Extended Data Figure 5.. Comparison of interaction between FimD plug domain and NTD (a) and between FimD CTDs and FimCF (b) in Conformers 1 and 2 by superimposing the two conformations.

The Plug domain and NTD of Conformer 1 are colored in salmon and orange, respectively, and Plug domain and NTD of Conformer 2 in cyan and magenta, respectively. In the right side, electron densities of regions involved in the interaction in Conformer I are shown in the top panel (FimD plug) and bottom panel (FimD NTD). Some amino acids have clear side-chain densities. (b) Superimposition of FimD_CTDs-FimCF in Conformers 1 (magenta). and 2 (colored as in Fig. 3). (c) Detailed interactions in Site 1. Extensive interactions are present in Conformer 2. Much weaker interactions are present in conformer 1 (between Q17 and T717). (d) Detailed interactions in Site 2 (marked in panel a). In Conformer 2, hydrophobic interactions exist between FimC L54 and FimD F766, and between I780 and A782. Much weaker interactions are present in Conformer 1. (e) The electron densities in regions involved in Sites 1 and 2 interactions between FimC and FimD in Conformer 1. Some amino acids have side-chain densities.

Extended Data Figure 6.. Sequence conservation of the interacting interface at the two extreme termini of the FimD usher.

Residues involved in FimD NTD and FimC interaction are labeled with circles, and residues in FimD NTD and FimD CTD2 interaction are labeled with triangles.

Extended Data Figure 7.. The interactions between FimD NTD and FimCF in Conformer 1 (a) and in modeled Conformer 3 (b).

There are no interactions between the FimD NTD and FimF subunit in either conformer. The interactions between the FimD N-tail and the FimC chaperone are essentially the same in the two conformers.

Figure 1.. The missing link in pilus biogenesis and cryo-EM of FimD-tip complex.

(a) FimD-tip components. LD: Lectin domain. PD: Pilin domain. N-tail: 24 residues preceding the FimD folded NTD. (b) A partial pilus biogenesis sketch. The red arrows and question marks highlight a key unknown step – the NTD-to-CTDs handover of the chaperone-subunit. DsbA catalyzes disulfide bond formation in a nascent subunit, a prerequisite for subunit recognition by FimC . (c,d) The cryo-EM 3D map of Conformer 1 at 4.0 Å resolution (c) and of Conformer 2 at 5.1 Å resolution (d), respectively. Subunits are individually colored as in (a).

Figure 2.. Atomic models of Conformers 1 and 2.

(a,b) Cryo-EM structures of the FimD-tip complex in Conformer 1 (a) and Conformer 2 (b), colored as in Fig. 1a. The dashed orange shape highlights the plug, which contacts the NTD in Conformer 2 but loses contact in Conformer 1 (see Extended Data Fig. 7). (c) Interactions between FimD N-tail and CTD2 in cartoon view. (d) Electron density for the FimD N-tail, with a local resolution of 4 Å. (e) FimD N-tail in cartoon view interacting with FimD CTD2 in surface-charge view, ranging from the blue positive to red negative charges.

Figure 3.. The FimD N-tail adopts three conformations during a subunit-incorporation cycle.

(a) Modeled Conformer 3 in which FimA (wheat) bound to a FimC’ chaperone (yellow) is being recruited by the FimD-tip complex. In the recruitment phase, the FimD N-tail is folded as a helical motif. (b) Comparison of FimD NTD and N-tail in Conformers 1 and 3 by superimposing the FimD β-barrel. (c) A 3-step NTD-to-CTDs handover mechanism of FimC-subunit at the usher, highlighting the three conformations of the FimD N-tail: disordered in Step 1, helical in Step 2, and a loop in Step 3. See text for details.