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

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.

Gram-negative pathogens commonly interact with their environment using long, linear, surface-exposed protein appendages called pili. In uropathogenic Escherichia coli, type 1 pili carry at their distal end a dedicated mannose-specific adhesin, FimH, that is responsible for the attachment of bacteria to the bladder epithelium and their subsequent internalization and biofilm-like organization inside the urothelial cells.

Type 1 pili are representative of a large class of non-covalently linked fibres on the surface of gram-negative bacteria, synthesized via the conserved chaperone/usher (CU) pathway[1,2,3]. Type 1 pili are composed of four different subunit types (FimH, FimG, FimF, and FimA). The adhesin FimH and two linker subunits FimG and FimF form a short flexible fibrillar tip that is attached to an extended rigid and helically wound rod of thousands of FimA subunits (Supplementary Fig. 1a)[4-6]. Subunits cross the inner membrane via the SecYEG secretory pathway. In the periplasm, folding and stability of the subunits require formation of a binary complex with the FimC chaperone[7,8]. Chaperone:subunit complexes are then targeted to the outer membrane usher, FimD, which catalyses the ordered polymerization of subunits and enables the translocation of the growing fibre across the outer membrane in a self-energized process[9,10].

All pilus subunits (or pilins) exhibit an incomplete Ig-like fold, characterized by the absence of the C-terminal β-strand[11-13] (Supplementary Fig. 1b), leaving a deep hydrophobic groove on the subunit surface (Supplementary Fig. 1c). As a result, pilus subunits are unstable on their own, unless in a chaperone:subunit complex or bound to an adjacent subunit within the pilus. Both chaperone:subunit and subunit:subunit interactions involve a fold-complementation mechanism whereby the subunit’s non-canonical Ig-fold is complemented in trans by, respectively, an extended β-strand in the N-terminal domain of the chaperone (strand G1) or a 10 to 20-residue long peptide extension at the N-terminus of the adjacent subunit (called the N-terminal extension or Nte)[11-14] (Supplementary Fig. 1b). During subunit polymerization, the chaperone donor strand binding the subunit’s hydrophobic groove (an interaction termed donor-strand complementation or DSC) is replaced by the Nte of the newly incorporated subunit in a process called donor-strand exchange (DSE)[11] (Supplementary Fig. 1b).

The structure of the translocation domain of the P pilus usher PapC in its inactive state revealed a 24-stranded β-barrel protein[15]. The loop between strands 6 and 7 of the β-barrel holds a 80-residue insertion that forms a plug domain that, in the non-engaged usher, resides in the barrel lumen, gating the usher channel shut. In addition to the translocation domain, ushers (~800 residues) contain a ~120-residue N-terminal domain (NTD) responsible for chaperone:subunit binding and recruitment[16-18] and a ~170 residue C-terminal domain (CTD) of poorly understood function[19,20] (Fig. 1a). How these domains cooperate to recruit chaperone:subunit complexes, catalyze subunit polymerization, and translocate the nascent pilus through the membrane is unknown. To provide insights into these processes, we present here the crystal structure of the FimD usher bound to its cognate FimC:FimH chaperone:adhesin substrate and that of the non-engaged FimD usher translocation domain.

Fig. 1

Structure of the FimD:FimC:FimH complex

a, schematic diagram of domain organization of FimH (FimHL, FimHP = lectin and pilin domain, respectively), FimC (FimCN and FimCC for N- and C-terminal domain, respectively) and FimD (see text). b, activity assay demonstrating that the purified FimD:FimC:FimH complex is functional. FimD:FimC:FimH was challenged at t=0 by the FimC:FimGS92C[A647] complex fluorescently labelled by Alexa 647 reacted on residue 92 of FimG (see position of residue 92 in Supplementary Fig. 2b). Intensity of the fluorescent FimG:FimH band (the DSE product) was used to assess the % progress of the DSE reaction. Inset: raw SDS-PAGE gel visualized as described in Methods. Each band represents a time point. c, side view ribbon representation of the FimD:FimC:FimH structure, with FimH in green, FimC in yellow and the FimD NTD, β-barrel, plug, CTD1 and CTD2 in blue, slate, magenta, cyan and purple, respectively. β1t, β6t and β7t, and β24t indicate the β-barrel strands (see secondary structure labelling nomenclature in Supplementary Fig. 3a) connecting the barrel to, respectively, the NTD, the plug and the CTDs.

A stoichiometric complex containing the type 1 pilus usher FimD bound to the FimC:FimH chaperone:adhesin complex (Fig. 1a) was purified and shown to be active (Fig. 1b). It was then crystallized and its structure determined to 2.8 Å resolution (Fig. 1c, Supplementary Fig. 2a, Supplementary Table 1, and Methods). Like PapC, FimD contains a 24-stranded β-barrel (residues 139-665), interrupted by a plug domain (residues 241-324) inserted in the periplasmic loop linking strands 6 and 7 (Figs. 1, 2, and topology diagram in Supplementary Fig. 3). However, in contrast to the PapC structure, which captured the non-activated, unbound translocation channel, the plug domain in the FimD:FimC:FimH complex now resides in the periplasm, underneath the translocation domain and next to the NTD (Fig. 1c; Supplementary Fig. 4). The usher NTD has been shown to form a binding site for chaperone:subunit complexes, including FimC:FimH[16-18]. In the FimD:FimC:FimH structure, however, the NTD lays idle, making no interactions with FimC (see below); the FimC:FimH complex instead is bound to two Ig-like domains formed at the usher C-terminus, CTD1 and CTD2 (residues 666-750 and 751-834, respectively).

Fig. 2

Channel conformations in apo and activated (FimC:FimH-engaged) FimD usher

a, top (left) and side (right) view ribbon representations of the superimposed apo-FimD (cyan) and activated FimD (slate) β-barrel. The plug domain in the channel lumen in apo FimD (magenta) rotates into the periplasm following FimD activation (pink). b, top view surface representation of the apo-FimD (left) and activated FimD (right, for clarity, showing only the translocation channel and FimH lectin domain, FimHL). The plug and FimHL are coloured magenta and green, respectively.

FimH is a two-domain protein (Fig. 1a), where the N-terminal lectin domain (residues 1-157; FimHL) is responsible for receptor binding, and the C-terminal or pilin domain (residues 158-279; FimHp) forms the interacting region with either the chaperone within the chaperone:adhesin complex in the periplasm or with the adjacent subunit (FimG) within the pilus[12]. In the ternary FimD:FimC:FimH complex, FimC stabilizes the FimH pilin domain via a typical DSC fold-complementation interaction, which remains unchanged compared to the FimC:FimH complex alone[12]. Remarkably, the FimH lectin domain inserts into the lumen of the translocation channel, traversing the entire length of the channel, its tip exposed on the extracellular side of the usher. FimD is the first transporter to be visualized with a substrate protein inserted through its lumen. The FimH pilin domain and the FimC chaperone are located underneath the pore.

Usher activation involves a large conformational change in the β-barrel domain

The FimC:FimH complex is the first chaperone:subunit complex to bind to the usher and is required to drive a conformational change in the latter that primes it for pilus biogenesis[10,21,22]. The molecular nature of this activation process is unknown. In order to get a direct comparison between the FimC:FimH-engaged form and the apo-form of the type 1 pilus usher, we crystallized the isolated FimD translocation domain (residues 124-663) and determined its structure to 3.0 Å resolution (Fig. 2 and Supplementary Fig. 5a). Apo-FimD closely resembles the structure of the PapC translocation domain (RMSD for corresponding Cα atoms of 1.7 Å). It is composed of a kidney-shaped 24-stranded β-barrel occluded by a plug domain (residues 241-324). The structure of the translocation domain in the FimC:FimH-engaged usher shows a dramatic conformational change in the β-barrel. The 24-stranded β-barrel rearranges from an oval-shaped pore with a 52 Å by 28 Å diameter to a near circular pore of 44 Å by 36 Å diameter (Cα to Cα distances; Fig. 2a, left panel). This large conformational rearrangement in the FimD translocation channel upon activation by FimC:FimH is unprecedented in β-barrel proteins, which were until now considered rigid structures.

In the apo-FimD, the translocation channel is completely sealed off by the plug domain (Fig. 2b, left panel). In the FimC:FimH -engaged complex, the plug domain is displaced into the periplasm, opening a circular channel of 32 Å now occupied by the FimH lectin domain (Fig. 2b, right panel). In apo-FimD the plug domain makes close contacts with the inner wall of the β-barrel, burying 2738 Å2 of surface area (Fig. 2b, left panel). In contrast, in the ternary complex, the β-barrel - FimH interface buries 1590 Å2 of surface area and includes fewer contacts with FimH compared to the β-barrel - plug interface in the apo form (Fig. 2b, right panel): only 6 β-barrel Cα atoms lay within 5 Å from FimH in FimD:FimC:FimH, compared to 39 β-barrel Cα atoms laying within 5 Å of the plug in apo-FimD. The more distant contact in the ternary complex structure likely provides room for the variability in subunit diameter among the different subunit types and also might facilitate translocation through the pore (Supplementary Figs. 5c, 5d).

The usher contains two chaperone:subunit binding sites

To date, the only region of the usher known to bind chaperone:subunit complexes is the usher N-terminal domain (NTD)[16,17,18]. The FimD:FimC:FimH structure now shows the existence of a second binding site on the usher, located at the C-terminal domains, CTD1 and CTD2 (Figs. 1c, 3a). The FimC:FimH complex contacts the FimD usher over a surface area of 3802 Å2. Outside the interaction of the FimD channel with the FimH lectin domain (see above), the most extensive interaction with the FimC:FimH complex is formed by the usher CTD1 (Fig. 3a; Supplementary Fig. 6a). CTD1 contacts the FimH lectin domain and FimC over a surface area of 621 Å2 and 422 Å2, respectively. Contact area between CTD2 and the FimC:FimH complex is 504 Å2 large and is primarily with FimC. Removal of the CTDs or of CTD2 alone or point mutations in CTD1 abrogate pilus biogenesis (see Li et al. (2010)[23] and this work (Supplementary Table 2)). Using electron paramagnetic resonance (EPR) spectroscopy we also demonstrate that subsequent subunits localize to the CTDs binding site after undergoing DSE (Fig. 3c and Supplementary Fig. 7). Moreover, these complexes are fully functional i.e. able to incorporate the next subunit into the nascent pilus (Supplementary Figs. 2b and 2c).

Fig. 3

FimC:FimH interactions with FimD in the FimD:FimC:FimH complex

a, side view of the ribbon representation of the FimC:FimH interface with the FimD CTDs and b, with the FimD plug and NTD, as found in the FimD:FimC:FimH complex. For clarity, just the respective FimD domains are shown. The boxed interfaces (a1: FimH-CTD1, a2: FimC-CTD2, b1: FimH-plug and b2: FimH-NTD) are described in the text and shown in detail in Supplementary Fig. 6. Color coding is as in Fig. 1. c, DEER measurement of the distance between two nitroxide spin labels, one on residue 756 of FimD (located in CTD2) in the FimD:FimC:FimH complex, and the other on residue 74 of FimC in the FimC:FimG complex (see details and controls in Methods and Supplementary Fig. 7; see also Supplementary Fig. 7 for results of distance measurements by EPR between residue 774 of FimD CTD2 and residue 74 of FimC). The Form factor (main graph; red line), the fit to the data using DeerAnalysis2010[38] (main graph; black line), and the distance distribution derived from the data (inset; black line) are shown. For comparison, we include the distance distribution predicted by MMM[39] from the crystal structure of FimD:FimC:FimH assuming that the position of FimC:FimG is similar to the previously bound chaperone-subunit complex FimC:FimH (green line) and the distance distribution from a model structure of FimD:FimC:FimH where FimC:FimG was positioned at the NTD as in Nishyama et al.[17] (cyan line; see Supplementary Fig. 7a). It can be seen that the vast majority of the distance distribution obtained experimentally overlaps with that predicted when FimC:FimG locates at the CTDs. A minor fraction corresponding to a distance around 3 nm suggests a conformational equilibrium in solution.

Other than its interaction with the CTDs, the FimC:FimH complex also comes into contact with the usher plug domain and the NTD (Fig. 3b; Supplementary Fig. 6b). The contact surface area between the plug and the FimH lectin domain is significant (474 Å2). Although the NTD is located within proximity of the FimH pilin domain, the small contact surface area of 189 Å2 and its low shape complementarity[24] of 0.45 indicates a weak interaction (Supplementary Fig. 6b). Notably, this contact zone does not overlap with the known, canonical chaperone:subunit binding site at the NTD (see below and Supplementary Fig. 6c).

When comparing the interface between FimD CTDs and FimC:FimH in the FimD:FimC:FimH structure with the interface between the FimD NTD and FimC:FimH in the structure of the NTD:FimC:FimHp complex reported by Nishiyama et al.[17], it becomes apparent that the binding sites overlap (Supplementary Fig. 8).

Thus, the usher contains two chaperone:subunit binding sites and the question arises whether these are mutually exclusive for chaperone:subunit binding or rather operate in concert, and if so, in what sequence.

A single usher protomer forms a pilus assembly machine

Chaperone/usher pili extend by step-wise addition of new chaperone:subunit complexes at the base of the growing fibre. Because the last incorporated chaperone:subunit complex is known to remain bound on the usher[18,22], the usher requires two chaperone:subunit binding sites for function. The FimD:FimC:FimH structure and the EPR data presented here demonstrate that subunits at the base of the fibre are bound to the CTDs, with the NTD lying idle. To investigate whether in the FimD:FimC:FimH complex the NTD is able to recruit the next chaperone:subunit complex, we superimposed the known structure of the FimD NTD bound to FimC:FimF[25] (the structure of the NTD:FimC:FimG complex is not available) onto the NTD in the FimD:FimC:FimH crystal structure (Figs. 4a, 4b). This superimposition demonstrates that the NTD in the FimD:FimC:FimH complex is available for recruitment of a chaperone:subunit complex without steric clashes with the FimC:FimH complex bound at the CTDs. The requirement of an accessible NTD was tested by an in vitro DSE experiment, where the chaperone:subunit binding site of the NTD of the purified FimD:FimC:FimH complex was blocked by a bulky molecule (Supplementary Fig. 9). The inactivation of the NTD results in a near loss of further subunit incorporation, suggesting that the NTD indeed acts as the recruitment site for new chaperone:subunit complexes[16-18].

Fig. 4

Chaperone:subunit incorporation cycle at the FimD usher

a, Side view of the FimD:FimC:FimH complex (FimC:FimH in surface representation) with a new chaperone:subunit complex (FimC’:FimG, yellow:orange, respectively) modelled at the NTD binding site (the model is from PDB:3BWU; i.e., based on the crystal structure of FimD NTD alone bound to FimC:FimF). b, clipped view of the FimC’:FimG – FimC:FimH contact zone (boxed area in a), showing positioning of the FimG N-terminal extension (FimG Nte; in red) above the P5 pocket in the FimC:FimH complex (FimC:FimH in yellow:green, the P5 pocket shown in light green).

The superimposition presented in Fig. 4a provides unique insights into the catalytic mechanism of a monomeric usher. The ability of the Nte of an incoming subunit to initiate the DSE reaction with the previously-assembled subunit is crucially dependent on a defined binding site in that subunit, called the P5 site[26] (Supplementary Fig. 1). The P5 site allows the incoming Nte to access the hydrophobic groove of the preceding subunit, allowing it to displace the chaperone donor strand in a step-wise zip-in-zip-out mechanism[26-29]. The in silico model of FimC:FimF docked at the NTD of the FimD:FimC:FimH complex shows that the newly recruited subunit comes into close proximity with the FimH pilin domain, representative for the subunit that resides at the base of the growing fibre (Fig. 4a). Strikingly, the Nte of the subunit bound at the NTD lays directly above the P5 pocket of the subunit bound at the CTDs, perfectly positioned to initiate the DSE reaction (Fig. 4b). Together, the active recruitment of new chaperone:subunit complexes to the usher NTD and their ideal positioning with respect to the penultimate chaperone:subunit complex located at the CTDs provide a rationale for the catalytic ability of the usher (Supplementary Fig. 10). In the proposed model for the catalytic cycle, the chaperone:subunit complex at the base of the growing pilus fibre resides at the usher’s CTDs. New subunits are recruited to the NTD and brought into ideal orientation to undergo DSE with the subunit bound at the CTDs (now the penultimate subunit; Supplementary Fig. 10, step 1). Upon DSE, the chaperone is displaced from the penultimate subunit and dissociates from the CTDs (Supplementary Fig. 10, step 2). To reset the assembly machinery for a new incorporation, the incoming chaperone:subunit complex would need to dissociate from the NTD and be transferred to the CTDs site, concomitantly pushing the penultimate subunit into the translocation channel (Supplementary Fig. 10, steps 3 and 4, respectively). How the hand-over of the chaperone:subunit complex from the usher’s NTD to the CTDs occurs remains speculative.

Conclusion

The crystal structure of FimD bound to FimC:FimH provides the remarkable view of a protein transporter caught in the act of secreting its cognate substrate. Together with the FimD translocator domain structure, it elucidates not only the mechanism of gating leading to FimH insertion into the FimD barrel, but also the subsequent steps of subunit polymerization and nascent pilus translocation. Pilicide compounds recently shown to inhibit pilus biogenesis target the interface between chaperone:subunit complexes and the usher NTD[30]. The crystal structure presented here unravels a complex choreography of domain motion and protein-protein interactions that will no doubt be of crucial importance in the design of additional compounds capable of disrupting type 1 pilus biogenesis and thus inhibiting cystitis, an infectious disease that plagues millions of individuals worldwide.

METHODS SUMMARY

Purification and crystallization

FimD:FimC:FimH with a Strep-tag at the C-terminus of FimD was purified as described previously with an additional Strep-tag affinity chromatography step[15]. After addition of trypsin (which removes 21 residues at the N-terminus of FimD and cleaves its β13-14 loop), the complex was crystallized by hanging-drop vapour diffusion. The 6×His tagged FimD translocation domain (residues 124 – 663) was purified by Ni-NTA affinity and size exclusion chromatography, and crystallized by hanging-drop vapour diffusion.

Structure determination and refinement

The crystals of the FimD:FimC:FimH complex contained two ternary complexes per asymmetric unit, related by a pseudotranslation. The chaperone:subunit (FimC:FimH) or usher domains, for which the structures (NTD) or structures of homologous domains (PapC translocation domain and plug, PapC CTD2) were available, were located individually using molecular replacement (MR), as implemented in Phaser[31] and Molrep[32]. CTD1 was built manually using Coot[33]. Refinement with Refmac[34,35] converged to a model with an Rfactor of 0.219 and an Rfree of 0.277. The structure of the FimD translocation domain was solved by MR with the equivalent PapC structure (PDB ID 2vqi) as a search model using Phaser[31]. The structure was built in Coot[33], and refined in Phenix[36] to an Rfactor of 0.229 and Rfree of 0.305.

DSE assay

The FimD:FimC:FimH complex was mixed with fluorescently-labelled FimC:FimG, where FimG was labelled with Alexa 647 on FimG residue 92. DSE progression was monitored by the appearance of the fluorescent FimG:FimH band on SDS-PAGE gels. For DSE experiments involving a FimD:FimC:FimH complex with a bulky molecule blocking NTD binding, FimD was reacted with Alexa 594 on residue 109.

EPR spectroscopy

The FimD:FimC:FimH complex was spin-labelled on residue 756 or residue 774 of FimD. The FimC:FimG complex was spin-labelled on residue 74 of FimC. Double Electron-Electron Resonance (DEER) measurements for distance determination were performed as described previously[37].