Catch-bond mechanism of the bacterial adhesin FimH

Abstract

Ligand-receptor interactions that are reinforced by mechanical stress, so-called catch-bonds, play a major role in cell-cell adhesion. They critically contribute to widespread urinary tract infections by pathogenic Escherichia coli strains. These pathogens attach to host epithelia via the adhesin FimH, a two-domain protein at the tip of type I pili recognizing terminal mannoses on epithelial glycoproteins. Here we establish peptide-complemented FimH as a model system for fimbrial FimH function. We reveal a three-state mechanism of FimH catch-bond formation based on crystal structures of all states, kinetic analysis of ligand interaction and molecular dynamics simulations. In the absence of tensile force, the FimH pilin domain allosterically accelerates spontaneous ligand dissociation from the FimH lectin domain by 100,000-fold, resulting in weak affinity. Separation of the FimH domains under stress abolishes allosteric interplay and increases the affinity of the lectin domain. Cell tracking demonstrates that rapid ligand dissociation from FimH supports motility of piliated E. coli on mannosylated surfaces in the absence of shear force.

Figure 1. FimH·DsG resembles fimbrial tip FimH.

(a) Preparation of the FimH·DsG complex by DSE. Left: reaction scheme of the DSE reaction, in which DsG displaces the FimC chaperone from the FimH pilin domain. Right: kinetics of the FimH·DsG complex formation at 37 °C, monitored by analytical gel filtration. DSE was initiated by mixing the FimC·FimH complex (15 μM) with excess DsG peptide (50 μM). Samples were removed after different incubation times, rapidly cooled on ice and immediately subjected to gel filtration. The reaction can be followed by the decrease in the FimC·FimH complex concentration and the simultaneous increase in the concentrations of FimH·DsG and free FimC (FimC and FimH·DsG coelute as a single peak at ∼12 ml). The chromatogram at the bottom shows that the FimC·FimH complex is stable against dissociation/aggregation under the chosen conditions. The rate constant of DSE estimated from these data is ∼0.5 M⁻¹ s⁻¹. (b) Structure of FimH^F18·DsG (lectin domain FimH_L, red; pilin domain FimH_P, yellow; DsG, blue; circle and square indicate N- and C termini, respectively). (c) FimH from the fimbrial tip structure (left, PDB ID: 3JWN (ref. 17); FimG, blue; FimF, green) is superposed onto FimH^F18·DsG based on their pilin domains (aa. 160–279), in the superposition (right) fimbrial FimH is shown in grey. (d) Close-up on the DsG peptide (stick representation) bound to FimH^F18·DsG with 2F_o–F_c electron density map. Backbone hydrogen bonds of the DsG peptide and β-strands 2 (β2) and 9 (β9) of FimH_P are indicated.

Figure 2. Crystallographic analysis of FimH conformational states.

(a) FimH^F18·DsG in the A_free (left) and in the A_bound states (FimH^F18·DsG·HM) in comparison with the S_bound state of FimH^K12·DsF·HM and the isolated FimH_L^F18·HM (right). The FimH_L, FimH_P, DsF and DsG are coloured in red, yellow, green and blue. The experimentally in crystallo trapped orientation of FimH_P in FimH^K12·DsF·HM and a modelled position based on a hinge motion stretching around Gly157 is indicated. A schematic representation for each crystal structure, similar to Fig. 1a, is given. The tip of the clamp loop and the C terminus of FimH_L are indicated as a circle and diamond, respectively. (b) Comparison of the conformation of the ligand-binding site in the A_bound (red) and S_bound (orange) states with the isolated lectin domain FimH_L (grey) and (c) comparison of the interdomain interface of the lectin domain.

Figure 3. The interdomain region in the S_bound state.

Close-up of the interdomain region of FimH^K12·DsF·HM in the A_bound form (left) and FimH^K12·DsF·HM in the S_bound state (right). A cartoon representation for each crystal structure, similar to Fig. 1a, is given. Key residues in the interface are shown as sticks. FimH_P, FimH_L and DsF are coloured in yellow, red and green, respectively.

Figure 4. Kinetics of HM binding and release by full-length FimH.

(a) Fluorescence spectra (excitation at 280 nm) of FimH_L^F18 (2 μM; red lines) and FimH^F18·DsG (2 μM; black lines) in the absence (solid lines) or presence of 200 μM HM (dotted lines). (b) Equilibrium titration of FimH^F18·DsG (2 μM) with HM, recorded via the fluorescence increase at 320 nm. The total concentration of HM is plotted against the recorded fluorescence signal. Data were fitted (solid line) according to equation (2) (cf. experimental section) and yielded a K_d value of 9.9±1.5 μM. (c) Stopped-flow fluorescence kinetics of HM binding to FimH^F18·DsG (1.0 μM), recorded via the fluorescence change above 320 nm. The HM concentration was varied between 0 and 50 μM. Five representative traces are shown (HM concentrations are given in μM). The fluorescence traces were globally fitted according to a second-order binding and first-order dissociation reaction (solid lines; Table 2). (d) Amplitudes of the reactions monitored in c, plotted against the total HM concentration. Data were fitted (solid line) according to equation (2), yielding a K_d value of 12±1 μM.

Figure 5. HM binding and release by the isolated FimH lectin domain FimH_L.

Analysis of FimH_L·HM interactions based on competition between HM and the synthetic fluorescent GN-FP-4 ligand. (a) HM binding to FimH_L analysed by displacement of GN-FP-4 from FimH_L variants as indicated. An equimolar mixture of FimH_L and GN-FP-4 (1 μM each) was incubated with different HM concentrations (10 nM–3.2 mM) for >18 h. GN-FP-4 displacement is monitored by a decrease in fluorescence polarization at 528±20 nm (excitation at 485 nm). Data were fitted (solid lines) according to a mechanism in which two ligands compete for the same binding site, with fixed K_d values for GN-FP-4 binding (cf. Table 2). (b) Kinetics of HM dissociation from FimH_L. A solution with equimolar concentrations of FimH_L and HM (3 μM each, guaranteeing >95% occupancy with HM) was mixed with excess GN-FP-4 (10 μM), and the decrease in GN-FP-4 fluorescence at 520 nm as a consequence of HM dissociation and GN-FP-4 binding was recorded (Supplementary Fig. 5f–j). The obtained first-order kinetics are independent of the GN-FP-4 concentration and thus directly monitor HM dissociation.

Figure 6. Cell-tracking analysis of bacterial motility on mannosylated surfaces.

E. coli cells piliated with FimH^F18 or the FimH^F18-Ala188Asp variants were tracked under static conditions in the absence of shear force. (a) The fraction of bacteria attached to mannose-coated (1M-BSA) or BSA-coated surface (negative control) at the beginning of the time-lapse movies (white bars) and after 5 min (black bars) are given. Bacterial motility on 1M-BSA was analysed in the absence and presence of HM. The delay between application of bacteria and movie recording was ∼1 min. (b) Fraction of tracked cells that were pre-attached (yellow; speed <0.5 μm s⁻¹), permanently attach (red), were mobile (white), transiently attach (green) or permanently detach (blue) during the entire observation time (5 min). Right: schematic depiction of the observed cell behaviour. FimH^F18-piliated E. coli show almost exclusively transient attachment events on 1M-BSA. FimH^F18-Ala188Asp-piliated E. coli show less transient attachment but enhanced permanent attachment to 1M-BSA. Transient and permanent attachment to 1M-BSA is significantly reduced in the presence of HM. For each experiment five to seven independent replicates were analysed.

Figure 7. Catch-bond mechanism of FimH-mediated cell adhesion.

(a) In the absence of tensile mechanical force, formation of the FimH-Uroplakin 1a (UPIa) complex comprises the highly dynamic transition of the A_free to the A_bound state. The reaction likely proceeds via a transient encounter complex (indicated in square brackets). The reaction of the encounter complex to A_bound is not rate-limiting and must have a half-life of less than 1 ms. Dissociation of the receptor from the FimH lectin domain in the A_bound state is promoted via dynamic allostery by the pilin domain that acts as a negative allosteric regulator. The reaction from A_bound to the encounter complex corresponds to k_off. Fast binding and release of UPIa by FimH enables bacterial motility on the bladder epithelium. (b) Shear force increases the population of the S_bound state of FimH, in which the pilin and lectin domains are separated. The dissociation of S_bound under shear force is slowed down up to 100,000-fold compared with A_bound. The indicated rate constants and half-lives correspond to the interaction between FimH^F18 and the model ligand HM. Rate limiting reactions are indicated by solid arrows, and fast, non-limiting reactions by dashed arrows.