Structural basis for mechanical force regulation of the adhesin FimH via finger trap-like beta sheet twisting

Abstract

The Escherichia coli fimbrial adhesive protein, FimH, mediates shear-dependent binding to mannosylated surfaces via force-enhanced allosteric catch bonds, but the underlying structural mechanism was previously unknown. Here we present the crystal structure of FimH incorporated into the multiprotein fimbrial tip, where the anchoring (pilin) domain of FimH interacts with the mannose-binding (lectin) domain and causes a twist in the beta sandwich fold of the latter. This loosens the mannose-binding pocket on the opposite end of the lectin domain, resulting in an inactive low-affinity state of the adhesin. The autoinhibition effect of the pilin domain is removed by application of tensile force across the bond, which separates the domains and causes the lectin domain to untwist and clamp tightly around the ligand like a finger-trap toy. Thus, beta sandwich domains, which are common in multidomain proteins exposed to tensile force in vivo, can undergo drastic allosteric changes and be subjected to mechanical regulation.

Figure 1. Overall structure

A. FimH-containing fimbrial tip crystal structure. B. The lectin domain docked to the pilin domain (black) in the fimbrial tips. C. The isolated lectin domain bound to butyl-mannose (black) as previously crystallized (1UWF,(Bouckaert et al., 2005)). In all panels of all figures, the large β-sheet is shown in purple, the split β-sheet in orange, the swing loop in pink (residues 22–35), the linker loop in light blue (151–158), the insertion loop in green (109–124), the clamp loop in cyan (8–16) the 3₁₀/α-helix in yellow (59–72), and remaining regions of the lectin domain in gray. The dashed lines indicate the length from the N-terminus (residue 1, orange sphere) to the C-terminus of the lectin domain (residue 158, blue sphere.) These and other structural cartoons were made with Pymol (Delano Scientific LLC.)

Figure 2. Effect of ligand and primary structure on conformation

In all structural cartoon panels, co-crystallized butyl mannose is shown in black sticks, while pocket-forming residues are shown as sticks with carbon atoms colored by loop identity (see Figure 1), oxygen atoms in red and nitrogen atoms in blue. Red dotted lines show hydrogen bonds described in the text. Black dotted lines indicate distances described in the text A,B. Top-view of the mannose-binding site in the two conformations. C. Solution affinity curve of tip and purified lectin domains binding to α-MM in Surface Plasmon Resonance (SPR) assays. D. Profiles of F-18 tips and purified lectin domains dissociating from mannosylated bovine serum albumin (man-BSA) in SPR assays. All curves are normalized to the response units at the start of the wash (108 for tip, 79 for LD^F18, and 101 for LD^J96) to indicate fraction remaining bound. E,F. Bottom-view of the two conformations of the interdomain regions with the mAb21-binding residues shown as spheres. G, Dilution curve of mAb21 binding to two variants of isolated lectin domain. H. Effect of mannose on mAb21 binding to LD^F18 and tip. (Data are represented as mean +/− SEM.)

Figure 3. NMR spectra of purified 1mM LD^F18

¹H-¹⁵N HSQC overlay of LD^F18 in the mannose-free (black) and mannose-bound (red) forms. The latter contains 1mM α-MM, which is a 1:1 ratio of protein to mannose. Large chemical shift differences are not observed between the two spectra, indicating that binding of mannose induces only small, local conformational changes. See also Figure S1.

Figure 4. Structural changes caused by pilin domain interaction

Colors and representations are the same as in Figures 1 and 3. The Cα atoms are shown as spheres for three β-sheet residues (36, 74, and 127) that are in a plane perpendicular to the field of view, as shown by the dotted black line. Residues 22 and 63 are also shown as spheres to show how they move relative to this plane and the other residues. A, B. View from side of swing loop. C, D. View from side of insertion loop. E, G. Propagation of conformational changes from the proximal region to the distal mannose-binding site. F, H. photograph of a cardboard folder with an orange page representing the split β-sheet and a purple page representing the large β-sheet in an unperturbed state (F) and with the lower left front edge pushed upward (H), to demonstrate the analogy for the page-turning mechanism of β-sandwich allostery.

Figure 5. Mutational Regulation

A. Binding of mAb21 to wildtype or disulfide-locked FimH in fimbriae, with and without α-MM or DTT. B. Number of bacteria expressing wildtype or disulfide-locked FimH binding to man-BSA surface with or without DTT.

Figure 6. Mechanical Regulation

Profiles of tip dissociating from man-BSA at various tensile forces, as measured by AFM. These survival plots show the fraction of interactions that are left as a function of time after force is applied. The BiACore® surface plasmon resonance (SPR) dissociation profiles for the tip and LD^F18 are superimposed for comparison.