Dynamic Sampling of Multiple Conformations in FimH Modulates Bacterial Adhesion

Abstract

Gram-negative bacteria express hundreds of diverse pili assembled by the chaperone-usher pathway (CUP) for surface colonization and biofilm formation. CUP pili are multi-subunit surface appendages tipped with two-domain adhesins that recognize distinct ligands through a receptor-binding domain while anchored to an adaptor subunit through a pilin domain. Uropathogenic E. coli utilize the type 1 pilus adhesin FimH to bind mannose, which decorates the bladder epithelium. This interaction is critical in urinary tract infection (UTI) pathogenesis. Bioinformatic studies have demonstrated that the composition of three positively selected residues (PSRs) in FimH impact its role in pathogenesis. However, the mechanism by which residues that lie outside the mannose-binding pocket alter the binding and virulence of FimH remains poorly understood. Crystallographic studies indicate that FimH adopts elongated and compact conformations. We postulated that PSRs in FimH allosterically modulate a dynamic equilibrium of multiple conformations in solution, which impacts function. To address this hypothesis, we first show by small-angle X-ray scattering that FimH alleles bound to FimG peptide, which reflects a tip-like setting, vary drastically in their average conformation. PSRs that promote extension of FimH conformation exhibit high affinity for mannose. Further, collision cross-section distributions measured by ion mobility mass spectrometry indicate that FimH alleles adopt altered equilibria of two major conformations. Congruently, preliminary analyses of molecular dynamics simulations suggest that PSRs modulate flux across a conformational landscape characterized by a constricted tense state and multiple, rapidly exchanging relaxed states. Currently, we are investigating the biophysical implications of multiple relaxed states with predicted functionality on bacterial attachment. Altogether, by integrating biophysical and computational methods to map the FimH conformer-function relationship, this work begins to unravel how a conformational phase switch regulates bacterial adhesion and how evolutionary processes fine-tune protein dynamics to guide host colonization.