Conformational switch of the bacterial adhesin FimH in the absence of the regulatory domain: Engineering a minimalistic allosteric system

Abstract

Edited by Wolfgang Peti For many biological processes such as ligand binding, enzymatic catalysis, or protein folding, allosteric regulation of protein conformation and dynamics is fundamentally important. One example is the bacterial adhesin FimH, where the C-terminal pilin domain exerts negative allosteric control over binding of the N-terminal lectin domain to mannosylated ligands on host cells. When the lectin and pilin domains are separated under shear stress, the FimH-ligand interaction switches in a so-called catch-bond mechanism from the low-to high-affinity state. So far, it has been assumed that the pilin domain is essential for the allosteric propagation within the lectin domain that would otherwise be conformationally rigid. To test this hypothesis, we generated mutants of the isolated FimH lectin domain and characterized their thermodynamic, kinetic, and structural properties using isothermal titration calorimetry, surface plasmon resonance, nuclear magnetic resonance, and X-ray techniques. Intriguingly, some of the mutants mimicked the conformational and kinetic behaviors of the full-length protein and, even in absence of the pilin domain, conducted the cross-talk between allosteric sites and the mannoside-binding pocket. Thus, these mutants represent a minimalistic allosteric system of FimH, useful for further mechanistic studies and antagonist design. The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S11, Tables S1-S2, supporting Extended Experimental procedures, and supporting Refs. 1-5. The atomic coordinates and structure factors (code 5MCA) have been deposited in the Protein Data Bank (http://wwpdb.org/).

Conformational stability of the bacterial adhesin, FimH, with an inactivating mutation

“…Exposing the interdomain region to water, which disrupts inhibition by the pilin domain, is thought to induce a conformational change in the lectin domain 18,20 . The conformational change has been described by the width of the β ‐sandwich fold, 14,17,21 as well as the putative allosteric pathway sites connecting the interdomain region to the binding pocket 12,20,22 …”

“…However, while the lectin domain with a single Arg‐60‐Pro amino acid mutation (HL mutant) is stabilized in the inactive conformation, 12 HL mutant undergoes an allostery‐like conformational change upon binding mannoside 22 . As a result, Rabbani et al 22 have proposed HL mutant as a “minimal model” for FimH allostery that is smaller, consists of a single domain, and is more experimentally tractable than full‐length FimH. Although the structure and function of the HL mutant have been well characterized, its dynamics have not yet been investigated experimentally or computationally.…”

“…The impact of the Arg‐60‐Pro mutation on the structure and function of HL mutant has also been well characterized. After Rodriguez et al 12 selected the Arg‐60‐Pro mutation to energetically favor the inactive state from a set of trial mutations tested in RosettaDesign, Rabbani et al 22 then confirmed that the backbone structure of HL mutant matches the lectin domain of H2 inactive using crystallography and chemical shift mapping from 1H‐15N‐HSQC NMR spectroscopy. The structural similarity between HL mutant and H2 inactive suggests that the β ‐bulge has stronger allosteric coupling to the clamp segment than the interdomain regions, which are missing in HL mutant.…”

RMSD analysis of structures of the bacterial protein FimH identifies five conformations of its lectin domain

“…The major conformational states observed for the lectin domain are one with a wide mannose binding site, another with a narrow site around the ligand, and a third intermediate conformation. The major conformations of the lectin domains have been variously labeled as “elongated” and “compressed,” “open” and “closed,” “low affinity” and “high affinity,” “R” and “T” states, and “loose” and “tight” . Adjectives describing the ligand binding site (“wide” and “narrow”) will be used here as labels for the major domain conformations.…”

Cell behavior of the highly sticky bacterium Acinetobacter sp. Tol 5 during adhesion in laminar flows

“…A strong flow, even laminar flow, can cause the detachment of bacterial cells from surfaces and the breakage of biofilms 5 . On the other hand, the adhesiveness of Escherichia coli to surfaces is enhanced through a conformational change of FimH under conditions of increased shear stress 6 – 8 . For Pseudomonas aeruginosa , the residence time of adhesion increases with increasing shear stress 9 .…”