Abstract
Critical molecular events that control conformational transitions in most allosteric proteins are ill-defined. The mannose-specific FimH protein of Escherichia coli is a prototypic bacterial adhesin that switches from an 'inactive' low-affinity state (LAS) to an 'active' high-affinity state (HAS) conformation allosterically upon mannose binding and mediates shear-dependent catch bond adhesion. Here we identify a novel type of antibody that acts as a kinetic trap and prevents the transition between conformations in both directions. Disruption of the allosteric transitions significantly slows FimH's ability to associate with mannose and blocks bacterial adhesion under dynamic conditions. FimH residues critical for antibody binding form a compact epitope that is located away from the mannose-binding pocket and is structurally conserved in both states. A larger antibody-FimH contact area is identified by NMR and contains residues Leu-34 and Val-35 that move between core-buried and surface-exposed orientations in opposing directions during the transition. Replacement of Leu-34 with a charged glutamic acid stabilizes FimH in the LAS conformation and replacement of Val-35 with glutamic acid traps FimH in the HAS conformation. The antibody is unable to trap the conformations if Leu-34 and Val-35 are replaced with a less bulky alanine. We propose that these residues act as molecular toggle switches and that the bound antibody imposes a steric block to their reorientation in either direction, thereby restricting concerted repacking of side chains that must occur to enable the conformational transition. Residues homologous to the FimH toggle switches are highly conserved across a diverse family of fimbrial adhesins. Replacement of predicted switch residues reveals that another E. coli adhesin, galactose-specific FmlH, is allosteric and can shift from an inactive to an active state. Our study shows that allosteric transitions in bacterial adhesins depend on toggle switch residues and that an antibody that blocks the switch effectively disables adhesive protein function.
Highlights
Receptor-ligand interactions are essential for cell adhesion, molecular transport, signal transduction, enzymatic catalysis, etc
We describe a monoclonal antibody against the mannose-specific bacterial adhesin, FimH, that blocks the conformational transition in both directions
We propose that the conformational trapping involves a steric block of the core-to-surface switching of certain residues which is critical for the allosteric transitions
Summary
Receptor-ligand interactions are essential for cell adhesion, molecular transport, signal transduction, enzymatic catalysis, etc. Most ligand-binding proteins adopt both ‘inactive’ and ‘active’ conformations and the transition between the two states is usually allosterically controlled. Ligand binding to LAS FimH induces conformational changes to an intermediate state in which the binding pocket is closed but the interdomain interaction is preserved [8] and the end-point HAS in which the LD and PD are separated is achieved via an allosteric transition [9,10,11]. Force-induced domain separation may occur on a faster timescale than the mannose-induced separation and is critical during FimH-mediated bacterial adhesion under physiological shear induced by flow conditions. The mannose-induced transition may occur on a slower timescale but likely plays a critical role during adhesion under low-shear, static, or equilibrium conditions. While the structural differences between the endpoint LAS and HAS of FimH are well defined, the key molecular events that underlie the allosteric transition remain undefined
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