Friday, April 26, 2024
4/26/2024
Abstract This proposal seeks to identify potential allosteric properties in adhesins of human enterobacterial pathogens - Escherichia coli, Klebsiella pneumoniae/oxytoca, Enterobacter spp, Proteus mirabilis, and Salmonella – that are assembled via a chaperone-usher pathway (CUP). To date, only the mannose-specific, type 1 fimbrial adhesin of E. coli, FimH, has been demonstrated to be an allosteric protein that can exist in alternative functional (active/inactive) conformations. This property allows bacteria that contain FimH as part of hair-like surface appendages, fimbriae or pili, to bind ligand presented on host cells rapidly from an inactive conformation and to remain bound for very long lifetimes under shear force by transiting to an active conformation. The long-lived (slow dissociation) binding involves formation of so-called `catch-bonds' that can be activated and become stronger under tensile mechanical force and involve an allosteric switch. To date no other bacterial adhesin has been demonstrated to be allosteric and to exist in alternative functional (active/inactive) conformations. To identify other adhesins that work via similar mechanisms, we will focus on adhesins that are part of fimbriae or pili and belong to the same CUP structural class as FimH. We recently identified a set of aliphatic or aromatic residues that act as molecular toggles that control the allosteric switch between active and inactive conformations by switching their orientation between the protein core and surface. It is possible to stabilize either active or inactive conformation of the adhesin by “surface locking” such toggles through substitution to hydrophilic charged residues. We will use putative analogs of the FimH toggles to identify the existence of allosteric states in other CUP adhesins that are homologous or non-homologous to FimH, using mutagenesis, various functional assays, and three types of structural analysis – NMR, X-ray crystallography, and cryo-EM. Success of our studies will contribute to understanding of general mechanisms of bacterial adhesion to host cells and, ultimately, to the design of optimized vaccines and small molecule inhibitors. If certain adhesins are found to be allosteric, in-depth analysis of their physiologically-relevant structure/functional properties and significance for pathogenesis as well as practical implementation of the findings will be the focus of future studies.
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