LrpCBA pilus proteins of gut-dwelling Ligilactobacillus ruminis: crystallization and X-ray diffraction analysis

Abstract

Adhesion to host surfaces for bacterial survival and colonization involves a variety of molecular mechanisms. Ligilactobacillus ruminis, a strict anaerobe and gut autochthonous (indigenous) commensal, relies on sortase-dependent pili (LrpCBA) for adherence to the intestinal inner walls, thereby withstanding luminal content flow. Here, the LrpCBA pilus is a promiscuous binder to gut collagen, fibronectin and epithelial cells. Structurally, the LrpCBA pilus displays a representative hetero-oligomeric arrangement and consists of three types of pilin subunit, each with its own location and function, i.e. tip LrpC for adhesion, basal LrpB for anchoring and backbone LrpA for length. To provide further structural insights into the assembly, anchoring and functional mechanisms of sortase-dependent pili, each of the L. ruminis pilus proteins was produced recombinantly for crystallization and X-ray diffraction analysis. Crystals of LrpC, LrpB, LrpA and truncated LrpA generated by limited proteolysis were obtained and diffracted to resolutions of 3.0, 1.5, 2.2 and 1.4 Å, respectively. Anomalous data were also collected from crystals of selenomethionine-substituted LrpC and an iodide derivative of truncated LrpA. Successful strategies for protein production, crystallization and derivatization are reported.