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.

Keywords: Ligilactobacillus ruminis; LrpCBA pilus; autochthonous commensals; backbone LrpA; basal LrpB; gut bacteria; intestine; pilins; tip LrpC.

Figure 1

Size-exclusion profiles and SDS–PAGE analysis of purified recombinant L. ruminis pilus proteins. (a) SeMet-LrpC. (b) LrpB. (c) LrpA. The elution volume peaks (as indicated) correspond to the monomeric form of each protein. An SDS–PAGE analysis of each protein (as indicated) after the final purification step is shown. The sizes (in kDa) of the molecular-weight markers (lane M) are indicated.

Figure 2

Crystallization of L. ruminis pilus proteins. (a) SeMet-LrpC crystals (monoclinic) grown in 0.1 M sodium HEPES pH 7.5, 15% PEG 20 000. (b) LrpB crystals (orthorhombic) grown in 0.2 M calcium acetate, 22% PEG 3350, 0.1 M l-proline. (c) LrpA crystals (trigonal) grown in 0.1 M sodium acetate pH 4.6, 15% PEG 3000, 5% polyvinylpyrrolidone K15.

Figure 3

X-ray diffraction patterns collected from crystals at synchrotron sources. (a) SeMet-LrpC. (b) LrpB. (c) LrpA. Circular dashed lines indicate resolution arcs.