The crystal structure of the N-terminal domain of the backbone pilin LrpA reveals a new closure-and-twist motion for assembling dynamic pili in Ligilactobacillus ruminis.

Sortase-dependent pili are long surface appendages that mediate attachment, colonization and biofilm formation in certain genera and species of Gram-positive bacteria. Ligilactobacillus ruminis is an autochthonous gut commensal that relies on sortase-dependent LrpCBA pili for host adherence and persistence. X-ray crystal structure snapshots of the backbone pilin LrpA were captured in two atypical bent conformations leading to a zigzag morphology in the LrpCBA pilus structure. Small-angle X-ray scattering and structural analysis revealed that LrpA also adopts the typical linear conformation, resulting in an elongated pilus morphology. Various conformational analyses and biophysical experiments helped to demonstrate that a hinge region located at the end of the flexible N-terminal domain of LrpA facilitates a new closure-and-twist motion for assembling dynamic pili during the assembly process and host attachment. Further, the incongruent combination of flexible domain-driven conformational dynamics and rigid isopeptide bond-driven stability observed in the LrpCBA pilus might also extend to the sortase-dependent pili of other bacteria colonizing a host.

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

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.