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.

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

“…Mass-spectrometric analysis, size-exclusion chromatography and SDS-PAGE analysis confirmed that the purified 49 kDa LrpA F was pure, homogeneous and in a monomeric form in solution (Prajapati et al, 2021). As part of the structure determination, a truncated and stable 30 kDa fragment of LrpA (LrpA T ) was also generated by limited proteolysis with subtilisin and was then purified using the aforementioned purification protocol for LrpA F (Prajapati et al, 2021). LrpA F was also purified by exchanging the buffer with 0.1 M sodium acetate pH 4.8, 150 mM NaCl during size-exclusion chromatography to check the effect of pH on the oligomerization state and conformational changes.…”

“…Size-exclusion chromatography was then performed using a HiPrep 26/60 Sephacryl S-200 column (GE Healthcare) with Tris buffer (see above). Mass-spectrometric analysis, size-exclusion chromatography and SDS-PAGE analysis confirmed that the purified 49 kDa LrpA F was pure, homogeneous and in a monomeric form in solution (Prajapati et al, 2021). As part of the structure determination, a truncated and stable 30 kDa fragment of LrpA (LrpA T ) was also generated by limited proteolysis with subtilisin and was then purified using the aforementioned purification protocol for LrpA F (Prajapati et al, 2021).…”

“…The unprocessed form of the L. ruminis (ATCC 25644) backbone pilin LrpA consists of 507 amino acids (UniProtKB ID E7FRT5). Mature full-length LrpA (LrpA F ) protein (residues 35-472) was recombinantly cloned and expressed in Escherichia coli BL21 (DE3) pLysS cells (Prajapati et al, 2021;Supplementary Table S1). Protein purification was performed as reported previously (Prajapati et al, 2021).…”

“…Mature full-length LrpA (LrpA F ) protein (residues 35-472) was recombinantly cloned and expressed in Escherichia coli BL21 (DE3) pLysS cells (Prajapati et al, 2021;Supplementary Table S1). Protein purification was performed as reported previously (Prajapati et al, 2021). Briefly, E. coli cells were cultivated in lysogeny broth (LB) growth medium supplemented with 50 mg ml À 1 kanamycin at 310 K until an optical density (OD 600 ) of 0.6 was reached.…”

“…The LrpCBA pilus displays an archetypal heterooligomeric structure consisting of the tip LrpC (123 kDa), basal LrpB (39 kDa) and backbone LrpA (49 kDa) pilins (Yu et al, 2015). As a note, the LrpCBA pilus constituents lack any significant sequence identity to other known pilin structures, including the SpaCBA and SpaFED pilins from L. rhamnosus GG (Prajapati et al, 2021). Not unusually (Kankainen et al, 2009;Rintahaka et al, 2014;von Ossowski et al, 2013), the LrpCBA pilus has a promiscuous binding specificity (arising from the tip adhesin LrpC) for different intestinal surface components, such as collagen, fibronectin and epithelial cells, although it has no affinity for mucus substrates (Yu et al, 2015).…”