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LrpCBA pilus proteins of gut-dwelling Ligilactobacillus ruminis: crystallization and X-ray diffraction analysis
Acta Crystallographica Section F ( IF 1.072 ) Pub Date : 2021-07-28 , DOI: 10.1107/s2053230x21007263
Amar Prajapati 1 , Airi Palva 2 , Ingemar von Ossowski 2 , Vengadesan Krishnan 1
Affiliation  

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.

中文翻译:

肠道内瘤胃乳杆菌的 LrpCBA 菌毛蛋白:结晶和 X 射线衍射分析

细菌生存和定殖对宿主表面的粘附涉及多种分子机制。瘤胃乳杆菌是一种严格厌氧菌和肠道原生共生菌,依靠分选酶依赖性菌毛 (LrpCBA) 粘附在肠内壁上,从而承受肠腔内容物的流动。在这里,LrpCBA 菌毛是肠道胶原蛋白、纤连蛋白和上皮细胞的混杂粘合剂。在结构上,LrpCBA菌毛表现出代表性的异源寡聚排列,由三种类型的菌毛蛋白亚基组成,每种亚基都有自己的位置和功能,即用于粘附的尖端LrpC、用于锚定的基底LrpB和用于长度的主干LrpA。为了进一步了解分选酶依赖性菌毛的组装、锚定和功能机制,重组生产了每种瘤胃乳杆菌菌毛蛋白,用于结晶和 X 射线衍射分析。获得了通过有限蛋白水解产生的 LrpC、LrpB、LrpA 和截短的 LrpA 晶体,并衍射分辨率分别为 3.0、1.5、2.2 和 1.4 Å。还从硒代蛋氨酸取代的 LrpC 和截短的 LrpA 的碘化物衍生物的晶体中收集了异常数据。报道了蛋白质生产、结晶和衍生化的成功策略。
更新日期:2021-08-04
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