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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Dimerization and interactions of Brucella suis VirB8 with VirB4 and VirB10 are required for its biological activity
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Dimerization and interactions of Brucella suis VirB8 with VirB4 and VirB10 are required for its biological activity

机译：猪布鲁氏菌VirB8与VirB4和VirB10的二聚化和相互作用是其生物学活性所必需的

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摘要

VirB8-like proteins are essential components of type IV secretion systems, bacterial virulence factors that mediate the translocation of effector molecules from many bacterial pathogens into eukaryotic cells. Based on cell biological, genetic, and x-ray crystallographic data, VirB8 was proposed to undergo multiple protein-protein interactions to mediate assembly of the translocation machinery. Here we report the results of a structure-function analysis of the periplasmic domain of VirB8 from the mammalian pathogen Brucella suis, which identifies amino acid residues required for three protein-protein interactions. VirB8 variants changed at residues proposed to be involved in dimerization, and protein-protein interactions were purified and characterized in vitro and in vivo. Changes at M102, Y105, and E214 affected the self-association as measured by analytical ultracentrifugation and gel filtration. The interaction with 8. suis VirB10 was reduced by changes at T201, and change at 8230 inhibited the interaction with VirB4 in vitro. The in vivo functionality of VirB8 variants was determined by complementation of growth in macrophages by a 8. suis virB8 mutant and by using a heterologous assay of type IV secretion system assembly in Agrobacterium tumefiaciens. Changes at Y105, T201, 8230, and at several other residues impaired the in vivo function of VirB8, suggesting that we have identified interaction sites of relevance in the natural biological context.
机译：VirB8样蛋白是IV型分泌系统的必需成分，IV型分泌系统是细菌毒力因子，介导效应子分子从许多细菌病原体进入真核细胞的转运。基于细胞生物学，遗传和X射线晶体学数据，VirB8被提议进行多种蛋白质相互作用，以介导转运机器的组装。在这里，我们报告了来自哺乳动物病原体猪布鲁氏菌的VirB8的周质结构域的结构功能分析的结果，该结果确定了三种蛋白质-蛋白质相互作用所需的氨基酸残基。拟参与二聚化的残基上的VirB8变体发生了变化，并在体外和体内对蛋白-蛋白相互作用进行了纯化和表征。 M102，Y105和E214处的变化会影响自缔合，这是通过分析超速离心和凝胶过滤测得的。与T8猪的VirB10的相互作用因T201的变化而减少，而8230的变化在体外抑制了与VirB4的相互作用。 VirB8变体的体内功能是通过8种猪suis virB8突变体在巨噬细胞中的生长互补以及通过使用根癌农杆菌中IV型分泌系统装配的异源测定来确定的。 Y105，T201、8230和其他几个残基的变化削弱了VirB8的体内功能，这表明我们已经确定了与自然生物学相关的相互作用位点。

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