Ramifications of kinetic partitioning on usher-mediated pilus biogenesis
Ramifications of kinetic partitioning on usher-mediated pilus biogenesis
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DOI：
10.1093/emboj/17.8.2177
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发表时间：
1998-04-15
期刊：
影响因子：
11.4
通讯作者：
Hultgren, SJ
中科院分区：
文献类型：
--
作者：
Saulino, ET;Thanassi, DG;Hultgren, SJ
多种革兰氏阴性细菌中不同黏附结构的生物发生依赖于伴侣/usher通路。对于usher蛋白如何在外膜上转运蛋白亚基，以及这些粘附结构如何发生组装，我们所知甚少。我们已经发现了usher蛋白调节1型菌毛有序组装的几种机制，特别是通过伴侣-粘附蛋白复合物与usher蛋白的关键相互作用。利用表面等离子体共振实时研究了伴侣-亚基复合物与引子的结合和解离事件，发现伴侣-粘附复合物与引子的结合最紧密、最快。这表明，伴侣-粘附复合物与usher的动力学分配是粘附素在菌毛中尖端定位的决定性因素。此外，我们鉴定并纯化了一种体内形成的伴侣-粘附-usher组装中间体。胰蛋白酶消化试验表明，该复合体中的引子处于改变的构象中，这在菌毛组装期间保持不变。数据支持一个模型，在该模型中，伴侣-粘附蛋白复合物与引子的结合稳定了引子的组装能力构象，并允许启动毛组装。
The biogenesis of diverse adhesive structures in a variety of Gram-negative bacterial species is dependent on the chaperone/usher pathway. Very little is known about how the usher protein translocates protein subunits across the outer membrane or how assembly of these adhesive structures occurs. We have discovered several mechanisms by which the usher protein acts to regulate the ordered assembly of type 1 pili, specifically through critical interactions of the chaperone-adhesin complex with the usher. A study of association and dissociation events of chaperone-subunit complexes with the usher in real time using surface plasmon resonance revealed that the chaperone-adhesin complex has the tightest and fastest association with the usher. This suggests that kinetic partitioning of chaperone-adhesin complexes to the usher is a defining factor in tip localization of the adhesin in the pilus, Furthermore, we identified and purified a chaperone-adhesin-usher assembly intermediate that was formed in vivo. Trypsin digestion assays showed that the usher in this complex was in an altered conformation, which was maintained during pilus assembly. The data support a model in which binding of the chaperone-adhesin complex to the usher stabilizes the usher in an assembly-competent conformation and allows initiation of pilus assembly.