Assembly and stoichiometry of the core structure of the bacterial flagellar type III export gate complex.
Assembly and stoichiometry of the core structure of the bacterial flagellar type III export gate complex.
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DOI：
10.1371/journal.pbio.2002281
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发表时间：
2017-08
期刊：
影响因子：
9.8
通讯作者：
Minamino T
中科院分区：
文献类型：
--
作者：
Fukumura T;Makino F;Dietsche T;Kinoshita M;Kato T;Wagner S;Namba K;Imada K;Minamino T
细菌鞭毛III型输出装置，这是所需的鞭毛组装超出细胞膜，由跨膜出口门复合物和细胞质ATP酶复合物。FlhA、FlhB、FliP、FliQ和FliR在基体MS环内形成门复合物，尽管FliO是肠道沙门氏菌中有效形成出口门所必需的。然而，目前还不清楚它们是如何形成门复合体的。在这里，我们报告说，FliP形成一个homohexameric环的直径为10 nm。丙氨酸取代保守的Phe-137，Phe-150，和Glu-178残基的周质域的FliP（FliPP）抑制FliP 6环的形成，抑制鞭毛蛋白输出。FliO形成了一个5 nm的环状结构，具有3个与FliP 6环结合的钳状结构。FliPP的晶体结构和基于结构的光交联实验表明，FliPP的Phe-150和Ser-156参与FliP-FliP相互作用，Phe-150，Arg-152，Ser-156和Pro-158负责FliP-FliO相互作用。FliP的过表达恢复了一个FliO突变体的运动性到野生型水平，这表明FliP 6环是出口门复合物中的一个功能单元，FliO不是最终门结构的一部分。共纯化测定揭示FlhA、FlhB、FliQ和FliR与FliO/FliP复合物相关。我们提出，出口门复合物的组装开始于FliP 6环形成的帮助下的FliO支架，其次是FliQ，FliR，和FlhB，最后FlhA在MS环形成。细菌鞭毛III型出口门复合物是一种膜包埋的纳米机器，负责鞭毛蛋白的出口，并存在于基体MS环中心孔内的一片膜中。在这项工作中，我们调查如何形成的出口门复杂的启动。输出门复合物由5种高度保守的跨膜蛋白组成：FlhA、FlhB、FliP、FliQ和FliR。每个亚基蛋白质组装成门在MS环形成过程中以良好的协调方式。跨膜蛋白FliO是S.肠，但不是必需的鞭毛蛋白输出。在这里，我们进行FliP的生化和结构分析，并提供直接的证据表明，FliP形成一个直径为10 nm的三聚体的二聚体结构。输出门复合物的组装开始于FliP 6环形成，在FliO支架的帮助下，随后是FliQ、FliR和FlhB，最后是在MS环形成期间的FlhA。鉴于鞭毛和毒力因子传递注射体机制之间的结构和功能相似性，我们提出注射体的FliP同系物的周质结构域可能是新型抗生素的良好靶点。
The bacterial flagellar type III export apparatus, which is required for flagellar assembly beyond the cell membranes, consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. FlhA, FlhB, FliP, FliQ, and FliR form the gate complex inside the basal body MS ring, although FliO is required for efficient export gate formation in Salmonella enterica. However, it remains unknown how they form the gate complex. Here we report that FliP forms a homohexameric ring with a diameter of 10 nm. Alanine substitutions of conserved Phe-137, Phe-150, and Glu-178 residues in the periplasmic domain of FliP (FliPP) inhibited FliP6 ring formation, suppressing flagellar protein export. FliO formed a 5-nm ring structure with 3 clamp-like structures that bind to the FliP6 ring. The crystal structure of FliPP derived from Thermotoga maritia, and structure-based photo-crosslinking experiments revealed that Phe-150 and Ser-156 of FliPP are involved in the FliP–FliP interactions and that Phe-150, Arg-152, Ser-156, and Pro-158 are responsible for the FliP–FliO interactions. Overexpression of FliP restored motility of a ∆fliO mutant to the wild-type level, suggesting that the FliP6 ring is a functional unit in the export gate complex and that FliO is not part of the final gate structure. Copurification assays revealed that FlhA, FlhB, FliQ, and FliR are associated with the FliO/FliP complex. We propose that the assembly of the export gate complex begins with FliP6 ring formation with the help of the FliO scaffold, followed by FliQ, FliR, and FlhB and finally FlhA during MS ring formation. The bacterial flagellar type III export gate complex is a membrane-embedded nanomachine responsible for flagellar protein export and exits in a patch of membrane within the central pore of the basal body MS ring. In this work, we investigate how formation of the export gate complex is initiated. The export gate complex is composed of 5 highly conserved transmembrane proteins: FlhA, FlhB, FliP, FliQ, and FliR. Each subunit protein assembles into the gate during MS ring formation in a well-coordinated manner. The transmembrane protein FliO is required for efficient assembly of the export gate complex in S. enterica but is not essential for flagellar protein export. Here we carry out biochemical and structural analyses of FliP and provide direct evidence suggesting that FliP forms a trimer-of-dimer structure with a diameter of 10 nm. The assembly of the export gate complex begins with FliP6 ring formation with the help of the FliO scaffold, followed by FliQ, FliR, and FlhB and finally FlhA during MS ring formation. Given the structural and functional similarities between the flagellar and the virulence-factor-delivering injectisome machineries, we propose that the periplasmic domain of FliP homologues of the injectisome could be a good target for novel antibiotics.
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DOI：
10.1107/s0907444905036693
发表时间：
2006-01-01
影响因子：
2.2
作者：
Evans, P
通讯作者：
Evans, P
影响因子：
3.7
作者：
McMurry JL;Minamino T;Furukawa Y;Francis JW;Hill SA;Helms KA;Namba K
通讯作者：
Namba K
影响因子：
3.7
作者：
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通讯作者：
Samatey FA
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
3.6
作者：
Fan, F;Ohnishi, K;Macnab, RM
通讯作者：
Macnab, RM