Residues of the cytoplasmic domain of MotA essential for torque generation in the bacterial flagellar motor.
Residues of the cytoplasmic domain of MotA essential for torque generation in the bacterial flagellar motor.
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MotA 胞质结构域的残基对于细菌鞭毛运动中扭矩的产生至关重要。
DOI：
10.1006/jmbi.1997.1316
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Blair,DF
中科院分区：
文献类型：
--
作者：
Zhou,J;Blair,DF
大肠杆菌的 MotA 蛋白是鞭毛的一个组成部分，与 MotB 一起在跨膜质子传导中发挥作用。 MotA 和 MotB 被认为形成鞭毛马达的定子。它们是完整的膜蛋白； MotA 在细胞质中具有一个大的结构域（约 22 kDa），而 MotB 的结构域小得多（约 3 kDa）。最近的研究表明，MotA 和/或 MotB 的细胞质部分可能存在于电机中产生扭矩的活性位点。为了验证 MotA 的细胞质结构域在扭矩产生中发挥作用的提议，并确定对功能最重要的氨基酸残基，我们对该结构域进行了突变分析。使用随机诱变，分离出 MotA 细胞质残基的许多突变，这些突变要么消除或削弱扭矩的产生。在大多数情况下，受影响的残基并不保守，并且许多替换涉及脯氨酸残基的丢失或增加，这表明这些突变通过改变蛋白质构象而不是直接影响活性位点的残基来破坏功能。使用定点诱变，MotA 细胞质结构域中的保守残基被单独替换，或者在带电残基的情况下以各种组合替换。结果确定了 MotA 的四个对运动功能很重要的残基。它们是位于细胞质结构域的 Arg90 和 Glu98，以及位于细胞质结构域和跨膜结构域之间的界面的 Pro173 和 Pro222。讨论了这些残基在扭矩产生中的可能作用。
The MotA protein of Escherichia coli is a component of the flagellum that functions, together with MotB, in transmembrane proton conduction. MotA and MotB are believed to form the stator of the flagellar motor. They are integral membrane proteins; MotA has a large (ca 22 kDa) domain in the cytoplasm, and MotB a much smaller one (ca 3 kDa). Recent work suggests that cytoplasmically located parts of MotA and/or MotB might be present at the active site for torque generation in the motor. To test the proposal that the cytoplasmic domain of MotA functions in torque generation, and to identify the amino acid residues most important for function, we have carried out a mutational analysis of this domain. Using random mutagenesis, many mutations of cytoplasmic residues of MotA were isolated, which either abolish or impair torque generation. In most cases the residues affected are not conserved, and many of the replacements involve loss or gain of a proline residue, which suggests that these mutations disrupt function by altering the protein conformation rather than by directly affecting residues of an active site. Using site-directed mutagenesis, the conserved residues in the cytoplasmic domain of MotA were replaced, either singly or, in the case of charged residues, in various combinations. The results identify four residues of MotA that are important for motor function. These are Arg90 and Glu98, located in the cytoplasmic domain, and Pro173 and Pro222, located at the interface between the cytoplasmic domain and the membrane-spanning domain. Possible roles for these residues in torque generation are discussed.