The N terminus of the flagellar switch protein, FliM, is the binding domain for the chemotactic response regulator, CheY
The N terminus of the flagellar switch protein, FliM, is the binding domain for the chemotactic response regulator, CheY
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DOI：
10.1006/jmbi.1998.1730
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发表时间：
1998-05-08
影响因子：
5.6
通讯作者：
Eisenbach, M
中科院分区：
文献类型：
--
作者：
Bren, A;Eisenbach, M
鼠伤寒沙门氏菌和相关细菌物种趋化过程中信号转导的一个关键事件是反应调节剂 CheY 的磷酸化形式（CheY 类似于 P）与位于其基部的鞭毛运动开关之间的相互作用。这种相互作用的结果是鞭毛旋转方向从默认的逆时针方向转变为顺时针方向。 CheY 与开关处的 P 相似的对接位点是蛋白质 FliM。本研究的目的是鉴定 FliM 的 CheY 结合域。我们克隆了 17 个 fliM 突变体，每个突变体都有转换缺陷并在不同位置有点突变，然后过表达并纯化了它们的产物。通过化学交联测定每种 FliM 突变蛋白的 CheY 结合能力。所有在 N 末端具有氨基酸取代的突变蛋白 FliM6LI、FliM7SY 和 FliM10EG 与 P 类似的 CheY 结合程度比野生型 FliM 低得多。 CheY 与其他突变蛋白的 P 结合相似，与野生型 FliM 相似。为了研究包含这三个突变的 FliM 结构域是否确实是 CheY 结合结构域，我们合成了由 FliM 的前 16 个氨基酸残基组成的肽，包括 FIM 的高度保守区域（残基 6 至 15）。该肽结合 CheY，并且在更大程度上结合与 P 类似的 CheY。它还与全长 FliM 竞争与 P 类似的 CheY。这些结果表明 FliM 的 CheY 结合结构域位于 N 末端，残基 1 至 16 内，并表明 FliM 单体可以形成完整的 CheY 结合位点。 (C) 1998 学术出版社有限公司。
A key event in signal transduction during chemotaxis of Salmonella typhimurium and related bacterial species is the interaction between the phosphorylated form of the response regulator CheY (CheY similar to P) and the switch of the flagellar motor,:located at its base. The consequence of this interaction is a shift in the direction of flagellar rotation from the default, counterclockwise, to clockwise. The docking site of CheY similar to P at the switch is the protein FliM. The purpose of this study was to identify the CheY-binding domain of FliM. We cloned 17fliM mutants, each defective in switching and having a point mutation at a different location, and then overexpressed and purified their products. The CheY-binding ability of each of the FliM mutant proteins was determined by chemical cross linking. All the mutant proteins with an amino acid substitution at the N terminus, FliM6LI, FliM7SY and FliM10EG, bound CheY similar to P to a much lesser extent than did wild-type FliM. CheY similar to P-binding of the other mutant proteins was similar to wild-type FliM. To investigate whether the FliM domain that includes these three mutations is indeed the CheY-binding domain, we synthesized a peptide composed of the first 16 amino acid residues of FliM, including a highly conserved region of FIM (residues 6 to 15). The peptide bound CheY and, to a larger extent, CheY similar to P. It also competed with full-length FliM on CheY similar to P. These results indicate that the CheY-binding domain of FliM is located at the N terminus, within residues 1 to 16, and suggest that FliM monomers can form a complete site for CheY binding. (C) 1998 Academic Press Limited.