Bren A, Eisenbach M
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, 76100, Israel.
J Mol Biol. 1998 May 8;278(3):507-14. doi: 10.1006/jmbi.1998.1730.
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 approximately 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 approximately P at the switch is the protein FliM. The purpose of this study was to identify the CheY-binding domain of FliM. We cloned 17 fliM 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 crosslinking. All the mutant proteins with an amino acid substitution at the N terminus, FliM6LI, FliM7SY and FliM10EG, bound CheY approximately P to a much lesser extent than did wild-type FliM. CheY approximately 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 FliM (residues 6 to 15). The peptide bound CheY and, to a larger extent, CheY approximately P. It also competed with full-length FliM on CheY approximately 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.
鼠伤寒沙门氏菌及相关细菌物种在趋化作用期间信号转导的一个关键事件是应答调节蛋白CheY的磷酸化形式（CheYP）与位于鞭毛马达基部的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。CheYP在开关处的对接位点是蛋白质FliM。本研究的目的是鉴定FliM的CheY结合结构域。我们克隆了17个fliM突变体，每个突变体在开关功能上存在缺陷且在不同位置有一个点突变，然后对其产物进行过表达和纯化。通过化学交联确定每个FliM突变蛋白的CheY结合能力。所有在N端有氨基酸替代的突变蛋白，即FliM6LI、FliM7SY和FliM10EG，与CheYP的结合程度比野生型FliM小得多。其他突变蛋白与CheYP的结合与野生型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，我们合成了一种由FliM的前16个氨基酸残基组成的肽，包括FliM的一个高度保守区域（第6至15位残基）。该肽与CheY结合，并且在更大程度上与CheYP结合。它还在CheYP上与全长FliM竞争。这些结果表明，FliM的CheY结合结构域位于N端，在第1至16位残基内，并表明FliM单体可以形成一个完整的CheY结合位点。
