The structures of T87I phosphono-CheY and T87I/Y106W phosphono-CheY help to explain their binding affinities to the FliM and CheZ peptides.
The structures of T87I phosphono-CheY and T87I/Y106W phosphono-CheY help to explain their binding affinities to the FliM and CheZ peptides.
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DOI：
10.1016/j.abb.2008.08.019
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发表时间：
2008-11-15
影响因子：
3.9
通讯作者：
Halkides CJ
中科院分区：
文献类型：
--
作者：
McAdams K;Casper ES;Matthew Haas R;Santarsiero BD;Eggler AL;Mesecar A;Halkides CJ
Chey是细菌趋化作用的反应调节因子。大肠杆菌Chey突变体T87I和T87I/Y106W在Asp57上可以磷酸化，但不能产生鞭毛的顺时针旋转。为了从结构上了解这一表型，我们合成了两个Chey-P突变体的稳定类似物：T87I Phono-Chey和T87I Phono-Chey。测定了来自鞭毛运动蛋白Flim和磷酸酶CHIZ的多肽对磷酸-Chey和两个突变体的解离常数。这些多肽与Phono-Chey的结合强度几乎与Chey-P一样强，但它们不能与T87I Phono-Chey或T87I/Y106W Phono-Chey结合，这意味着突变蛋白在体内不能紧密结合FILM或CHEZ。T87I膦-Chey和T87I/Y106W膦-Chey的结构分别被解析到1.8？和2.4？I87的体积增加迫使Y106或W106的侧链进入更易溶于溶剂的构象，从而封闭了肽结合部位。
CheY is a response regulator in bacterial chemotaxis. E. coli CheY mutants T87I and T87I/Y106W CheY are phosphorylatable on Asp57 but unable to generate clockwise rotation of the flagella. To understand this phenotype in terms of structure, stable analogs of the two CheY-P mutants were synthesized: T87I phosphono-CheY and T87I phosphono-CheY. Dissociation constants for peptides derived from flagellar motor protein FliM and phosphatase CheZ were determined for phosphono-CheY and the two mutants. The peptides bind phosphono-CheY almost as strongly as CheY-P; however, they do not bind T87I phosphono-CheY or T87I/Y106W phosphono-CheY, implying that the mutant proteins cannot bind FliM or CheZ tightly in vivo. The structures of T87I phosphono-CheY and T87I/Y106W phosphono-CheY were solved to resolutions of 1.8 Å and 2.4 Å, respectively. The increased bulk of I87 forces the side chain of Y106 or W106, into a more solvent-accessible conformation, which occludes the peptide-binding site.
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