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首页> 外文期刊>Journal of bacteriology >Proposed Signal Transduction Role for Conserved CheY Residue Thr87, a Member of the Response Regulator Active-Site Quintet
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Proposed Signal Transduction Role for Conserved CheY Residue Thr87, a Member of the Response Regulator Active-Site Quintet

机译：保守的CheY残基Thr87的拟议信号传导作用

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CheY serves as a structural prototype for the response regulator proteins of two-component regulatory systems. Functional roles have previously been defined for four of the five highly conserved residues that form the response regulator active site, the exception being the hydroxy amino acid which corresponds to Thr87 in CheY. To investigate the contribution of Thr87 to signaling, we characterized, genetically and biochemically, several cheY mutants with amino acid substitutions at this position. The hydroxyl group appears to be necessary for effective chemotaxis, as a Thr→Ser substitution was the only one of six tested which retained a Che+ swarm phenotype. Although nonchemotactic, cheY mutants with amino acid substitutions T87A and T87C could generate clockwise flagellar rotation either in the absence of CheZ, a protein that stimulates dephosphorylation of CheY, or when paired with a second site-activating mutation, Asp13→Lys, demonstrating that a hydroxy amino acid at position 87 is not essential for activation of the flagellar switch. All purified mutant proteins examined phosphorylated efficiently from the CheA kinase in vitro but were impaired in autodephosphorylation. Thus, the mutant CheY proteins are phosphorylated to a greater degree than wild-type CheY yet support less clockwise flagellar rotation. The data imply that Thr87 is important for generating and/or stabilizing the phosphorylation-induced conformational change in CheY. Furthermore, the various position 87 substitutions differentially affected several properties of the mutant proteins. The chemotaxis and autodephosphorylation defects were tightly linked, suggesting common structural elements, whereas the effects on self-catalyzed and CheZ-mediated dephosphorylation of CheY were uncorrelated, suggesting different structural requirements for the two dephosphorylation reactions.
机译：CheY作为两组分调节系统的响应调节蛋白的结构原型。先前已经对形成应答调节活性位点的五个高度保守残基中的四个残基中的四个定义了功能性作用，只是羟基氨基酸对应于CheY中的Thr87。为了研究Thr87对信号转导的贡献，我们在遗传和生物化学上表征了几个在该位置具有氨基酸取代的 cheY 突变体。羟基似乎是有效趋化性所必需的，因为Thr→Ser取代是六个测试中唯一保留Che + 群表型的一个。尽管是非趋化性的，但氨基酸替换为T87A和T87C的 cheY 突变体可以在不存在CheZ（一种刺激CheY磷酸化的蛋白质）的情况下或与第二个位点激活突变配对时产生顺时针鞭毛旋转Asp13→Lys，表明87位的羟基氨基酸对于鞭毛开关的激活不是必需的。所有纯化的突变蛋白均在体外有效地从CheA激酶磷酸化，但在自身去磷酸化中受损。因此，与野生型CheY相比，突变型CheY蛋白的磷酸化程度更高，但支持的顺时针鞭毛旋转较少。数据表明，Thr87对于产生和/或稳定CheY的磷酸化诱导的构象变化很重要。此外，87位的各种取代差异地影响了突变蛋白的几种特性。化学趋化性和自体去磷酸化缺陷紧密相连，表明共同的结构元素，而对CheY的自催化和CheZ介导的去磷酸化的影响是不相关的，这表明两个去磷酸化反应的结构要求不同。

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