Proposed signal transduction role for conserved CheY residue Thr87, a member of the response regulator active-site quintet
Proposed signal transduction role for conserved CheY residue Thr87, a member of the response regulator active-site quintet
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DOI：
10.1128/jb.180.14.3563-3569.1998
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发表时间：
1998-07-01
影响因子：
3.2
通讯作者：
Bourret, RB
中科院分区：
文献类型：
--
作者：
Appleby, JL;Bourret, RB
CheY作为双组分调节系统的反应调节蛋白的结构原型。功能作用先前已被定义为四个高度保守的残基，形成的反应调节剂活性位点，例外的是羟基氨基酸，其对应于Thr 87在CheY，为了调查的贡献Thr 87信号，我们的特点，遗传和生化，几个cheY突变体的氨基酸取代在这个位置。羟基似乎是有效趋化性所必需的，因为Thr->Ser取代是六个测试的保留Che（+）群表型的唯一一个。尽管具有非趋化性，但具有氨基酸取代T87 A和T87 C的cheY突变体可以在不存在CheZ（一种刺激CheY去磷酸化的蛋白质）的情况下或当与第二个位点激活突变Asp 13->Lys配对时产生顺时针鞭毛旋转，这表明87位的羟基氨基酸对于鞭毛开关的激活不是必需的，所有纯化的突变体蛋白质检查磷酸化有效地从CheA激酶在体外，但受损的自去磷酸化。因此，突变体CheY蛋白被磷酸化的程度比野生型CheY Set支持较少的顺时针鞭毛旋转。这些数据暗示Thr 87对于产生和/或稳定磷酸化诱导的CheY构象变化是重要的。此外，不同的位置87取代差异影响突变蛋白的几个特性。趋化性和自身去磷酸化缺陷紧密相连，这表明共同的结构元素，而对自催化和CheZ介导的CheY去磷酸化的影响是不相关的，这表明两个去磷酸化反应的不同结构要求。
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 Set 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.