THE RESPONSE REGULATORS CHEB AND CHEY EXHIBIT COMPETITIVE-BINDING TO THE KINASE CHEA
THE RESPONSE REGULATORS CHEB AND CHEY EXHIBIT COMPETITIVE-BINDING TO THE KINASE CHEA
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DOI：
10.1021/bi00045a003
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发表时间：
1995-11-14
期刊：
影响因子：
2.9
通讯作者：
WEIS, RM
中科院分区：
文献类型：
--
作者：
LI, JY;SWANSON, RV;WEIS, RM
细菌化学感受信号通路的自磷酸化激酶CheA向两种反应调节蛋白CheY或受体甲基酯酶（切布）中的任一种提供磷酰基。用等温滴定量热法，证明了CheA和由氨基酸残基1-233组成的CheA片段（CheA（1-233））在298 K下分别以相似的解离常数2.0和1.2 μ M与CheY结合，表明CheY结合位点完全在1-233氨基酸位点内。切布与CheA（1-233）结合的K-D为3.2 μ M，也与完整的CheA结合，其亲和力相同。发现CheY与切布竞争结合至CheA（1-233），尽管CheY与切布的调节结构域之间的序列同一性水平低。在两组独立的实验中确定CheY和切布结合的竞争性质：用CheY滴定CheB-CheA（1-233）络合物或用CheY-CheA（1-233）络合物滴定切布的滴定实验，和竞争性亲和层析实验，其使用Ni-NTA螯合树脂作为组氨酸标记的CheA（1-233）的复合物的亲和基质，片段和CheY或切布。还研究了磷酸化、结合位点突变和活性位点突变的影响，以探讨CheY中构象变化作为CheY-CheA相互作用的调节机制的影响。发现在过量EDTA存在下，磷酸化的CheY对CheA（1-233）的亲和力降低2倍，并且6 mM Mg 2+进一步降低磷酸化的CheY对CheA（1-233）的亲和力（约100 μ mol/L）。3倍），尽管Mg 2+本身对切布或CheY与CheA的相互作用没有影响（1-233）。因此，数据表明磷酸化的CheY在生理条件下对CheA具有显著较低的亲和力。磷酸化可能会引起显着的构象变化，降低的CheY-CheA相互作用的强度的想法，支持的相对值的相关常数测定的CheY活性位点和结合位点突变体。CheY中远离磷酸化位点的结合位点突变（A103 V）使K-a减少10倍，而活性位点突变产生适度（2倍）减少。
The autophosphorylating kinase CheA of the bacterial chemosensory signaling pathway donates a phosphoryl group to either of two response regulator proteins, CheY or the receptor methylesterase (CheB). With isothermal titration calorimetry, it was demonstrated that CheA and a CheA fragment composed of amino acid residues 1-233 (CheA(1-233)) bound to CheY with similar dissociation constants of 2.0 and 1.2 mu M at 298 K respectively, indicating that the CheY binding site is wholly within the 1-233 amino acid locus. CheB bound to CheA(1-233) With a K-D Of 3.2 mu M, and also bound to intact CheA with the same affinity. CheY was found to compete with CheB for binding to CheA(1-233), in spite of the low level of sequence identity between CheY and the regulatory domain of CheB. The competitive nature of CheY and CheB binding was determined in two independent sets of experiments: titration experiments in which either a CheB-CheA(1-233) complex was titrated with CheY or CheB was titrated with a CheY-CheA(1-233) complex, and competitive affinity chromatography experiments that used Ni-NTA-chelating resin as an affinity matrix for complexes of the histidine-tagged CheA(1-233) fragment and CheY or CheB. The effects of phosphorylation, binding-site mutations, and active-site mutations were also studied to probe the influence of conformational changes in CheY as a regulatory mechanism of CheY-CheA interactions. Phosphorylated CheY, in the presence of excess EDTA, was found to have a 2-fold lower affinity for CheA(1-233), and 6 mM Mg2+ further reduced the affinity of phosphorylated CheY for CheA(1-233) (ca. 3-fold), although Mg2+ On it, own had no effect on the interactions of either CheB or CheY with CheA(1-233) The data thus indicate that phosphorylated CheY has a significantly lower affinity for CheA under physiological conditions. The idea that phosphorylation may induce a significant conformational change, reducing the strength of the CheY-CheA interactions, is supported by the relative values of the association constants measured for CheY active-site and binding-site mutants. A binding-site mutation (A103V) in CheY, which is remote from the site of phosphorylation produced a 10-fold reduction in K-a, whereas active-site mutations produced a modest (2-fold) reduction.