Towards understanding a molecular switch mechanism:: Thermodynamic and crystallographic studies of the signal transduction protein CheY
Towards understanding a molecular switch mechanism:: Thermodynamic and crystallographic studies of the signal transduction protein CheY
复制标题
DOI：
10.1006/jmbi.2000.4507
复制
发表时间：
2000-10-20
影响因子：
5.6
通讯作者：
Párraga, A
中科院分区：
文献类型：
--
作者：
Solà, M;López-Hernández, E;Párraga, A
信号转导蛋白CheY显示出α/β平行多肽折叠，包括高度不稳定的螺旋α 4和带强电荷的活性位点。阻碍α 4在不同的晶体结构中具有不同的位置和构象，表明它是一个移动的片段。此外，这种螺旋的不稳定性被认为具有功能意义，因为它涉及与递质蛋白激酶CheA、靶蛋白FliM和磷酸酶CheZ的蛋白质-蛋白质接触。CheY的活性位点包括三个天冬氨酸残基和一个赖氨酸残基的簇，所有这些残基都参与蛋白质活化所需的Mg 2+的结合。两个步骤来研究磷酸化后CheY的激活机制：首先，我们独立地用丙氨酸取代活性位点中的三个天冬氨酸残基;其次，在螺旋α 4中设计了几个突变，以增加其稳定性水平并改善其对蛋白质核心的包装。这些突变蛋白的结构和热力学分析提供了活性位点区域和螺旋α 4之间联系的进一步证据，并有助于理解活性位点的微小运动如何传递和放大到蛋白质表面。(C)北京大学出版社.
The signal transduction protein CheY displays an alpha/beta -parallel polypeptide folding, including a highly unstable helix alpha4 and a strongly charged active site. Helix alpha4 has been shown to adopt various positions and conformations in different crystal structures, suggesting that it is a mobile segment. Furthermore, the instability of this helix is believed to have functional significance because it is involved in protein-protein contacts with the transmitter protein kinase CheA, the target protein FliM and the phosphatase CheZ. The active site of CheY comprises a cluster of three aspartic acid residues and a lysine residue, all of which participate in the binding of the Mg2+ needed for the protein activation. Two steps were followed to study the activation mechanism of CheY upon phosphorylation: first, we independently substituted the three aspartic acid residues in the active site with alanine; second, several mutations were designed in helix alpha4, both to increase its level of stability and to improve its packing against the protein core. The structural and thermodynamic analysis of these mutant proteins provides further evidence of the connection between the active-site area and helix alpha4, and helps to understand how small movements at the active site are transmitted and amplified to the protein surface. (C) 2000 Academic Press.