Solà M, López-Hernández E, Cronet P, Lacroix E, Serrano L, Coll M, Párraga A
Institut de Biologia Molecular de Barcelona, CSIC, Jordi Girona 18-26, 08034, Barcelona, Spain.
J Mol Biol. 2000 Oct 20;303(2):213-25. doi: 10.1006/jmbi.2000.4507.
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 Mg(2+) 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 alpha 4, 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 alpha 4, and helps to understand how small movements at the active site are transmitted and amplified to the protein surface.
信号转导蛋白CheY呈现出α/β平行的多肽折叠结构，包括一个高度不稳定的α4螺旋和一个电荷密集的活性位点。α4螺旋在不同的晶体结构中呈现出不同的位置和构象，表明它是一个可移动的片段。此外，该螺旋的不稳定性被认为具有功能意义，因为它参与了与信号转导蛋白激酶CheA、靶蛋白FliM和磷酸酶CheZ的蛋白质-蛋白质相互作用。CheY的活性位点由三个天冬氨酸残基和一个赖氨酸残基组成，它们都参与蛋白质激活所需的Mg(2+)的结合。研究CheY磷酸化激活机制分两步进行：首先，我们将活性位点的三个天冬氨酸残基独立替换为丙氨酸；其次，在α4螺旋中设计了几个突变，以提高其稳定性并改善其与蛋白质核心的堆积。对这些突变蛋白的结构和热力学分析进一步证明了活性位点区域与α4螺旋之间的联系，并有助于理解活性位点的微小运动是如何传递并放大到蛋白质表面的。
