Boukhvalova Marina, VanBruggen Ricaele, Stewart Richard C
Department of Cell Biology and Molecular Genetics and Graduate Program in Molecular and Cellular Biology, University of Maryland, College Park, Maryland 20742, USA.
J Biol Chem. 2002 Jun 28;277(26):23596-603. doi: 10.1074/jbc.M202288200. Epub 2002 Apr 18.
Chemotactic responses of Escherichia coli to aspartic acid are initiated by a ternary protein complex composed of Tar (chemoreceptor), CheA (kinase), and CheW (a coupling protein that binds to both Tar and CheA and links their activities). We used a genetic selection based on the yeast two-hybrid assay to identify nine cheW point mutations that specifically disrupted CheW interaction with CheA but not with Tar. We sequenced these single point mutants and purified four of the mutant CheW proteins for detailed biochemical characterizations that demonstrated the weakened affinity of the mutant CheW proteins for CheA, but not for Tar. In the three-dimensional structure of CheW, the positions affected by these mutations cluster on one face of the protein, defining a potential binding interface for interaction of CheW with CheA. We used a similar two-hybrid approach to identify four mutation sites that disrupted CheW binding to Tar. Mapping of these "Tar-sensitive" mutation sites and those from previous suppressor analysis onto the structure of CheW defined an extended surface on a face of the protein that is adjacent to the CheA-binding surface and that may serve as an interface for CheW binding to Tar.
大肠杆菌对天冬氨酸的趋化反应由一种三元蛋白复合物引发，该复合物由Tar（化学感受器）、CheA（激酶）和CheW（一种偶联蛋白，它与Tar和CheA都结合并连接它们的活性）组成。我们基于酵母双杂交试验进行了遗传筛选，以鉴定九个cheW点突变，这些突变特异性地破坏了CheW与CheA的相互作用，但不影响其与Tar的相互作用。我们对这些单点突变体进行了测序，并纯化了四个突变的CheW蛋白用于详细的生化特性分析，结果表明突变的CheW蛋白对CheA的亲和力减弱，但对Tar的亲和力未受影响。在CheW的三维结构中，受这些突变影响的位置聚集在蛋白质的一个面上，确定了CheW与CheA相互作用的潜在结合界面。我们使用类似的双杂交方法鉴定了四个破坏CheW与Tar结合的突变位点。将这些“Tar敏感”突变位点以及先前抑制子分析中的位点映射到CheW的结构上，确定了蛋白质一个面上的一个扩展表面，该表面与CheA结合表面相邻，可能作为CheW与Tar结合的界面。
