Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
J Mol Biol. 2011 Jun 17;409(4):483-95. doi: 10.1016/j.jmb.2011.03.040. Epub 2011 Apr 2.
Characterizing protein-protein interactions in a biologically relevant context is important for understanding the mechanisms of signal transduction. Most signal transduction systems are membrane associated and consist of large multiprotein complexes that undergo rapid reorganization--circumstances that present challenges to traditional structure determination methods. To study protein-protein interactions in a biologically relevant complex milieu, we employed a protein footprinting strategy based on isotope-coded affinity tag (ICAT) reagents. ICAT reagents are valuable tools for proteomics. Here, we show their utility in an alternative application--they are ideal for protein footprinting in complex backgrounds because the affinity tag moiety allows for enrichment of alkylated species prior to analysis. We employed a water-soluble ICAT reagent to monitor cysteine accessibility and thereby to identify residues involved in two different protein-protein interactions in the Escherichia coli chemotaxis signaling system. The chemotaxis system is an archetypal transmembrane signaling pathway in which a complex protein superstructure underlies sophisticated sensory performance. The formation of this superstructure depends on the adaptor protein CheW, which mediates a functionally important bridging interaction between transmembrane receptors and histidine kinase. ICAT footprinting was used to map the surfaces of CheW that interact with the large multidomain histidine kinase CheA, as well as with the transmembrane chemoreceptor Tsr in native E. coli membranes. By leveraging the affinity tag, we successfully identified CheW surfaces responsible for CheA-Tsr interaction. The proximity of the CheA and Tsr binding sites on CheW suggests the formation of a composite CheW-Tsr surface for the recruitment of the signaling kinase to the chemoreceptor complex.
在生物相关环境中描绘蛋白质-蛋白质相互作用对于理解信号转导的机制非常重要。大多数信号转导系统都与膜相关，由经历快速重组的大型多蛋白复合物组成——这种情况给传统的结构确定方法带来了挑战。为了在生物相关的复杂环境中研究蛋白质-蛋白质相互作用，我们采用了基于同位素编码亲和标签（ICAT）试剂的蛋白质足迹策略。ICAT 试剂是蛋白质组学的有价值工具。在这里，我们展示了它们在替代应用中的实用性——它们是复杂背景下蛋白质足迹分析的理想选择，因为亲和标签部分允许在分析之前富集烷基化物种。我们使用水溶性 ICAT 试剂来监测半胱氨酸的可及性，从而鉴定出大肠杆菌趋化信号系统中两种不同蛋白质-蛋白质相互作用涉及的残基。趋化系统是一种典型的跨膜信号通路，其中复杂的蛋白质超结构是复杂感官性能的基础。这种超结构的形成取决于衔接蛋白 CheW，它介导跨膜受体和组氨酸激酶之间的功能重要桥接相互作用。ICAT 足迹法用于绘制 CheW 与大型多功能组氨酸激酶 CheA 以及天然大肠杆菌膜中的跨膜化学感受器 Tsr 相互作用的表面。通过利用亲和标签，我们成功鉴定了 CheW 表面与 CheA-Tsr 相互作用有关的部分。CheA 和 Tsr 结合位点在 CheW 上的接近性表明形成了一个复合的 CheW-Tsr 表面，用于将信号激酶募集到化学感受器复合物。
