Protein footprinting in a complex milieu: identifying the interaction surfaces of the chemotaxis adaptor protein CheW.
Protein footprinting in a complex milieu: identifying the interaction surfaces of the chemotaxis adaptor protein CheW.
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DOI：
10.1016/j.jmb.2011.03.040
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发表时间：
2011-06-17
影响因子：
5.6
通讯作者：
Kiessling LL
中科院分区：
文献类型：
--
作者：
Underbakke ES;Zhu Y;Kiessling LL
在生物学相关的背景下表征蛋白质-蛋白质相互作用对于理解信号转导机制非常重要。大多数信号转导系统都是膜相关的，由大型多蛋白复合物组成，这些复合物会经历快速重组，这对传统的结构测定方法提出了挑战。为了研究生物相关的复杂环境中蛋白质-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重要工具。在这里，我们展示了它们在替代应用中的实用性——它们是复杂背景中蛋白质足迹的理想选择，因为亲和标签部分允许在分析之前富集烷基化物质。我们采用水溶性 ICAT 试剂来监测半胱氨酸的可及性，从而识别大肠杆菌趋化信号系统中参与两种不同蛋白质-蛋白质相互作用的残基。趋化系统是一种典型的跨膜信号传导途径，其中复杂的蛋白质超结构是复杂的感觉性能的基础。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功能上重要的桥接相互作用。 ICAT 足迹法用于绘制与大的多域组氨酸激酶 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 CheW 表面。通过利用亲和标签，我们成功地识别了负责 CheA 和 Tsr 相互作用的 CheW 表面。 CheW 上 CheA 和 Tsr 结合位点的接近表明形成了复合 Tsr-CheW 表面，用于将信号激酶招募到化学感受器复合物。
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, multi-protein complexes that undergo rapid reorganization, circumstances that present challenges to traditional structure determination methods. To study protein–protein interactions in 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 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 complex protein superstructure underlies sophisticated sensory performance. The formation of this superstructure depends upon the adaptor protein CheW, which mediates a functionally important bridging interaction between the transmembrane receptors and the histidine kinase. ICAT footprinting was used to map the surfaces of CheW that interact with the large, multi-domain histidine kinase CheA as well as the transmembrane chemoreceptor Tsr in native E. coli membranes. By leveraging the affinity tag, we successfully identified CheW surfaces responsible for CheA and Tsr interaction. The proximity of the CheA and Tsr binding sites on CheW suggests the formation of a composite Tsr-CheW surface for recruitment of the signaling kinase to the chemoreceptor complex.
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影响因子：
3.6
作者：
Briegel, Ariane;Ding, H. Jane;Jensen, Grant J.
通讯作者：
Jensen, Grant J.
影响因子：
5.6
作者：
Cliff, Matthew J.;Alizadeh, Tooba;Waltho, Jonathan P.
通讯作者：
Waltho, Jonathan P.
DOI：
10.1073/pnas.092071899
发表时间：
2002-05-14
影响因子：
11.1
作者：
Ames, P;Studdert, CA;Parkinson, JS
通讯作者：
Parkinson, JS
DOI：
10.1073/pnas.86.4.1208
发表时间：
1989-02-01
影响因子：
11.1
作者：
BORKOVICH, KA;KAPLAN, N;SIMON, MI
通讯作者：
SIMON, MI
影响因子：
15
作者：
Hamel, DJ;Dahlquist, FW
通讯作者：
Dahlquist, FW