The source of high signal cooperativity in bacterial chemosensory arrays
The source of high signal cooperativity in bacterial chemosensory arrays
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DOI：
10.1073/pnas.1600216113
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发表时间：
2016-03-22
影响因子：
11.1
通讯作者：
Parkinson, John S.
中科院分区：
文献类型：
--
作者：
Pinas, German E.;Frank, Vered;Parkinson, John S.
大肠杆菌化学感觉系统由大量跨膜化学受体组成，这些跨膜化学受体与专用的组氨酸激酶CHEA和连接蛋白CHEW有关，CHEA活性与受体控制有关。激酶活性对受体配体占有率变化的响应可以是高度协同的，反映了多个CHEA和受体分子的变构偶联。最近的结构和功能研究导致了一种工作模型，在该模型中，受体核心复合体是信号的最小单位，通过Chew和CheA的P5结构域之间独特的界面2相互作用连接成六角阵列。为了测试这个阵列模型，我们构建并鉴定了CHEA和CHECH突变体，这些突变体在关键界面2残基上有氨基酸替换。突变蛋白在体内的界面2特异性交联试验中被证明是有缺陷的，并形成了分散在细胞膜周围的信号复合体，而不是像野生型化学传感阵列那样聚集在细胞极点。在体内，Interface2突变体对引诱剂刺激的反应下调了CheA的活性，但协同作用比野生型要小得多。此外，含有荧光团标记受体的突变细胞表现出更大的基础各向异性，这种各向异性对吸引剂刺激的反应迅速发生变化，这与松散排列的受体的简单变化一致。我们得出结论，界面2损伤破坏了核心复合体之间的重要网络连接，阻止了受体在大型变构团队中运行。这项工作证实了接口2在组织化学感觉阵列、将簇状阵列引导到细胞极点以及产生其高度协作的信号特性方面的关键作用。
The Escherichia coli chemosensory system consists of large arrays of transmembrane chemoreceptors associated with a dedicated histidine kinase, CheA, and a linker protein, CheW, that couples CheA activity to receptor control. The kinase activity responses to receptor ligand occupancy changes can be highly cooperative, reflecting allosteric coupling of multiple CheA and receptor molecules. Recent structural and functional studies have led to a working model in which receptor core complexes, the minimal units of signaling, are linked into hexagonal arrays through a unique interface 2 interaction between CheW and the P5 domain of CheA. To test this array model, we constructed and characterized CheA and CheW mutants with amino acid replacements at key interface 2 residues. The mutant proteins proved defective in interface 2-specific in vivo cross-linking assays, and formed signaling complexes that were dispersed around the cell membrane rather than clustered at the cell poles as in wild type chemosensory arrays. Interface 2 mutants down-regulated CheA activity in response to attractant stimuli in vivo, but with much less cooperativity than the wild type. Moreover, mutant cells containing fluorophore-tagged receptors exhibited greater basal anisotropy that changed rapidly in response to attractant stimuli, consistent with facile changes in loosely packed receptors. We conclude that interface 2 lesions disrupt important network connections between core complexes, preventing receptors from operating in large, allosteric teams. This work confirms the critical role of interface 2 in organizing the chemosensory array, in directing the clustered array to the cell poles, and in producing its highly cooperative signaling properties.