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首页> 外文期刊>Biochemistry >TRANSMEMBRANE SIGNALING BY THE ASPARTATE RECEPTOR - ENGINEERED DISULFIDES REVEAL STATIC REGIONS OF THE SUBUNIT INTERFACE
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TRANSMEMBRANE SIGNALING BY THE ASPARTATE RECEPTOR - ENGINEERED DISULFIDES REVEAL STATIC REGIONS OF THE SUBUNIT INTERFACE

机译：铝酸盐受体的跨膜信号-动力消解亚单位界面的暴露静态区。

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摘要

Ligand binding to the periplasmic domain of the transmembrane aspartate receptor generates an intramolecular conformational change which spans the bilayer and ultimately signals the cytoplasmic CheA histidine kinase, thereby triggering chemotaxis. The receptor is a homodimer stabilized by the interface between its two identical subunits: the present study investigates the role of the periplasmic and transmembrane regions of this interface in the mechanism of transmembrane signaling. Free cysteines and disulfide bonds are engineered into selected interfacial positions, and the resulting effects on the transmembrane signal are assayed by monitoring in vitro regulation of kinase activity. Three of the 14 engineered cysteine pairs examined, as well as six of the 14 engineered disulfides, cause perturbations of the interface structure which essentially destroy transmembrane regulation of the kinase. The remaining 11 cysteine pairs, and eight engineered disulfides covalently linking the two subunits at locations spanning positions 18-75, are observed to retain significant transmembrane kinase regulation. The eight functional disulfides positively identify adjacent faces of the two N-terminal helices in the native receptor dimer and indicate that large regions of the periplasmic and transmembrane subunit interface remain effectively static during the transmembrane signal. The results are consistent with a model in which the subunit interface plays a structural role, while the second membrane-spanning helix transmits the ligand-induced signal across the bilayer to the kinase binding domain. The effects of engineered cysteines and disulfides on receptor methylation in vitro are also measured, enabling direct comparison of the in vitro, methylation and phosphorylation assays.
机译：配体与跨膜天冬氨酸受体周质结构域的结合会产生跨双层的分子内构象变化，并最终向细胞质CheA组氨酸激酶发出信号，从而触发趋化性。该受体是通过其两个相同亚基之间的界面稳定的同型二聚体：本研究调查了该界面的周质和跨膜区域在跨膜信号传导机制中的作用。将游离半胱氨酸和二硫键工程化到选定的界面位置，并通过监测激酶活性的体外调节来分析对跨膜信号的最终影响。检查的14个工程半胱氨酸对中的3个以及14个工程二硫化物中的6个引起了界面结构的扰动，这实质上破坏了激酶的跨膜调节。观察到剩余的11个半胱氨酸对和8个工程化的二硫键在跨18-75位的位置共价连接两个亚基，保留了明显的跨膜激酶调节作用。八个功能性二硫键可肯定地识别天然受体二聚体中两个N末端螺旋的相邻面，并表明周质和跨膜亚基界面的大部分区域在跨膜信号期间仍保持有效的静态。结果与其中亚基界面起结构作用的模型一致，而第二跨膜螺旋将配体诱导的信号跨双层传输至激酶结合域。还测量了工程化的半胱氨酸和二硫化物对体外受体甲基化的影响，从而可以直接比较体外，甲基化和磷酸化测定。

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