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锁定/解锁二硫键可确定天冬氨酸受体的跨膜信号螺旋结构。

Lock on/off disulfides identify the transmembrane signaling helix of the aspartate receptor.

作者信息

Chervitz S A, Falke J J

机构信息

Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.

出版信息

J Biol Chem. 1995 Oct 13;270(41):24043-53. doi: 10.1074/jbc.270.41.24043.

Abstract

The aspartate receptor of the bacterial chemotaxis pathway regulates the autophosphorylation rate of a cytoplasmic histidine kinase in response to ligand binding. The transmembrane signal, which is transmitted from the periplasmic aspartate-binding domain to the cytoplasmic regulatory domain, is carried by an intramolecular conformational change within the homodimeric receptor structure. The present work uses engineered cysteines and disulfide bonds to probe the nature of this conformational change, focusing in particular on the role of the second transmembrane alpha-helix. Altogether 26 modifications, consisting of 13 cysteine pairs and the corresponding disulfide bonds, have been introduced into the contacts between the second transmembrane helix and adjacent helices. The effects of these modifications on the transmembrane signal have been quantified by in vitro assays which measure (i) ligand binding, (ii) receptor-mediated regulation of kinase activity, and (iii) receptor methylation. All three parameters are observed to be highly sensitive to perturbations of the second transmembrane helix. In particular, 13 of the 26 modifications (6 cysteine pairs and 7 disulfides) significantly increase or decrease aspartate affinity, while 15 of the 26 modifications (6 cysteine pairs and 10 disulfides) destroy transmembrane kinase regulation. Importantly, 3 of the perturbing disulfides are found to lock the receptor in the "on" or "off" signaling state by covalently constraining the second transmembrane helix, demonstrating that it is possible to use engineered disulfides to lock the signaling function of a receptor protein. A separate aspect of the study probes the thermal motions of the second transmembrane helix: 4 disulfides designed to trap large amplitude twisting motions are observed to disrupt function but form readily, suggesting that the helix is mobile. Together the results support a model in which the second transmembrane helix is a mobile signaling element responsible for communicating the transmembrane signal.

摘要

细菌趋化途径中的天冬氨酸受体响应配体结合来调节细胞质组氨酸激酶的自磷酸化速率。跨膜信号从周质天冬氨酸结合结构域传递到细胞质调节结构域,由同二聚体受体结构内的分子内构象变化携带。本研究利用工程化的半胱氨酸和二硫键来探究这种构象变化的本质,特别关注第二个跨膜α螺旋的作用。总共26种修饰,包括13对半胱氨酸对和相应的二硫键,已被引入到第二个跨膜螺旋与相邻螺旋之间的接触部位。这些修饰对跨膜信号的影响已通过体外测定进行量化,这些测定测量(i)配体结合,(ii)受体介导的激酶活性调节,以及(iii)受体甲基化。观察到所有这三个参数对第二个跨膜螺旋的扰动都高度敏感。特别是,26种修饰中的13种(6对半胱氨酸对和7个二硫键)显著增加或降低了天冬氨酸亲和力,而26种修饰中的15种(6对半胱氨酸对和十10个二硫键)破坏了跨膜激酶调节。重要的是,发现3个干扰性二硫键通过共价约束第二个跨膜螺旋将受体锁定在“开启”或“关闭”信号状态,表明可以使用工程化二硫键来锁定受体蛋白的信号功能。该研究的另一个方面探究了第二个跨膜螺旋的热运动:观察到4个设计用于捕获大幅度扭曲运动的二硫键破坏了功能但易于形成,表明该螺旋是可移动的。这些结果共同支持了一个模型,其中第二个跨膜螺旋是一个负责传递跨膜信号的可移动信号元件。

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