LOCK ON OFF DISULFIDES IDENTIFY THE TRANSMEMBRANE SIGNALING HELIX OF THE ASPARTATE RECEPTOR
LOCK ON OFF DISULFIDES IDENTIFY THE TRANSMEMBRANE SIGNALING HELIX OF THE ASPARTATE RECEPTOR
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DOI：
10.1074/jbc.270.41.24043
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发表时间：
1995-10-13
影响因子：
4.8
通讯作者：
FALKE, JJ
中科院分区：
文献类型：
--
作者：
CHERVITZ, SA;FALKE, JJ
细菌趋化途径的天冬氨酸受体调节胞质组氨酸激酶响应配体结合的自磷酸化速率。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。目前的工作使用工程化的半胱氨酸和二硫键来探测这种构象变化的性质，特别关注第二跨膜α-螺旋的作用。总共26个修饰，由13个半胱氨酸对和相应的二硫键组成，已被引入到第二跨膜螺旋和相邻螺旋之间的接触。这些修饰对跨膜信号的影响已通过体外测定进行定量，所述体外测定测量（i）配体结合，（ii）受体介导的激酶活性调节，和（iii）受体甲基化。所有这三个参数被观察到是高度敏感的扰动的第二跨膜螺旋。特别是，26项修改中有13项（6个半胱氨酸对和7个二硫化物）显著增加或降低天冬氨酸亲和力，而26个修饰中的15个修饰（5个半胱氨酸对和10个二硫化物）破坏跨膜激酶调节，重要的是，发现其中3种干扰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，这表明可以使用工程化的二硫化物来锁定受体蛋白的信号传导功能。该研究的一个单独方面探索了第二跨膜螺旋的热运动：观察到设计用于捕获大幅度扭转运动的4个二硫化物破坏功能但易于形成，表明螺旋是移动的。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跨膜信号传递的移动的信号元件。
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 (5 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.