Coupling between the N- and C-terminal domains influences transducin-alpha intrinsic GDP/GTP exchange.
Coupling between the N- and C-terminal domains influences transducin-alpha intrinsic GDP/GTP exchange.
复制标题
N 端和 C 端结构域之间的偶联影响转导蛋白-α 内在 GDP/GTP 交换。
DOI：
10.1021/bi992156l
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发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
Artemyev,NO
中科院分区：
文献类型：
--
作者：
Muradov,KG;Artemyev,NO
异源三聚体G蛋白α亚基的N-末端区域代表了主要的Gβγ接触位点之一，并参与了与G蛋白偶联受体的相互作用。为了探讨转导素-α N端结构域在G蛋白功能中的作用，我们研究了一种嵌合Gtiα亚基（Ns-Gtiα）与光激活视紫红质（R*）的相互作用。相对于Ns-Gtiα，Gtiα显示出略高的R* 刺激的GTPγS结合率，表明Gtα N端序列适度参与了受体的识别。但Ns-Gtiα的内禀核苷酸交换速率（kapp= 0.014 min-1）明显快于Gtiα（kapp= 0.0013 min-1）。在相互嵌合体Nt-Gsα中，Gsα的42个N端残基被Gtα残基取代，其内在GTPγ S结合率（kapp= 0.0075 min-1）显著低于Gsα（kapp = 0.028 min-1）。Gtα N-末端区域内的残基Val 30（Gsα中的His 41）与C-末端残基Ile 339相互作用。为了验证嵌合Gα亚基中观察到的内在核苷酸交换率变化可能归因于这种相互作用的假设，制备了Gtiα Val 30 His、Gtiα Ile 339 Ala和Ns-Gtiα His 41 Val突变体，并分析了基础GTPγS结合。Gtiα Val 30 His和Gtiα Ile 339 Ala的GTPγS结合率较亲本蛋白增加（kapp分别为0.010和0.009 min-1），而Ns-Gtiα His 41 Val的GTPγS结合率较亲本蛋白降低（kapp= 0.0011 min-1）。这些结果表明，Gtα的N-末端和C-末端结构域之间的偶联对于在未受刺激的转导蛋白中维持低核苷酸交换速率是重要的。
The N-terminal regions of the heterotrimeric G-protein α-subunits represent one of the major Gβγ contact sites and have been implicated in an interaction with G-protein-coupled receptors. To probe the role of the N-terminal domain of transducin-α in G-protein function, a chimeric Gtiα subunit with the 31 N-terminal Gtα residues replaced by the corresponding 42 residues of Gsα (Ns-Gtiα) has been examined for the interaction with light-activated rhodopsin (R*). Gtiα displayed a somewhat higher R*-stimulated rate of GTPγS binding relative to Ns-Gtiα, suggesting modest involvement of the Gtα N-terminal sequence in recognition of the receptor. However, the intrinsic rate of nucleotide exchange in Ns-Gtiα was significantly faster (kapp= 0.014 min-1) than that in Gtiα (kapp= 0.0013 min-1) as judged by the GTPγS binding rates. Substitution of 42 N-terminal residues of Gsα by the Gtα residues in a reciprocal chimera, Nt-Gsα, had an opposite effectnotable reduction in the intrinsic GTPγS-binding rate (kapp= 0.0075 min-1) in comparison with Gsα (kapp= 0.028 min-1). Residue Val30 (His41 in Gsα) within the N-terminal region of Gtα interacts with the C-terminal residue, Ile339. To test the hypothesis that observed changes in the intrinsic nucleotide exchange rate in chimeric Gα subunits might be attributed to this interaction, GtiαVal30His, GtiαIle339Ala, and Ns-GtiαHis41Val mutants have been made and analyzed for basal GTPγS binding. GtiαVal30His and GtiαIle339Ala had increased GTPγS binding rates (kapp= 0.010 and 0.009 min-1, respectively), whereas Ns-GtiαHis41Val had a decreased GTPγS binding rate (kapp= 0.0011 min-1) relative to their parent proteins. These results suggest that the coupling between the N-terminal and C-terminal domains of Gtα is important for maintaining a low nucleotide exchange rate in unstimulated transducin.