Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, Tamil Nadu, India.
FEBS J. 2017 Aug;284(15):2464-2481. doi: 10.1111/febs.14143. Epub 2017 Jul 2.
Deciphering the mechanism of activation of heterotrimeric G proteins by their cognate receptors continues to be an intriguing area of research. The recently solved crystal structure of the ternary complex captured the receptor-bound α-subunit in an open conformation, without bound nucleotide has improved our understanding of the activation process. Despite these advancements, the mechanism by which the receptor causes GDP release from the α-subunit remains elusive. To elucidate the mechanism of activation, we studied guanine nucleotide-induced structural stability of the α-subunit (in response to thermal/chaotrope-mediated stress). Inherent stabilities of the inactive (GDP-bound) and active (GTP-bound) forms contribute antagonistically to the difference in conformational stability whereas the GDP-bound protein is able to switch to a stable intermediate state, GTP-bound protein loses this ability. Partial perturbation of the protein fold reveals the underlying influence of the bound nucleotide providing an insight into the mechanism of activation. An extra stable, pretransition intermediate, 'empty pocket' state (conformationally active-state like) in the unfolding pathway of GDP-bound protein mimics a gating system - the activation process having to overcome this stable intermediate state. We demonstrate that a relatively more complex conformational fold of the GDP-bound protein is at the core of the gating system. We report capturing this threshold, 'metastable empty pocket' conformation (the gate) of α-subunit of G protein and hypothesize that the receptor activates the G protein by enabling it to achieve this structure through mild structural perturbation.
解析异源三聚体 G 蛋白与其同源受体的激活机制仍然是一个有趣的研究领域。最近解决的三元复合物晶体结构捕捉到了受体结合的 α 亚基处于开放构象，没有结合核苷酸，这提高了我们对激活过程的理解。尽管取得了这些进展，但受体如何导致 GDP 从 α 亚基释放的机制仍然难以捉摸。为了阐明激活机制，我们研究了鸟嘌呤核苷酸诱导的 α 亚基结构稳定性（响应热/变构介质介导的应激）。无活性（GDP 结合）和活性（GTP 结合）形式的固有稳定性拮抗地有助于构象稳定性的差异，而 GDP 结合蛋白能够切换到稳定的中间状态，GTP 结合蛋白则失去这种能力。对蛋白质折叠的部分扰动揭示了结合核苷酸的潜在影响，为激活机制提供了深入的了解。在 GDP 结合蛋白的解折叠途径中，一个额外稳定的、预过渡中间态“空口袋”状态（构象活性态样）模拟了一个门控系统 - 激活过程必须克服这个稳定的中间态。我们证明，GDP 结合蛋白相对更复杂的构象折叠是门控系统的核心。我们报告了捕获这个阈值，即 G 蛋白 α 亚基的“亚稳定空口袋”构象（门），并假设受体通过允许 G 蛋白通过温和的结构扰动达到这种结构来激活 G 蛋白。
