<br>RhoGDI 磷酸化调控 RhoA GTP 酶的分子机制,Biophysical Journal

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RhoGDI 磷酸化调控 RhoA GTP 酶的分子机制
Biophysical Journal ( IF 3.2 ) Pub Date : 2023-11-21 , DOI: 10.1016/j.bpj.2023.11.018
Krishnendu Sinha ₁ , Amit Kumawat ₁ , Hyunbum Jang ₂ , Ruth Nussinov ₃ , Suman Chakrabarty ₁

Affiliation

Rho 特异性鸟嘌呤核苷酸解离抑制剂（RhoGDI）在 Rho 家族 GTP 酶的调节中起着至关重要的作用。它们作为负调节因子，通过与 GTP 酶的失活 GDP 结合状态形成复合物来阻止 Rho GTP 酶的激活。从 RhoGDI 结合复合物中释放 Rho GTP 酶是 Rho GTP 酶激活所必需的。生化研究提供了“磷酸化密码”的证据，其中 RhoGDI 的某些特定残基的磷酸化选择性地释放其 GTP 酶伴侣（RhoA、Rac1、Cdc42 等）。这项工作试图了解这种特异性磷酸化诱导的结合亲和力降低背后的分子机制。使用对 RhoA-RhoGDI 复合物的野生型和磷酸化状态的几微秒长原子分子动力学模拟，我们提出了一种基于分子相互作用的复合物解离机理模型。磷酸化会诱导主要的结构变化，特别是在 RhoA 带正电荷的多碱基区（PBR）和 RhoGDI 带负电荷的 N 末端区域，这些区域对结合亲和力的贡献最大。分子力学泊松-玻尔兹曼表面积结合能计算显示，在 RhoGDI 的 RhoA 特异性位点，磷酸化相互作用显著减弱。相反，Rac1 特异性位点的磷酸化不会显著影响整体结合亲和力，这证实了磷酸化密码的存在。RhoA 特异性磷酸化导致 RhoA 的 PBR 与 RhoGDI 的 N 端区域之间的接触数量减少，这表现为结合亲和力的降低。利用氢键占有分析和能量扰动网络，我们提出了一种变构响应的机理模型，即以氢键和盐桥的重排和重新布线的形式，从磷酸化位点向 PBR 和埋藏的香叶基序的远程信号传播。我们的结果强调了特异性静电相互作用在磷酸化密码表现中的关键作用。

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Molecular mechanism of regulation of RhoA GTPase by phosphorylation of RhoGDI

Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) play a crucial role in the regulation of Rho family GTPases. They act as negative regulators that prevent the activation of Rho GTPases by forming complexes with the inactive GDP-bound state of GTPase. Release of Rho GTPase from the RhoGDI-bound complex is necessary for Rho GTPase activation. Biochemical studies provide evidence of a "phosphorylation code," where phosphorylation of some specific residues of RhoGDI selectively releases its GTPase partner (RhoA, Rac1, Cdc42, etc.). This work attempts to understand the molecular mechanism behind this specific phosphorylation-induced reduction in binding affinity. Using several microseconds long atomistic molecular dynamics simulations of the wild-type and phosphorylated states of the RhoA-RhoGDI complex, we propose a molecular-interaction-based mechanistic model for the dissociation of the complex. Phosphorylation induces major structural changes, particularly in the positively charged polybasic region (PBR) of RhoA and the negatively charged N-terminal region of RhoGDI that contribute most to the binding affinity. Molecular mechanics Poisson-Boltzmann surface area binding energy calculations show a significant weakening of interaction on phosphorylation at the RhoA-specific site of RhoGDI. In contrast, phosphorylation at a Rac1-specific site does not affect the overall binding affinity significantly, which confirms the presence of a phosphorylation code. RhoA-specific phosphorylation leads to a reduction in the number of contacts between the PBR of RhoA and the N-terminal region of RhoGDI, which manifests a reduction of the binding affinity. Using hydrogen bond occupancy analysis and energetic perturbation network, we propose a mechanistic model for the allosteric response, i.e., long-range signal propagation from the site of phosphorylation to the PBR and buried geranylgeranyl group in the form of rearrangement and rewiring of hydrogen bonds and salt bridges. Our results highlight the crucial role of specific electrostatic interactions in manifestation of the phosphorylation code.

更新日期：2023-11-21

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