Department of Chemical and Biological Sciences, S. N. Bose National Centre for Basic Sciences, Kolkata, India.
Computational Structural Biology Section, Frederick National Laboratory for Cancer Research in the Cancer Innovation Laboratory, National Cancer Institute, Frederick, Maryland.
Biophys J. 2024 Jan 2;123(1):57-67. doi: 10.1016/j.bpj.2023.11.018. Epub 2023 Nov 21.
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.
Rho 特异性鸟嘌呤核苷酸解离抑制剂（RhoGDIs）在 Rho 家族 GTP ases 的调节中发挥着关键作用。它们作为负性调节剂，通过与 GTPase 的无活性 GDP 结合状态形成复合物，防止 Rho GTPase 的激活。RhoGDI 结合复合物中 Rho GTPase 的释放对于 Rho GTPase 的激活是必要的。生化研究提供了“磷酸化密码”的证据，其中 RhoGDI 的一些特定残基的磷酸化选择性地释放其 GTPase 伴侣（RhoA、Rac1、Cdc42 等）。这项工作试图理解这种特定的磷酸化诱导的结合亲和力降低背后的分子机制。通过对野生型和磷酸化状态的 RhoA-RhoGDI 复合物进行数微秒长的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机制模型，用于解释复合物的解离。磷酸化诱导了主要的结构变化，特别是在 RhoA 的正电荷多碱性区域（PBR）和 RhoGDI 的带负电荷的 N 端区域，这些区域对结合亲和力的贡献最大。分子力学泊松-玻尔兹曼表面区域结合能计算表明，在 RhoGDI 的 RhoA 特异性位点磷酸化时，相互作用显著减弱。相比之下，在 Rac1 特异性位点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，从而导致结合亲和力降低。通过氢键占有率分析和能量扰动网络，我们提出了一种变构响应的机制模型，即通过氢键和盐桥的重排和重新布线，从磷酸化位点到 PBR 和隐蔽的香叶基香叶基基团的长程信号传递。我们的结果强调了特定静电相互作用在磷酸化密码表现中的关键作用。
