Department of Chemical Engineering and Polymer Research Center, Bogazici University, Istanbul, Turkey.
Department of Mathematics, Physics and Electrical Engineering, Northumbria University, Newcastle, United Kingdom.
Biophys J. 2021 Mar 2;120(5):866-876. doi: 10.1016/j.bpj.2021.01.016. Epub 2021 Jan 28.
Rac1 is a small member of the Rho GTPase family. One of the most important downstream effectors of Rac1 is a serine/threonine kinase, p21-activated kinase 1 (PAK1). Mutational activation of PAK1 by Rac1 has oncogenic signaling effects. Here, although we focus on Rac1-PAK1 interaction by atomic-force-microscopy-based single-molecule force spectroscopy experiments, we explore the effect of active mutations on the intrinsic dynamics and binding interactions of Rac1 by Gaussian network model analysis and molecular dynamics simulations. We observe that Rac1 oncogenic mutations are at the hinges of three global modes of motion, suggesting the mechanical changes as potential markers of oncogenicity. Indeed, the dissociation of wild-type Rac1-PAK1 complex shows two distinct unbinding dynamic states that are reduced to one with constitutively active Q61L and oncogenic Y72C mutant Rac1, as revealed by single-molecule force spectroscopy experiments. Q61L and Y72C mutations change the mechanics of the Rac1-PAK1 complex by increasing the elasticity of the protein and slowing down the transition to the unbound state. On the other hand, Rac1's intrinsic dynamics reveal more flexible GTP and PAK1-binding residues on switches I and II with Q61L, Y72C, oncogenic P29S and Q61R, and negative T17N mutations. The cooperativity in the fluctuations of GTP-binding sites around the p-loop and switch I decreases in all mutants, mostly in Q61L, whereas some PAK1-binding residues display enhanced coupling with GTP-binding sites in Q61L and Y72C and within each other in P29S. The predicted binding free energies of the modeled Rac1-PAK1 complexes show that the change in the dynamic behavior likely means a more favorable PAK1 interaction. Overall, these findings suggest that the active mutations affect intrinsic functional dynamic events and alter the mechanics underlying the binding of Rac1 to GTP and upstream and downstream partners including PAK1.
Rac1 是 Rho GTPase 家族的一个小成员。Rac1 的最重要下游效应物之一是丝氨酸/苏氨酸激酶，p21 激活激酶 1（PAK1）。Rac1 对 PAK1 的突变激活具有致癌信号作用。在这里，虽然我们通过基于原子力显微镜的单分子力谱实验专注于 Rac1-PAK1 相互作用，但我们通过高斯网络模型分析和分子动力学模拟探索了活性突变对 Rac1 的固有动力学和结合相互作用的影响。我们观察到 Rac1 致癌突变位于三个全局运动模式的铰链处，这表明机械变化可能是致癌性的潜在标志物。事实上，野生型 Rac1-PAK1 复合物的解离显示出两种不同的非结合动态状态，而具有组成性活性 Q61L 和致癌性 Y72C 突变的 Rac1 则减少到一种，这是通过单分子力谱实验揭示的。Q61L 和 Y72C 突变通过增加蛋白质的弹性并减缓向非结合状态的转变，改变 Rac1-PAK1 复合物的力学性质。另一方面，Rac1 的固有动力学揭示了带有 Q61L、Y72C、致癌性 P29S 和 Q61R 以及负 T17N 突变的开关 I 和 II 上的 GTP 和 PAK1 结合残基更具弹性。所有突变体中，p 环和开关 I 周围 GTP 结合位点的波动协同性降低，主要在 Q61L 中，而在 Q61L 和 Y72C 中，一些 PAK1 结合残基与 GTP 结合位点之间的相互作用增强，并且在 P29S 中彼此之间的相互作用增强。建模的 Rac1-PAK1 复合物的预测结合自由能表明，动态行为的变化可能意味着 PAK1 相互作用更有利。总体而言，这些发现表明，活性突变影响内在功能动态事件，并改变 Rac1 与 GTP 以及上下游伙伴（包括 PAK1）结合的力学基础。
