The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.
Case Western Reserve University, Department of Physiology and Biophysics, Cleveland, Ohio 44106, United States.
J Phys Chem B. 2022 Sep 1;126(34):6408-6418. doi: 10.1021/acs.jpcb.2c03435. Epub 2022 Aug 17.
Orientational probability densities, = exp(-) (, local potential), of bond-vectors in proteins provide information on structural flexibility. The related conformational entropy, = -∫(ln )dΩ - ln ∫dΩ, provides the entropic contribution to the free energy of the physical/biological process studied. We have developed a new method for deriving and from MD simulations, using the N-H bond as probe. Recently we used it to study the dimerization of the Rho GTPase binding domain of Plexin-B1 (RBD). Here we use it to study RBD binding to the small GTPase Rac1. In both cases 1 μs MD simulations have been employed. The RBD has the ubiquitin fold with four mostly long loops. L3 is associated with GTPase binding, L4 with RBD dimerization, L2 participates in interdomain interactions, and L1 has not been associated with function. We find that RBD-Rac1 binding renders L1, L3, and L4 more rigid and the turns β/α and α/β more flexible. By comparison, RBD dimerization renders L4 more rigid, and the α-helices, the β-strands, and L2 more flexible. The rigidity of L1 in RBDRAC is consistent with L1-L3 contacts seen in previous MD simulations. The analysis of the L3-loop reveals two states of distinct flexibility which we associate with involvement in slow conformational exchange processes differing in their rates. Overall, the N-H bonds make an unfavorable entropic contribution of (5.9 ± 0.9) kJ/mol to the free energy of RBD-Rac1 binding; they were found to make a favorably contribution of (-7.0 ± 0.7) kJ/mol to the free energy of RBD dimerization. In summary, the present study provides a new perspective on the impact of Rac1 binding and dimerization on the flexibility characteristics of the RBD. Further studies are stimulated by the results of this work.
蛋白质中键向量的取向概率密度，(=\exp(-))（，局部势能），提供了结构灵活性的信息。相关的构象熵，(=!-\int(\ln),d\Omega-\ln!\int d\Omega)，为所研究的物理/生物学过程的自由能提供了熵贡献。我们开发了一种从 MD 模拟中推导出(和)的新方法，使用 N-H 键作为探针。最近我们用它来研究 Plexin-B1（RBD）Rho GTPase 结合域的二聚化。在这里，我们用它来研究 RBD 与小 GTPase Rac1 的结合。在这两种情况下，都使用了 1 μs 的 MD 模拟。RBD 具有泛素折叠结构，有四个主要的长环。L3 与 GTPase 结合有关，L4 与 RBD 二聚化有关，L2 参与了结构域间相互作用，而 L1 尚未与功能相关联。我们发现，RBD-Rac1 结合使 L1、L3 和 L4 更加刚性，β/α 和 α/β 转角更加灵活。相比之下，RBD 二聚化使 L4 更刚性，α-螺旋、β-链和 L2 更灵活。RBD-RAC 中的 L1 刚性与之前 MD 模拟中观察到的 L1-L3 接触一致。对 L3 环的分析揭示了两种明显不同的灵活性状态，我们将其与涉及不同速率的缓慢构象交换过程相关联。总的来说，N-H 键对 RBD-Rac1 结合的自由能有不利的熵贡献((5.9\pm0.9)kJ/mol)；它们对 RBD 二聚化的自由能有有利的贡献((-7.0\pm0.7)kJ/mol)。总之，本研究为 Rac1 结合和二聚化对 RBD 灵活性特征的影响提供了新的视角。这项工作的结果激发了进一步的研究。
