Gohlke Holger, Kiel Christina, Case David A
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
J Mol Biol. 2003 Jul 18;330(4):891-913. doi: 10.1016/s0022-2836(03)00610-7.
Absolute binding free energy calculations and free energy decompositions are presented for the protein-protein complexes H-Ras/C-Raf1 and H-Ras/RalGDS. Ras is a central switch in the regulation of cell proliferation and differentiation. In our study, we investigate the capability of the molecular mechanics (MM)-generalized Born surface area (GBSA) approach to estimate absolute binding free energies for the protein-protein complexes. Averaging gas-phase energies, solvation free energies, and entropic contributions over snapshots extracted from trajectories of the unbound proteins and the complexes, calculated binding free energies (Ras-Raf: -15.0(+/-6.3)kcal mol(-1); Ras-RalGDS: -19.5(+/-5.9)kcal mol(-1)) are in fair agreement with experimentally determined values (-9.6 kcal mol(-1); -8.4 kcal mol(-1)), if appropriate ionic strength is taken into account. Structural determinants of the binding affinity of Ras-Raf and Ras-RalGDS are identified by means of free energy decomposition. For the first time, computationally inexpensive generalized Born (GB) calculations are applied in this context to partition solvation free energies along with gas-phase energies between residues of both binding partners. For selected residues, in addition, entropic contributions are estimated by classical statistical mechanics. Comparison of the decomposition results with experimentally determined binding free energy differences for alanine mutants of interface residues yielded correlations with r(2)=0.55 and 0.46 for Ras-Raf and Ras-RalGDS, respectively. Extension of the decomposition reveals residues as far apart as 25A from the binding epitope that can contribute significantly to binding free energy. These "hotspots" are found to show large atomic fluctuations in the unbound proteins, indicating that they reside in structurally less stable regions. Furthermore, hotspot residues experience a significantly larger-than-average decrease in local fluctuations upon complex formation. Finally, by calculating a pair-wise decomposition of interactions, interaction pathways originating in the binding epitope of Raf are found that protrude through the protein structure towards the loop L1. This explains the finding of a conformational change in this region upon complex formation with Ras, and it may trigger a larger structural change in Raf, which is considered to be necessary for activation of the effector by Ras.
本文给出了蛋白质 - 蛋白质复合物H - Ras/C - Raf1和H - Ras/RalGDS的绝对结合自由能计算及自由能分解结果。Ras是细胞增殖和分化调控中的核心开关。在我们的研究中，我们考察了分子力学（MM）-广义玻恩表面积（GBSA）方法估算蛋白质 - 蛋白质复合物绝对结合自由能的能力。通过对从未结合蛋白质和复合物轨迹中提取的快照求平均气相能、溶剂化自由能和熵贡献，计算得到的结合自由能（Ras - Raf：-15.0(±6.3)kcal mol⁻¹；Ras - RalGDS：-19.5(±5.9)kcal mol⁻¹），如果考虑适当的离子强度，与实验测定值（-9.6 kcal mol⁻¹；-8.4 kcal mol⁻¹）相当吻合。通过自由能分解确定了Ras - Raf和Ras - RalGDS结合亲和力的结构决定因素。首次在这种情况下应用计算成本较低的广义玻恩（GB）计算来划分溶剂化自由能以及气相能在两个结合伙伴残基之间的分配。此外，对于选定的残基，通过经典统计力学估算熵贡献。将分解结果与界面残基丙氨酸突变体的实验测定结合自由能差异进行比较，Ras - Raf和Ras - RalGDS的相关系数r²分别为0.55和0.46。分解的扩展揭示了距离结合表位达25埃远的残基可对结合自由能有显著贡献。发现这些“热点”在未结合蛋白质中表现出较大的原子波动，表明它们位于结构上较不稳定的区域。此外，热点残基在复合物形成时局部波动的减小幅度明显大于平均值。最后，通过计算相互作用的成对分解，发现源自Raf结合表位的相互作用途径穿过蛋白质结构伸向环L1。这解释了与Ras形成复合物时该区域构象变化的发现，并且可能引发Raf中更大的结构变化，这被认为是Ras激活效应器所必需的。
