Yildiz Muslum
Department of Molecular Biology and Genetics, Gebze Technical University, Kocaeli, Turkey.
Proteins. 2025 Jun 26. doi: 10.1002/prot.70006.
The protein complex comprising the SH3 domain and DLC1 proteins plays a vital role in various cellular processes and diseases, including cancer. Essential dynamics for the stability of this complex, which cannot be elucidated by static X-ray crystal structures, have significant implications for understanding cellular physiology and critical diseases. We thoroughly investigated this complex using advanced molecular dynamics, Adaptively Biased Force MD (ABF-MD), and conventional MD (cMD) simulation methods. Radial distribution function (RDF) calculations demonstrate that the interaction between the two proteins is highly specific, as all mutations exhibit a single peak, indicating no additional interacting sites. The probabilities of two key interactions, Glu298-Arg1114 and Lys292-Leu1239, were observed to increase in cancer-related mutations but not in other mutations known to disrupt complex formation. Using a Markov State Model (MSM), we identified a key intermediate in the wild type that was absent in other variants. Correlation analysis of deviations in distances among key interacting residues showed values greater than 0.95, indicating cooperativity among interacting residues. cMD simulations also revealed increased distance values between interacting residues in complex-disrupting mutations, but not in cancer-related mutations. Principal component analysis (PCA) further revealed significant conformational changes, indicating important distinct conformations potentially involved in complex formation. Specifically, the loop region between residues 1236-1261 exhibits distinct conformations upon mutations among variants. This distinct conformation, particularly in the L1267D mutation, leads to the displacement of the SH3 domain from the binding site, which may contribute to complex destabilization. Additionally, PCA analysis suggests that complex-disrupting mutations significantly increase the ability of the loop region to explore different conformations compared to the wild type. In contrast, the cancer-related mutation, V1227M, does not significantly affect protein flexibility or its capacity to stay in a stable conformation. The binding energy analysis reveals that the wild-type DLC1 complex has moderate stability (-8.87 ± 1.31 kcal/mol), and the V1227M variant shows the most stable binding (-6.89 ± 1.04 kcal/mol) among other mutants. In contrast, L1267D, R1114A, and R1114E variants exhibit weaker binding affinities (-5.89 ± 1.01, -3.18 ± 1.04, and - 0.58 ± 1.11 kcal/mol, respectively), indicating reduced complex stability.
由SH3结构域和DLC1蛋白组成的蛋白质复合物在包括癌症在内的各种细胞过程和疾病中起着至关重要的作用。这种复合物稳定性的基本动力学无法通过静态X射线晶体结构阐明，对理解细胞生理学和重大疾病具有重要意义。我们使用先进的分子动力学、自适应偏置力分子动力学（ABF-MD）和传统分子动力学（cMD）模拟方法对该复合物进行了深入研究。径向分布函数（RDF）计算表明，两种蛋白质之间的相互作用具有高度特异性，因为所有突变都表现出一个单一峰，表明没有其他相互作用位点。观察到两种关键相互作用Glu298-Arg1114和Lys292-Leu1239在癌症相关突变中的概率增加，但在已知会破坏复合物形成的其他突变中没有增加。使用马尔可夫状态模型（MSM），我们在野生型中鉴定出一个关键中间体，而在其他变体中不存在。关键相互作用残基之间距离偏差的相关性分析显示值大于0.95，表明相互作用残基之间具有协同性。cMD模拟还揭示了在破坏复合物的突变中相互作用残基之间的距离值增加，但在癌症相关突变中没有增加。主成分分析（PCA）进一步揭示了显著的构象变化，表明可能参与复合物形成的重要不同构象。具体而言，残基1236-1261之间的环区域在变体之间的突变时表现出不同的构象。这种不同的构象，特别是在L1267D突变中，导致SH3结构域从结合位点移位，这可能导致复合物不稳定。此外，PCA分析表明，与野生型相比，破坏复合物的突变显著增加了环区域探索不同构象的能力。相比之下，癌症相关突变V1227M对蛋白质柔韧性或其保持稳定构象的能力没有显著影响。结合能分析表明，野生型DLC1复合物具有中等稳定性（-8.87±1.31千卡/摩尔），V1227M变体在其他突变体中表现出最稳定的结合（-6.89±1.04千卡/摩尔）。相比之下，L1267D、R1114A和R1114E变体表现出较弱的结合亲和力（分别为-5.89±1.01、-3.18±1.04和-0.58±1.11千卡/摩尔），表明复合物稳定性降低。
