Khan Abbas, Ali Syed Shujait, Zahid Muhammad Ammar, Abdelsalam Shahenda Salah, Albekairi Noorah, Al-Zoubi Raed M, Shkoor Mohanad, Wei Dong-Qing, Agouni Abdelali
Department of Pharmaceutical Sciences, College of Pharmacy, QU Health, Qatar University, Doha, Qatar.
Center for Biotechnology and Microbiology, University of Swat, Swat, Pakistan.
Proteins. 2025 Mar;93(3):684-701. doi: 10.1002/prot.26759. Epub 2024 Nov 5.
The RAF1-RAP1A interaction activates the MAPK/ERK pathway which is very crucial in the carcinogenesis process. This protein complex influences tumor formation, proliferation, and metastasis. Understanding aberrant interactions driven by clinical mutations is vital for targeted therapies. Hence, the current study focuses on the screening of clinically reported substitutions in the RAF1 and RAP1A genes using predictive algorithms integrated with all-atoms simulation, essential dynamics, and binding free energy methods. Survival analysis results revealed a strong association between RAF1 and RAP1A expression levels and diminished survival rates in cancer patients across different cancer types. Integrated machine learning algorithms showed that among the 134 mutations reported for these 2 proteins, only 13 and 35 were classified as deleterious mutations in RAF1 and RAP1P, respectively. Moreover, one mutation in RAF1 reported elevated levels of binding between RAF1 and RAP1P while in RAP1A, 7 mutations were reported to increase the binding affinity. The high-binding mutations, P34Q and V60F, were subjected to protein-protein coupling which confirmed the increase in the binding affinity. Wild-type and mutant RAF1-RAP1P bound complexes were subjected to molecular simulation investigation, revealing enhanced structural stability, increased compactness, and stabilized residue fluctuations of the mutant systems in contrast to the wild-type. In addition, hydrogen bonding analysis revealed a variation in the binding paradigm which further underscores the impact of these substitutions on the coupling of RAF1 and RAP1A. Principal component analysis (PCA) and free energy landscape (FEL) evaluation further determined dynamical variations in the wild-type and mutant complexes. Finally, the Gibbs free energy for each complex was estimated and found to be -71.94 ± 0.38 kcal/mol for the wild-type, -95.57 ± 0.37 kcal/mol for the V60F, and -85.76 ± 0.72 kcal/mol for P34Q complex. These findings confirm the effect of these variants on increasing the binding affinity of RAF1 to RAP1P. These mutations can therefore be targeted for cancer therapy to modulate the activity of the MAPK/ERK signaling pathway.
RAF1与RAP1A的相互作用激活了MAPK/ERK通路，该通路在致癌过程中至关重要。这种蛋白质复合物影响肿瘤的形成、增殖和转移。了解由临床突变驱动的异常相互作用对于靶向治疗至关重要。因此，当前的研究重点是使用与全原子模拟、主成分动力学和结合自由能方法相结合的预测算法，筛选RAF1和RAP1A基因中临床报告的替换。生存分析结果显示，RAF1和RAP1A的表达水平与不同癌症类型的癌症患者生存率降低之间存在强烈关联。综合机器学习算法表明，在这两种蛋白质报告的134个突变中，RAF1和RAP1A中分别只有13个和35个被分类为有害突变。此外，报告的RAF1中的一个突变显示RAF1与RAP1A之间的结合水平升高，而在RAP1A中，有7个突变报告增加了结合亲和力。对高结合突变P34Q和V60F进行了蛋白质-蛋白质偶联，证实了结合亲和力的增加。对野生型和突变型RAF1-RAP1A结合复合物进行了分子模拟研究，结果显示与野生型相比，突变系统的结构稳定性增强、紧凑性增加且残基波动稳定。此外，氢键分析揭示了结合模式的变化，这进一步强调了这些替换对RAF1和RAP1A偶联的影响。主成分分析（PCA）和自由能景观（FEL）评估进一步确定了野生型和突变型复合物的动力学变化。最后，估计每个复合物的吉布斯自由能，发现野生型为-71.94±0.38 kcal/mol，V60F为-95.57±0.37 kcal/mol，P34Q复合物为-85.76±0.72 kcal/mol。这些发现证实了这些变体对增加RAF1与RAP1A结合亲和力的作用。因此，这些突变可作为癌症治疗的靶点，以调节MAPK/ERK信号通路的活性。
