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Updated: Jun 8, 2025
Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods
Published on: July 17, 2019
Exploring the Dynamic Interplay of Deleterious Variants on the RAF1-RAP1A Binding in Cancer: Conformational Analysis,
Abbas Khan1, Syed Shujait Ali2, Muhammad Ammar Zahid1
1Department of Pharmaceutical Sciences, College of Pharmacy, QU Health, Qatar University, Doha, Qatar.
Abstract:
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.
Insights
This study identifies specific RAF1 and RAP1A gene mutations that enhance protein binding, potentially driving cancer by activating the MAPK/ERK pathway. These findings offer new targets for cancer therapy.
Area of Science:
- Molecular Biology
- Computational Biology
- Cancer Research
Background:
- The RAF1-RAP1A interaction is crucial for the MAPK/ERK pathway, a key player in cancer development, influencing tumor formation, proliferation, and metastasis.
- Understanding how clinical mutations affect this interaction is vital for developing targeted cancer therapies.
Purpose of the Study:
- To screen clinically reported RAF1 and RAP1A gene substitutions using predictive algorithms and molecular simulations.
- To investigate the impact of these mutations on RAF1-RAP1A binding affinity and their association with cancer patient survival.
Main Methods:
- Utilized predictive algorithms, all-atoms simulation, essential dynamics, and binding free energy calculations.
- Performed survival analysis, machine learning classification of mutations, and molecular dynamics simulations (PCA, FEL).
- Assessed changes in structural stability, compactness, residue fluctuations, and hydrogen bonding in wild-type versus mutant complexes.
Main Results:
- Survival analysis indicated a correlation between RAF1/RAP1A expression and reduced survival rates in cancer patients.
- Identified 13 deleterious mutations in RAF1 and 35 in RAP1A out of 134 reported.
- Discovered specific mutations (e.g., RAF1 P34Q, V60F; RAP1A 7 mutations) that significantly increase RAF1-RAP1A binding affinity.
- Molecular simulations showed enhanced stability and compactness in mutant complexes, with altered binding dynamics and increased binding free energy compared to wild-type.
Conclusions:
- Certain RAF1 and RAP1A mutations demonstrably increase binding affinity, potentially promoting cancer via the MAPK/ERK pathway.
- These high-binding mutations represent promising therapeutic targets for modulating RAF1-RAP1A interaction and treating cancer.
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