Free energy and flexibility analysis of autoinhibited human BRAF

The RAF serine/threonine protein kinases function as direct effectors of RAS in the intracellular transmission of extracellular growth signals, and they are key targets for drug discovery given the high incidence of oncogenic mutations in RAF and other components of this signaling pathway. In its inactive state, RAF is held in an autoinhibited conformation in the cytosol through a combination of intramolecular interactions and binding to a regulatory 14-3-3 protein dimer. Activation of RAF is initiated by its interaction with membrane-localized, GTP-bound RAS, which induces conformational changes that release RAF from its autoinhibited state. However, the molecular mechanisms governing RAF activation remain incomplete, largely due to the challenges in experimentally capturing intermediate conformational states in this process. To address this gap, we developed a comprehensive all-atom model of BRAF based on existing cryo-EM structures. Using this model, we performed extensive molecular dynamics simulations to evaluate the stability and free energy landscape of autoinhibited BRAF in solution. Our analysis reveals conformational flexibility within the autoinhibited complex, suggesting that this dynamic behavior may play a role in facilitating BRAF activation upon engagement with membrane-bound RAS.