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Updated: Apr 23, 2026
Author Spotlight: Integrating BRET-Based Assays and Rare Mutation Analysis to Decipher RAF Kinase Regulation in Live Cells
Published on: March 1, 2024
Free Energy and Flexibility Analysis of Autoinhibited Human BRAF
Jeremy O B Tempkin1, Fikret Aydin1, Sebnem Essiz2
1Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California 94550, United States.
Abstract:
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 the 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 the membrane-bound RAS.
Insights
RAF serine/threonine protein kinases are crucial for cell signaling and cancer drug discovery. New computational models reveal flexibility in inactive BRAF, potentially aiding its activation by RAS.
Area of Science:
- Molecular biology
- Cell signaling
- Biochemistry
Background:
- RAF serine/threonine protein kinases are key effectors of RAS in transmitting extracellular growth signals.
- RAF kinases are critical drug targets due to frequent oncogenic mutations in cancer.
- RAF proteins exist in an autoinhibited state, stabilized by intramolecular interactions and 14-3-3 proteins.
Purpose of the Study:
- To investigate the molecular mechanisms of RAF activation, particularly BRAF.
- To explore the conformational dynamics of autoinhibited BRAF.
- To understand how BRAF's inactive state facilitates activation by RAS.
Main Methods:
- Development of a comprehensive all-atom computational model of BRAF.
- Utilizing cryo-electron microscopy (cryo-EM) structures for model building.
- Performing extensive all-atom molecular dynamics simulations.
Main Results:
- The study evaluated the stability and free energy landscape of autoinhibited BRAF in solution.
- Analysis revealed significant conformational flexibility within the autoinhibited BRAF complex.
- This inherent flexibility may be crucial for BRAF activation upon RAS binding.
Conclusions:
- The dynamic nature of autoinhibited BRAF is a key characteristic.
- Conformational flexibility likely plays a role in the transition to the active state.
- Understanding these dynamics offers insights into RAF-mediated signaling and therapeutic strategies.
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