Many driver mutations in cancer are specific in that they occur at significantly higher rates than – presumably – functionally alternative mutations. For example, V600E in the BRAF hydrophobic activation segment (AS) pocket accounts for >95% of all kinase mutations. While many hypotheses tried to explain such significant mutation patterns, conclusive explanations are lacking. Here, we use experimental and in silico structure-energy statistical analyses, to elucidate why the V600E mutation, but no other mutation at this, or any other positions in BRAF’s hydrophobic pocket, is predominant. We find that BRAF mutation frequencies depend on the equilibrium between the destabilization of the hydrophobic pocket, the overall folding energy, the activation of the kinase and the number of bases required to change the corresponding amino acid. Using a random forest classifier, we quantitatively dissected the parameters contributing to BRAF AS cancer frequencies. These findings can be applied to genome-wide association studies and prediction models.
Mutations in the gene that encodes a protein called BRAF are commonly found in certain cancers, such as melanomas. The same BRAF mutation is found in nearly all of these cancers. This mutation causes the 600th amino acid in the BRAF protein – an amino acid called a valine – to be replaced with another amino acid, a glutamate.
BRAF is a type of enzyme called a kinase, and it transmits signals inside cells to promote cell growth. Kinases work by adding a phosphate group to other proteins to alter their activity. The structure of the BRAF kinase contains a pocket-like shape, and the valine at position 600 sits buried inside this pocket when the enzyme is inactive. The “valine-to-glutamate” mutation (often called V600E for short) disrupts the interactions that create this pocket. This in turn results in a permanently active form of BRAF and uncontrolled cell growth. However, it remains unclear why the valine-to-glutamate mutation is so much more common in cancer cells than any other mutation that could affect the pocket in BRAF.
To address this question, Kiel et al. used a computational tool to generate three-dimensional models for all the different amino acid substitutions that could occur in BRAF’s pocket. Each mutation was then assessed to see how it might destabilize the structure of BRAF. Only the mutations that affected the 600th amino acid were predicted to be able to open the pocket without destabilizing the part of the enzyme that adds phosphate groups to other proteins.
Kiel et al. validated their computational predictions by introducing normal or mutant versions of the BRAF-encoding gene into human cells grown in the laboratory. These experiments showed that a mutation that introduced an amino acid called histidine into position 600 could activate BRAF as much the valine-to-glutamate mutation. Kiel et al. suggest that this “valine-to-histidine” substitution is not found in cancers because it requires three changes to the DNA sequence of the BRAF gene, whereas the valine-to-glutamate substitution only requires one.
The results underscore the importance of considering changes at both the DNA and protein level when attempting to understand why certain cancer-causing mutations are more common than others.