Molecular mechanism of KRAS binding to BRAF and its regulation by the BRAF-specific region.

MAPK pathway activation requires recruitment of RAF kinases by GTP-bound RAS through the RAS-binding domain (RBD) and cysteine-rich domain (CRD). BRAF uniquely contains an N-terminal BRAF-specific region (BSR), but the structural basis of KRAS engagement and BSR function remain unclear. Here, we report the structure of KRAS bound to BRAF(RBD-CRD) and compare it with KRAS-CRAF(RBD-CRD). Although the RBD engages KRAS through a conserved mechanism, the BRAF(CRD) adopts a hinge-like displacement away from KRAS, forming fewer contacts and contributing less to binding than CRAF(CRD). Structural analysis, biophysical measurements, and AlphaFold3 modeling show that the BSR does not directly contact KRAS but instead regulates RBD accessibility through a cis-acting mechanism that requires linkage to the RBD. Without the CRD, the BSR-RBD linker restricts KRAS binding, whereas CRD inclusion stabilizes BSR positioning and partially relieves inhibition. Disease-associated mutations further support a regulatory role for BRAF(CRD), defining an isoform-specific mechanism regulating RAF activation.