Divergent CRD-Dependent Mechanisms Govern RAS Isoform-Selective Recruitment of CRAF and ARAF

RAF kinases interpret signals from the three major RAS isoforms to initiate MAPK pathway activation, yet the molecular logic that governs isoform-specific RAS recruitment and the early events that relieve RAF autoinhibition are not yet fully understood. In particular, how the modular N-terminal regulatory architecture of CRAF and ARAF, anchored by the multifunctional cysteine-rich domain (CRD), discriminates among HRAS, KRAS, and NRAS has remained a central unresolved question. Here, we combine quantitative biophysical measurements with structural and dynamic analyses to define how RAS isoform identity and CRD engagement shape the earliest steps of RAF activation. These studies reveal unexpectedly divergent modes of RAS recognition between CRAF and ARAF and expose previously unappreciated functions of the CRD in modulating RAS affinity and intramolecular regulatory contacts. We further identify a direct link between RAS binding and destabilization of RAF autoinhibition, providing mechanistic insight into how RAS initiates the transition from an inactive monomer to an activation-competent assembly. Finally, we show that emerging KRAS inhibitors variably perturb KRAS-CRAF interactions, offering insight into how these therapeutics influence early RAS-RAF signaling events. Together, this work uncovers distinct biophysical principles that govern RAS-RAF selectivity and reveals a regulatory role for the CRD that reframes our understanding of RAF activation and its dysregulation in RAS-driven cancers. Significance: Proteins in the RAS-RAF signaling pathway control cell growth and are frequently mutated in cancer. Despite their importance, how different RAS proteins selectively recruit RAF kinases has remained incompletely understood. This study reveals that the cysteine-rich regulatory region of RAF plays a central role in distinguishing RAS isoforms and controlling RAF activation. These insights clarify early steps in MAPK signaling and may guide the development of improved therapies targeting RAS-driven cancers.