KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation

Abstract

The first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we present crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures reveal that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutations at the KRAS-CRD interface result in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

Biogenesis of P-TEFb in CD4+ T cells to reverse HIV latency is mediated by protein kinase C (PKC)-independent signaling pathways

“…In turn GTP-bound Ras allosterically binds SOS leading to its full activation. This creates a positive Ras-GTP-SOS feedback loop that results in a conformational change in Ras which permits robust formation of Ras-Raf complexes at the membrane [71, 95].…”

“…The copyright holder for this preprint (which this version posted April 26, 2021. ; https://doi.org/10.1101/2021.04.26.441433 doi: bioRxiv preprint GTP-SOS feedback loop that results in a conformational change in Ras which permits robust formation of Ras-Raf complexes at the membrane [71,95].…”

Drug resistance in targeted cancer therapies with RAF inhibitors

“…The engagement of RTKs with their ligands triggers their kinase activity and results in the autophosphorylation of intracellular carboxyl-tails, which serve as docking sites for recruiting Grb2 and Sos1 to plasma membrane [86] . Further, active Sos1 proteins on the plasma membrane exert their GEF activity towards RAS, which in turn actives RAS proteins to recruit RAF/MEK heterodimers to the plasma membrane through the RBDs of RAF proteins [87][88][89] . Since active RAS proteins form oligomers or nanoclusters, RAF proteins are enriched on the plasma membrane, which promotes their side-to-side dimerization [90][91][92][93] .…”

Crystal Structure Reveals the Full Ras–Raf Interface and Advances Mechanistic Understanding of Raf Activation

“…While the interaction between Ras and Raf-RBD has been well studied, the transformation potential of Raf requires interaction with Ras through both the Raf-RBD and Raf-CRD [2,18,19]. The recent crystal structure of KRas-CRaf_RBD_CRD (PDB ID 6XI7) has contributed to our understanding of the weaker micromolar affinity interaction between Ras and the CRD [20]. However, questions remain about a possible role of Ras dimerization and the function of the CRD in this context.…”

“…Recent cryo-EM structures of inactive and active BRaf/14-3-3 and BRaf/MEK1/14-3-3 complexes have shed light into this mechanism revealing a critical role for the Raf-CRD in stabilizing the autoinhibited state [32,37,38]. It has been noted that the Raf-RBD is exposed in the complex with Ras, allowing its interaction with Ras to displace the CRD from the kinase/14-3-3 complex [20]. From there, the proposed model for Ras activation of Raf is built on the premise that the CRD must both bind Ras and insert into the membrane upon release from its autoinhibition role, leading to a signaling state in which Ras is monomeric on the membrane [20].…”