## 4 Discussion
Of the RasG12V dependent activating variants of Raf-CRD, Q156, K157 and Q166, T167 are pairs of hydrophilic residues exposed to solvent that follow one of the cysteine residues in the two loops containing the Zn\({}^{2+}\)-binding pockets. Residue N140 is also exposed to solvent and follows H139 that coordinates one of the Zn\({}^{2+}\) ions. The mechanism through which mutations of these residues lead to RasG12V-dependent activation of the kinase is currently not known. However, residue K157 is at the beginning of one of the segments thought to interact with the membrane. If this interaction were important for activity, one would expect that the K157A variant would inhibit rather than activate signaling through Raf. The remaining activating residues in the alanine-scanning mutagenesis study, R143, T145 and H175, make interactions across the dimer interface in the Ras-Raf-RBD_CRD dimer and K171 is part of the packing that stabilizes T145. We have shown that the CRD interactions across the dimer interface are dynamic in the Ras-Raf-RBD_CRD dimer. The activating variants other than F151A and D153A have no effect on WT Ras signaling. However, specific mutations at the dimer interface may synergize with RasG12V to stabilize the dynamic CRD for more efficient information transfer through the allosteric pathways with direct access to the active site and the Ras-Raf-RBD interface. Interestingly, variants associated with RASopathies are also weakly activating [78; 79]. In particular, BRaf T241P and Q257R are found in individuals with LEOPARD syndrome and cardiofacialcutaneous (CFC) syndrome, respectively [78]. These residues mediate contacts with the 14-3-3 scaffold dimer in the autoinhibited state. They correspond to T145 and N161 in CRaf, and in the context of the activated Ras-Raf dimer are located at the interface between the CRD and loop 8 across the Ras-Raf-RBD_CRD dimer interface (Figure 2b,c). The active BRaf/MEK1/14-3-3 structure, with a dephosphorylated 14-3-3-binding motif on the CR2 [32], displays a shift of the 14-3-3 dimer with respect to the Raf-kinase domain relative to the inactive state, accompanied by conformational rearrangement of the Raf-kinase C-terminus, which now redirects away from the Ras-binding surface of the Raf-CRD (Figure 4b). The absence of the Raf-RBD from the inactive BRaf cryo-EM reconstruction suggests it does not contribute toward stabilizing the autoinhibited state and therefore must remain accessible for Ras-binding [32]. This is consistent with a highly dynamic RBD observed by MD simulations [80]. Indeed, by aligning the CRaf-CRD from our Ras-Raf-RBD_CRD structure with the BRaf-CRD in the inactive complex, we observe that the short Raf-RBD_CRD linker is sufficient for the Raf-RBD to be accessible for interaction with Ras (Figure 4c and Figure S2c). We propose that Ras-binding to the Raf-RBD results in the dissociation of the 14-3-3 subunit from the CR2-binding site and redirection of the Raf C-terminus. This would expose the CR2 site for dephosphorylation, preventing association of the 14-3-3 dimer to reestablish Raf autoinhibition. This is consistent with an increase in Raf-kinase activity in cells upon dephosphorylation of the CR2 site [5; 29; 33]. The displacement of 14-3-3 from the CR2-binding site would promote its interaction with a second Raf kinase to facilitate kinase domain dimerization and further expose the Raf-CRD for extraction (Figure 4b,c), resulting in collapse of the autoinhibited conformation and progression toward Raf activation. Our proposed model for Ras-mediated activation of the MAPK pathway (Figure 4c) is similar to the recently published model [20] in the mechanism by which Raf-RBD binds to Ras. However, the two models deviate in that, in our model, binding of Raf-RBD to Ras promotes Ras dimerization, permitting allosteric modulation of the Galectin-binding regions on the Raf-RBD, to form a platform of Ras-Raf and Galectin dimers for signal amplification (Figure 3e).