## 4 Discussion
We have used the Protein Interfaces Surfaces and Assemblies (PISA) [49] website at the European Bioinformatics Institute to calculate the surface area buried in the KRas-CRaf-RBD_CRD dimer interface (PDB ID 6 XI7) as 1005 A\({}^{2}\), with a \(\Delta^{1}\)G \(p\)-value of 0.496, just within the cutoff indicative of an interaction-specific interface unlikely to be an artifact of crystal packing (see Methods section). It has been recently demonstrated that mutations of Raf-CRD residues at the primary Ras-Raf-CRD interface only marginally affect the affinity of the Ras-Raf RBD_CRD complex, dominated by the interaction with Raf-RBD, while these same mutations lead to reduction in the kinase activity in cells [20]. This is consistent with S177A, T182A and M183A variants at the primary Ras-binding site of the CRD inhibiting kinase activity in a previous alanine-scanning mutagenesis study aimed at probing binding epitopes on the surface of the Raf-CRD [74]. Interestingly, point mutations K144A, L160A and R164A also weakly inhibit Raf activation, with the double mutant K144A/L160A completely abrogating signal through Raf [74]. Here, we have two possible interpretations for these results. It can be argued that the inhibitory effects of the K144, L160 and R164 variants are due to disruption of the CRD interaction with the membrane. However, the data are also consistent with disruption of key interactions between the CRD and Ras loop 8 and ExSAK motifs across the dimer interface due to destabilization of the hydrophobic core maintained by key residues in the 143-151 and 157-164 segments of the CRD as described above. Figure 4: Proposed Mechanism for Ras-mediated activation of Raf. (**a**) Residues involved in the primary Ras–Raf-CRD interaction (pink) do not overlap with Raf-CRD residues used to stabilize contacts with the kinase domain (purple) and 14-3-3 proteins (wheat) in the autoinhibited state. (**b**) Comparison of Raf-kinase domain (purple) and 14-3-3 (wheat) conformations in the inactive (PDB ID 6 NYB) and active Raf structure (PDB ID 6Q0). Note the shift in the 14-3-3 dimer in the absence of the Raf CR2 motif and release of the Raf-CRD. (**c**) Proposed structural model for the Ras-mediated release of Raf autoinhibition and activation of MAPK signaling. Raf-RBD (cyan) is exposed in the autoinhibited state, disordered in the cryo-EM structure, likely due to flexibility in its linker to the CRD (dark green). The high-affinity binding of Raf-RBD to Ras places the exposed area of the CRD in its Ras-binding site, while steric hindrance is expected to displace the Raf C-terminus (purple) and promote the shift of the 14-3-3 dimer (wheat) towards its active state interaction with a second Raf kinase domain (purple). These two features of the Ras–Raf interaction are shown in steps 1 and 2. Binding of Raf-RBD concurrently promotes dimerization of the complex as shown in step 3, which we propose is a key step in binding Galectin dimers to build a platform for signal amplification (see Figure 3e). We propose that steps 1, 2 and 3 occur concertedly to activate Raf. In addition to the inhibitory mutations, the alanine-scanning mutagenesis study identified a number of activating mutations [74], all of which are in residues at or near the binding interface with 14-3-3 and Raf-kinase domain in the autoinhibited state. However, most of these variants, N140A, R143A, T145A, Q156A, K157A, Q166A, T167A, K171A, H175A are activating only in the context of RasG12V, suggesting a Ras dependence on the resulting increased Raf activity. The variants F151A and D153A have significant effects in the context of WT Ras, with a drastic increase in the basal Raf activity that the other mutants do not show [74]. These two residues make key interactions in the inactive state of the kinase [32] and activation is likely due to release of autoinhibition in the absence of Ras.