## 4 Discussion
This dimer model, which is derived from crystal structures and remains to be confirmed experimentally on the membrane, would preclude the CRD insertion into the membrane. We thus question the presumed membrane-binding function of the Raf-CRD in MAPK signaling, characterized solely in the context of monomeric Ras-Raf-RBD_CRD complexes [24; 26; 27]. It has been shown in live cells that the CRD is important for the recruitment of Raf to the membrane in the presence of activated Ras [72]. However, the study does not observe insertion of the CRD in the membrane, leaving open other possible mechanisms through which the CRD can have an effect. We also point out that experimental results where the Ras farnesyl group interacts in vitro with the CRD in solution [2; 73], are countered by experiments in cells that suggest that the farnesyl group does not contribute to the Ras-Raf-RBD_CRD interaction [74] and recent SPR experiments showing that the farnesyl group does not affect the affinity between KRas and CRaf-RBD_CRD [20]. Thus, it is not clear whether the farnesyl group is involved in the Ras-Raf-RBD_CRD interaction. The SPR experiments that accompany the KRas-CRaf-RBD_CRD structures validate the primary interaction between Ras and the CRD as critical for signaling through MAPK [20], consistent with common interactions in the two alternate models discussed here. We now know that the surface of the Raf-CRD contains non-overlapping binding sites for Ras (in the monomer of the complex), the Raf kinase domain and the scaffold protein 14-3-3 (Figure 4a). The CRD segments involved in the latter interactions have hydrophobic and positively charged residues that are thought to insert into the membrane once the CRD binds Ras and is released from its autoinhibited state [20; 21; 24; 75; 76]. They include residues 143-RKTFKLLAF-151 and 157-KFLLNGFR-164, with the intermittiting residues 152-CDICN-156 along with H173 and C176 coordinating the first Zn\({}^{2+}\)-binding site at the N-terminal end of the helix that forms the primary Ras-binding surface of the CRD (Figure 1). These segments are found in the interactions across the Ras dimer interface in our model that provides an alternative role for the CRD in the activated state of Raf. Although these regions are dynamic in NMR structures of the CRD both in solution [3] and in nanodiscs [24], in the crystal structure of the KRas-CRaf-RBD_CRD complex (PDB ID 6 XI7), residues 142-146 and 160-164 are part of a \(\beta\)-sheet that presents R143, T145, L160 and N161 for interaction with loop 8 and the ExSAK nucleotide-binding motif of the opposing Ras protomer in the dimer (Figure S9a), consistent with our simulations. L160 in particular makes van der Waals contact with the aliphatic portion of Ras K147 of the ExSAK motif across the dimer interface, which in turn interacts with Ras F28 in switch I. Both K147 and F28 are in direct contact with the guanine base of the nucleotide [77]. Several of the Raf-CRD residues in the two segments mentioned above form a core layer of hydrophobic interactions in the CRD that includes packing of L160 at the dimer interface, F151, F158, L159, F146 and the aliphatic portions K144 and R164 (Figure S9b). K144 and R164 have the charged portion of their side chains exposed to solvent, capping the end of this hydrophobic cluster opposite from L160. Thus, with the exception of K144 and R164, the polar groups in the supposed membrane-binding segments are arranged at the interface between the CRD and Ras, while the hydrophobic ones form a second layer stabilizing the core structure that presents the Ras-interacting residues. The question of whether the ordering of these key CRD segments is an artifact of our MD simulation model (HRas-CRaf-RBD_CRD) or crystallization (KRas-CRaf-RBD_CRD), or a biologically relevant consequence of dimerization is at the core of the present discussion.