## 1 Introduction  
Ras interacts with Raf kinase through Raf's N-terminal Ras-binding (RBD) and cysteine-rich (CRD) domains to drive cell proliferation through the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) pathway [1; 2; 3]. Mutations in Ras and Raf are major drivers of human cancers [4; 5] and in spite of great efforts over the last 20 years, the mechanism for Ras-mediated activation of Raf remains elusive. This gap in our basic understanding of the pathway has limited our ability to explore novel strategies to develop drugs against Ras-driven cancers. We previously solved crystal structures for wild-type Ras and its Q61L mutant bound to the Raf-RBD, revealing that this low-nanomolar-affinity complex occurs through electrostatic interactions at switch I (residues 30-40 in Ras) and that the oncogenic mutant RasQ61L has global impacts on the dynamics of the Ras-Raf complex [6; 7]. Most recently, we showed that the Raf-RBD promotes dimerization of the Ras G domain [8], consistent with data supporting the requirement for Ras dimerization in the activation of the Ras-Raf-MEK-ERK signaling pathway [9; 10; 11; 12]. Interestingly, both our HRas-CRaf-RBD (PDB ID 4G0N) and HRasQ61L/CRaf-RBD (PDB ID 4G3X) structures contain the Ras dimer with helices a4 and a5 at the interface, generated by taking the Ras-Raf-RBD complex in the asymmetric unit through a 2-fold crystallographic symmetry operation. Our molecular dynamics (MD) simulations of the dimer of the HRas-CRaf-RBD complex show that dimerization allosterically links the RBD sites for the scaffold protein Galectin-1 at the two ends of the dimer, suggesting a model in which the coupling of Ras-Raf dimers with Galectin dimers forms a signaling platform for kinetic proof reading and signal amplification [8]. This is consistent with the previously published observation that the Galectin-1 interaction with Raf is associated with an increase in HRas nanoclustering [13]. Galectins are part of a large family of \(\beta\)-galactosidase-binding animal lectins that have highly homologous carbohydrate recognition domains and are multifunctional, with activities modulated by interactions with saccharides or peptides/proteins [14]. It is as scaffold proteins inside the cell that Galectin-1 and Galectin-3 have been associated with HRas and KRas activities, respectively [15; 16]. The demonstration that this activity is through direct interaction between Galectin-1 dimers and CRaf-RBD was shown for HRas nanoclustering and signaling [13]. However, Galectin-3 readily dimerizes in the absence of glycans [17] and one can envision an analogous role in its signaling through KRas. 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 6 XI7) 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. The Raf-CRD requires coordination of two zinc ions and has been shown to interact with various phospholipids and supported lipid bilayers in vitro [3; 21; 22; 23]. This interaction is driven primarily by electrostatic attraction between the positively charged CRD and negative phospholipid headgroups on the membrane [22]. The CRD membrane-binding role has further been studied by MD simulations and recently supported by NMR-guided characterization of nanodisc-bound KRas-CRaf-RBD_CRD complexes [24; 25; 26; 27; 28]. However, interaction of the CRD with the membrane has only been observed in vitro or in silico in the absence of other signaling components, with no evidence that this interaction is biologically relevant in cells.