## 4 Discussion  
Ras nanoclustering on the membrane modulates Ras activation of MAPK signaling, but the significance of Ras dimers as functional signaling units is still not fully resolved in the literature [64]. Weak binding is predicted for Ras dimers in the absence of other factors [25; 64; 65; 66; 67], putting in question its viability as a robust signaling unit. However, we have recently shown that Raf-RBD promotes robust dimerization of Ras on supported lipid bilayers even at very low surface densities, in contrast with Ras in the absence of Raf-RBD, for which no dimers can be detected [8; 68]. This is consistent with observation of dimeric Ras in live cells [12] and with experiments that suggest a requirement for Ras dimerization in the activation of Raf [9; 10; 11; 12]. The recently published crystal structures of the KRas-CRaf-RBD_CRD complex, each with one complex monomer in the asymmetric unit in two different crystal forms (PDB IDs 6 XI7 and 6 XHB), led authors to suggest a model in which Ras acts as a monomer in the complex with Raf [20], building on an emerging model for Ras activation of Raf [69]. Here, the kinase domains of two nearby complexes dimerize to activate the signal without the need for the Ras molecules to dimerize. This model is consistent with years of research showing interaction of the CRD with the membrane in vitro and in silico [3; 21; 22; 23; 24; 25; 26; 27; 28]. Interestingly, one of the published crystal forms (PDB ID 6 XI7) has the dimer in the crystal with a structure that converges on our model of the dimer built from the crystal structure of the HRas-CRaf-RBD_CRD (PDB ID 7 JHP). Here, we offer an alternative model of Raf activation in which Ras dimerization upon binding Raf-RBD is a key step, with placement of the CRD at the base of the dimer interface, allosterically connecting elements of \(\alpha\)5, the interswitch region and two highly conserved elements of the nucleotide-binding site: the NKxD and ExSAK motifs. Within the bigger picture of signaling through Ras-Raf-MEK-ERK, the primary function of Ras in our model is not in promoting Raf dimerization _per se_, but in providing an essential building block for a signaling platform in which Ras-Raf dimers couple with Galectin dimers, leading to synchronized activation of multiple Raf kinase dimers, stabilized by 14-3-3, for signal amplification (Figure 3e). Binding of short oligosaccharides to Galectin-1 has recently been shown to promote allosteric connections to the dimer interface [70]. Given that the proposed site for Raf-RBD partially overlaps the glycan ligand-binding site [13], the binding of HRas-CRaf-RBD_CRD to Galectin-1 as we propose could result in allosteric connections across the entire signaling platform. Galectin-3, known to be monomeric when bound to glycans, readily self-dimerizes in the apo form [17], such that one could envision an analogous KRAS-Raf-RBD-Galectin-3 signaling platform. The Raf-CRD in this context is centrally and flexibly located to affect regulation both through diversifying the paths of allosteric connections in the signaling complex and possibly affecting active site function, such as GTP hydrolysis, through its proximity to the nucleotide-binding pocket. We recognize that the specific interface through which Ras dimerizes has not yet been unequivocally determined [64]. However, both the HRas-CRaf-RBD (PDB ID 4G0N) and the KRas-CRaf-RBD_CRD (PDB ID 6 XI7) dimer structures with the \(\alpha\)4-\(\alpha\)5 Ras dimer interface, as well as the many studies supporting Ras dimerization through this interface [8; 9; 50; 51; 71], point to our proposed model for the dimer. Given this dimer, we can hypothesize its placement with respect to the membrane in a signaling context. The evidence supporting an approximately perpendicular orientation of \(\alpha\)3, \(\alpha\)4 and \(\alpha\)5 with respect to the membrane [8; 50], and the fact that Ras is tethered to the membrane through the HVR following the C-terminal end of \(\alpha\)5, places the dimer with the Raf-CRD away from the membrane.