# Ras Multimers on the Membrane: Many Ways for a Heart-to-Heart Conversation
## 6 Scaffold and Effector Proteins in Ras Dimerization
While the discrepancies among these studies potentially arose from the different experimental and computational setups, the results thus far demonstrate the impact of binding partners on Ras conformation and dimerization on the membrane. The Raf1 CRD binds anionic lipids such as PS and PA and enhances Raf binding to Ras [201; 202; 203]. Compared with the conformations of Ras alone on the membrane [145], the additional interaction between Raf1 with anionic lipids seem to alter the conformational dynamics significantly (Figures 5 and 8). The [PERSON] and [PERSON] groups have used computational simulations to show that monomers of Ras-Raf RBD-CRD complexes sample multiple conformations on PS-containing membranes, with the Raf CRD alternating between membrane proximal and distal configurations [203; 204]. Both studies suggest the presence of conformations where two or more of the three Ras dimer helices (\(\alpha\)3, \(\alpha\)4, and \(\alpha\)5) are well exposed, although these dimerization compatible conformations were not observed in the PRE-NMR study [198]. Figure 7: Ras conformations in complexes with binding partners. (**A**) Crystal structure of GTP-bound KRas G-domain in complex with Raf1 RBD-CRD, showing its orientations in approximation with membrane modeled in, as in ref [197]. (**B**) NMR structures of full length KRas-GTP in complex with Raf1 RBD-CRD, showing two different conformations of the complex relative to the nanodisc membrane (rendered as gray balls), with the left conformation being dominant at low complex concentrations. (**C**) NMR structure of full length KRas-GTP dimer on nanodiscs. (**D**) Potential dimer configuration present in the crystal structure of (A) (PDB 6 XI7), as proposed by [PERSON] et al. [200]. The two Raf1 RBDs (blue) could serve as docking sites for Galectin-1 dimers, leading to formation of Ras nanoclusters [115]. The different proteins domains and motifs are labeled the same way as in (**A**). The HVRs in (**B**,**C**) are either partially buried in or visually blocked by the membrane. Another interesting insight from the studies discussed above is the role of Raf in stabilizing Ras dimers. Raf is known to dimerize through its kinase domain after binding to Ras [54; 205] and in so doing may stabilize Ras-GTP multimers [206]. In the [PERSON] et al. work, adding Raf1 RBD to HRas or KRAS-GTP enabled robust dimerization on supported lipid bilayers and to a lesser extent, in solution [115]. Thus, the stabilization effect may be mediated via both the RBD (initial binding) and the Raf kinase domain (upon Raf dimerization, which follows Ras dimerization). As will be discussed below, this enhancement may synergize with other mechanisms such as scaffold mechanisms to ensure robust dimerization of the inherently weak Ras dimer where needed. In this regard, it will be interesting to see whether other Ras effectors can do the same through similar mechanisms. Of note, while bearing sequence homologies, the RBDs of Raf1, PI3K, and RalGDS, bind Ras with somewhat different orientations and contacts [207; 208; 209]. Scaffold proteins could further alter how Ras forms dimers, alone or in complex with signaling partners. Based on the high-resolution structures, [PERSON] et al. also proposed that Galectin dimers, such as those of Galectin-1, can connect proximal Ras-RBD dimers into even larger and more stable signaling complexes to give rise to membrane condensates, which were implicated in Ras activation [211]. This provides an alternative model for Ras assembly into large multimers [115]. Indeed, Galectin-1 exists as dimers in the cytosol and has been shown to bind Raf RBD and stabilize HRas nanoclusters [212]. Another recent study provided an alternative mechanism for making large KRAS-Raf signaling clusters ('signalosomes'), where Galectin-3 served as a scaffold [213]. Galectin-3, which exists in solution as a mixture of monomers and pentamers [214], was previously implicated in augmenting KRAS-GTP nanocluster formation and signaling [215; 216].