# Ras Multimers on the Membrane: Many Ways for a Heart-to-Heart Conversation
## 6 Scaffold and Effector Proteins in Ras Dimerization  
In addition to the structural elements within membrane nanodomains, extrinsic factors such as Ras-binding proteins may also regulate how Ras dimers are constructed and function. Since Ras exerts its biological activities via interactions with effectors and other signaling partners, understanding Ras dimers in complex with signaling partners and scaffold proteins is as important as (if not more than) understanding how Ras dimerizes on its own. To date, there has not been a detailed structure of full-length and fully modified Ras in complex with full-length effectors such as Raf or PISK on a membrane. Nevertheless, exciting advances have been made in solving the structure of Ras in complex with the Raf RBD or both the Raf RBD and the cysteine-rich domain (CRD). Using X-ray crystallography, [PERSON] et al. solved the structure of KRas G-domain complexed with a peptide comprising both the RBD and CRD of Raf1. The structure revealed extensive interactions of KRas with both the RBD and the CRD, with the CRD close to the \(\beta\)2-loop-\(\beta\)3 and the \(\alpha\)5 helix of KRas. Aided by NMR spectroscopy, the authors modeled how this complex interacts with the membrane and proposed a model where the \(\alpha\)4 and \(\alpha\)5 helices of KRas as well as the CRD of Raf1 make close contacts with the membrane [197]. By fitting the structure to the possible KRas conformations observed in large-scale MD simulations [145], a membrane:KRas:Raf1 RBD-CRD model was inferred (Figure 7A). In this model, KRas adopts a conformation with its \(\alpha\)4-\(\alpha\)5 region juxtaposed to the membrane, similar to that shown in Figure 5B (left). This conformation enables good contacts between the CRD and the membrane but precludes dimerization at the \(\alpha\)4-\(\alpha\)5 (membrane occlusion) or the \(\beta\)2-\(\beta\)3 (blocked by the Raf1 CRD) interface. It also resembles one of the two observed conformations of KRas:Raf1 RBD-CRD on nanodiscs using PRE-NMR by [PERSON] et al. (Figure 7B, left) [198], although the folding of Raf1 CRD differs significantly between the two. Dimerization via the \(\alpha\)3-\(\alpha\)4 dimer interface might still be possible, as suggested in a simulation study by [PERSON] et al. [199]. In the PRE-NMR study, another, less populated conformation might also be compatible with Ras dimerization at both the \(\alpha\)4-\(\alpha\)5 and the \(\alpha\)3-\(\alpha\)4 interfaces (Figure 7B, right). Indeed, at increased KRas and Raf1 RBD-CRD concentrations, the equilibrium seems to shift towards the second conformation [198]. All these KRas conformations, however, deviate significantly from that seen in the KRas dimers observed on nanodiscs with PRE-NMR (see Figure 7C) [92]. In another study, [PERSON] et al. demonstrated that the presence of Raf RBD induces robust dimerization of both KRas-GTP and HRas-GTP on artificial membranes containing 20% PS, where Ras dimerization likely takes place at the \(\alpha\)4-\(\alpha\)5 interface. Here, binding of Raf RBD causes an allosteric effect extending from the switch region (Ras-RBD binding site) to the \(\alpha\)4-\(\alpha\)5 dimer interface, which may have helped stabilize the \(\alpha\)4-\(\alpha\)5 exposed conformation to promote formation of the corresponding dimer [115]. [PERSON] et al. obtained the crystal structure of HRas (G-domain)/Raf RBD-CRD, which is nearly identical to that of KRas (G-domain)/Raf RBD-CRD [197], and proposed an alternative model for the tertiary Ras-Ras/Raf-Raf complex based on a \(\alpha\)4-\(\alpha\)5 Ras dimer. In this model, the Raf CRD is located between the two Ras protomers and kept away from the membrane [200]. Interestingly, the authors pointed out that this dimer model is present in the crystal (between two-unit cells) from which the KRas-GTP:Raf1 RBD-CRD structure was obtained [197] (Figure 7D).