# Ras Multimers on the Membrane: Many Ways for a Heart-to-Heart Conversation
## 3 Ras Dimerization at Variable G-domain Interfaces  
To better understand how Ras dimers form and function in cells, a critical step is to define the Ras-Ras dimer interface. Both experimental and computational approaches have been employed to identify the motifs and residues responsible for Ras dimer formation. On the computation side, homology or data-driven modeling [111; 112] as well as template-based modeling [113] are widely used to generate physically fitting dimer structures, while molecular dynamics (MD) simulations extent the knowledge on the stability and interactions of the potential interfaces [91; 114; 115]. Recently machine learning approacheswere coupled with the above to generate more data with increased accuracy [116]. On the experimental side, recent studies using FTIR, NMR, FRET, and EM in combination with point mutations and functional assays have been used to test and refine the predicted dimer interfaces [71; 84; 85; 91; 92; 97; 117]. Current efforts mostly focused on identifying Ras-Ras contacts at the G-domain [81; 82]. A surprising finding from data thus far is the ability of Ras to dimerize at several different G-domain interfaces. The main three interfaces identified by several studies are as follows; two interfaces consist of \(\alpha\) helices \(\alpha\)4-\(\alpha\)5 and \(\alpha\)3-\(\alpha\)4, and the last one uses the region encompassing \(\beta\)2-\(\beta\)3. We briefly describe these dimer interfaces below. The \(\alpha\)4-\(\alpha\)5 dimer interface mostly covers the \(\alpha\)4 and \(\alpha\)5 helices, the last two helices before the HVR. The helices correspond to G-domain residues 126-138 and 151-166, respectively. The detailed structural representation of this interface is shown in Figure 3A (left). The interface was originally proposed by [PERSON] et al. in the context of NRas dimers [84] and later identified computationally for KRas by [PERSON] et al. [117], [PERSON] et al. [70], and [PERSON] et al. [118]. This interface has now been experimentally tested for KRas [65; 71; 92] and NRas [85]. Ras-Ras interactions at this interface give rise to a dimer where the two promoters are'standing' on their \(\alpha\)4 and \(\alpha\)5 helices relative to the membrane plane, leaving the effector region (switch I and II) well exposed. This dimer interface is therefore thought to be compatible with effector recruitment. This interface was targeted by the NS1 monobody [98], which blocks dimerization, signaling, and pancreatic tumorigenesis by mutant KRas [109; 110]. The second interface is the \(\alpha\)3-\(\alpha\)4 dimer interface, which consists of another set of \(\alpha\)-helices. It partially overlaps with the \(\alpha\)4-\(\alpha\)5 interface and roughly covers the G-domain residues 86-104 and 126-138. [PERSON] et al. proposed this interface combining biophysical experimental techniques and structure prediction algorithms [91] (Figure 3A, middle). They further confirmed the predictions using dynamic light scattering, FRET and NMR experiments. Another study from the same group showed that charge reversal mutations in two critical residues on this interface decrease the dimerization [97]. [PERSON] et al. [117] and [PERSON] et al. [70] also presented evidence for this interface. The third Ras dimer interface involves two \(\beta\) sheets, namely \(\beta\)2 (residues 38-43) and \(\beta\)3 (residues 52-54), as well as part of the switch I (residues D33, I36, E37, and D38) (Figure 3A, right). This interface is more distinct than the previous two, both in its location on the Ras surface and in that it has some overlap with the switch I region, which is involved in effector binding. [PERSON] et al. first proposed this interface in 2015 [91], where they named the corresponding dimer a '\(\beta\)-homodimer' to distinguish it from the '\(\alpha\)-homodimer' (\(\alpha\)3-\(\alpha\)4).