# Ras Multimers on the Membrane: Many Ways for a Heart-to-Heart Conversation
## 4 Membrane Interactions Regulate Ras Enrichment, Orientation, and Dimerization
(**A**) KRas G12D molecules exhibit three diffusion states on the cell membrane, a fast (F) state, an intermediate (N) state, and an immobile (I) state, with state transitions between the F:N and N:I states but not F:I states. Analysis of this model suggests a net flow of Ras molecules from F to N to I states, and KRas is internalized while in the I state and recycled back to the membrane in regions that support the F state. (**B**) Mapping the locations of the three diffusion states revealed a nested organization of the membrane domains that confers each state, with each nanodomain that entraps Ras (immobile, red) living inside another, intermediate domain (blue), whereas most the membrane supports fast and free diffusion (green) of Ras. (**C**) Model depicting how Ras (yellow circles) diffuses on the membrane, frequently encountering the intermediate domains (light blue) and occasionally trapped in structures (red) within the latter, where they presumably form dimers and nanoclusters. (**A,B**) adapted from [130]. environment, for example, may stabilize certain conformations, exposing some dimer interfaces and occluding others. The KRas HVR binds both PS and PIP2 with electrostatic interactions, which enables the HVR to adopt an orientation semi-parallel to the membrane (Figure 5A). The KRas HVR also specifically recognizes PS, and the presence of PS promotes the transition of the KRas HVR from a non-folded to a folded, helical conformation [138]. Since the KRas HVR is connected to the \(\alpha\)5 helix, this may rigidify the linkage between KRas and the membrane and influence the overall orientation of KRas. The Ras G-domains also interact with anionic lipids in the membrane; depending on the set of residues involved, the overall orientation of Ras will be very different [141; 145]. Experimental and computational studies show that KRas adopts many orientations on PS-containing membranes. The identified orientations vary somewhat among the studies. Two of the commonly observed orientations involve extensive membrane contacts of the Ras G-domain dimer interfaces: one at \(\alpha\)4-\(\alpha\)5 (Figure 5B, left) and the other \(\beta\)2-\(\beta\)3 (Figure 5B, right) [144; 145; 146; 148; 149; 150]. The \(\alpha\)3-\(\alpha\)4 dimer interface could also be buried in the membrane (not depicted in Figure 5) [145]. In another well populated (perhaps dominant) set of orientations, KRas takes a membrane-distal conformation (with the \(\alpha\)5'standing') in which all dimer interfaces are exposed (Figure 5B, middle) [148; 150]. Each orientation  
Figure 5: Membrane interactions dictate Ras orientations on the membrane, thus impacting accessibility of the dimer interfaces. (**A**) Interaction of KRas HVRs with different lipid moieties on the membrane. Two different HVR binding modes to the PIP2-enriched (black diamonds) membrane were observed, while one HVR binding mode was identified for PS-enriched (gray triangles) membrane [146; 147]. Lysine residues playing an important role in those interactions are shown with stick representation, and the residue numbers are listed below the HVR structures. (**B**) Subset of possible conformations sampled by the G-domain on anionic (PIP2 or PS) membranes. All G-domain orientations were rendered to approximation based on literature [144; 145; 146; 148; 149; 150]. exhibits a good degree of fluctuation, and Ras may transition between orientational states with relatively small kinetic barriers [148; 150]. KRas binds to PIP2-containing membranes much more strongly than to PS-containing membranes despite the fact that the KRas HVR can specifically recognize PS [143; 144; 147]. Interactions between PIP2 and the KRas G-domain involve an overlapping set of basic residues as in the case of PS, giving rise to two similar KRas orientations on the membrane (Figure 5B). However, the salt bridges between these residues and PIP2 apparently last longer (some up to ~160 ns in simulations) than those with PS (up to ~10 ns) [144].