GTP-Dependent K-Ras Dimerization

Abstract

SUMMARY Ras proteins recruit and activate effectors, including Raf, that transmit receptor-initiated signals. Monomeric Ras can bind Raf; however, activation of Raf requires its dimerization. It has been suspected that dimeric Ras may promote dimerization and activation of Raf. Here we show that the GTP-bound catalytic domain of K-Ras4B, a highly oncogenic splice variant of the K-Ras isoform, forms stable homodimers. We observe two major dimer interfaces. The first, highly populated β-sheet dimer interface is at the Switch I and effector binding regions, overlapping Raf’s, PI3K’s, RalGDS’ and additional effectors’ binding surfaces. This interface has to be inhibitory to such effectors. The second, helical interface also overlaps some effectors’ binding sites. This interface may promote Raf‘s activation. Our data reveal how Ras self-association can regulate effector binding and activity, and suggest that disruption of the helical dimer interface by drugs may abate Raf’s signaling in cancer.

Common mechanisms of catalysis in small and heterotrimeric GTPases and their respective GAPs

“…Similarly, a role for dimerization and nanocluster formation in signaling (Solman et al, 2015;Roob et al, 2016), possibly due to orientation changes of the G-domain (Mazhab-Jafari et al, 2015), was also reported by other groups. Specifically, Muratcioglu et al (2015) reported that K-Ras dimer formation is nucleotide dependent using dynamic light scattering and nuclear magnetic resonance (NMR) spectroscopy. Of note, this dimer formation by the Ras proteins provides a novel drug target (Santos, 2014; The N-Ras dimer with an interface consisting of residues from the β2, β3 loop, helix 4, and helix 5 as obtained by MD simulations (Güldenhaupt et al, 2012).…”

“…Subsequently, several Ras dimer interfaces, both similar and different to ours, were proposed based on MD simulations, mainly for K-Ras Prakash et al, 2016;Sayyed-Ahmad et al, 2016), and currently, there is no consensus on the detailed dimer interface. Some proposals are questionable from a physiological point of view; for example, dimerization including the effector domain ( Muratcioglu et al, 2015) would not lead to the observed increased signaling (Nan et al, 2013(Nan et al, , 2015. While measurements in cells have revealed that Ras forms nanoclusters and/ or dimers, and FRET between Ras molecules is in agreement with this (Solman et al, 2015), a detailed investigation of the interaction remains challenging.…”

Plasma membrane regulates Ras signaling networks

“…N-Ras also dimerizes in the GDP-bound inactive state in POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) bilayers. 71 Dimerization of K-Ras4B has been studied by NMR 74 and by photoactivated localization microscopy. 75 These studies identified contributions from the catalytic and HVR domains in dimer formation.…”

Seeing is believing: Ras dimers observed in live cells

“…More recently, two groups using molecular-dynamic modeling and a variety of biophysical methods have reported dimerization of the G domains of N-Ras (6) and H-Ras (7) when tethered via C-terminal lipid modifications to artificial phospholipid bilayers. Finally, Nussinov and colleagues (8), using similar techniques, very recently reported two different modes of dimerization of bacterially expressed, and therefore unprocessed, K-Ras4B in solution, one involving α-helices 3 and 4 and the other involving β-sheet interactions of the effector binding region. Importantly, both modes of dimerization were dependent on GTP binding.…”

“…First, the HVR is disordered and has a charge of +8 at physiological pH, making the mechanism of dimerization difficult to understand. Second, all of the recent biophysical studies that show dimerization reveal the dimerization interface to be in the catalytic G domain (6)(7)(8).…”