The Ras G Domain Lacks the Intrinsic Propensity to Form Dimers

Abstract

Ras GTPase is a molecular switch controlling a number of cellular pathways including growth, proliferation, differentiation, and apoptosis. Recent reports indicated that Ras undergoes dimerization at the membrane surface through protein-protein interactions. If firmly established this property of Ras would require profound reassessment of a large amount of published data and modification of the Ras signaling paradigm. One proposed mechanism of dimerization involves formation of salt bridges between the two GTPase domains (G domains) leading to formation of a compact dimer as observed in Ras crystal structures. In this work, we interrogated the intrinsic ability of Ras to self-associate in solution by creating conditions of high local concentration through irreversibly tethering the two G domains together at their unstructured C-terminal tails. We evaluated possible self-association in this inverted tandem conjugate via analysis of the time-domain fluorescence anisotropy and NMR chemical shift perturbations. We did not observe the increased rotational correlation time expected for the G domain dimer. Variation of the ionic strength (to modulate stability of the salt bridges) did not affect the rotational correlation time in the tandem further supporting independent rotational diffusion of two G domains. In a parallel line of experiments to detect and map weak self-association of the G domains, we analyzed NMR chemical shifts perturbations at a number of sites near the crystallographic dimer interface. The nearly complete lack of chemical shift perturbations in the tandem construct supported a simple model with the independent G domains repelled from each other by their overall negative charge. These results lead us to the conclusion that self-association of the G domains cannot be responsible for homodimerization of Ras reported in the literature.

Membrane-associated Ras dimers are isoform-specific: K-Ras dimers differ from H-Ras dimers

“…In solution, the HVRs in the dimer interact strongly with each other, and the collapsed HVR backbones embrace the hydrophobic cluster formed by the prenyl groups. Membrane anchoring through the prenyl lipid moieties is not sufficient for productive and stable dimers [67]. Attachment to the phospholipids provides further stabilization, biases the monomer [40] and dimer organization and effector-binding site orientation, and constrains fluctuations, which promotes dimerization.…”

The disordered hypervariable region and the folded catalytic domain of oncogenic K-Ras4B partner in phospholipid binding

“…The HVR can be sequestered by the globular catalytic domain of GDP-bound K-Ras4B or released from the sequestered state when the catalytic domain binds GTP [4]. It can specifically interact with the calcium modulator protein calmodulin [5–7], selectively associate with plasma membrane lipids [8,9], critically contribute to dimer formation at the membrane [10–14], become phosphorylated by protein kinase C (PKC) [15]; and be segregated at spatially distinct plasma membrane signaling platforms [16]. …”

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

“…Consequently, it is difficult to prove direct Ras-Ras interactions in cells. Recently, an NMR study did not detect dimerization using the G-domain without the anchor region and without a bilayer (Kovrigina et al, 2015). This indicates that interactions within the hypervariable region (HVR) and/or membrane attachment anchor are required for dimerization.…”