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.

Figure 1

Effect of ionic strength on rotational correlation times of the G domains in Ras181 (black circles), Ras-2-Ras (red circles), and Ras-11-Ras (blue circles) at 20°C (top) and 37°C (bottom). Dashed lines connect data points for the same protein sample to guide the eye. The correlation time of a monomeric G domain Ras166 at low salt and 20°C is shown in the top panel with an open circle. Correlation times measured with two independent preparations of Ras181 at 37°C (black circles) are shown separately to demonstrate reproducibility of the measurements. Error bars represent 95% confidence intervals. Vertical black bars show the expected range of rotational correlation times for the Ras conjugates if the G domains formed tight dimers at low salt condition (see Supporting Material for details of this estimate). The arrows indicate anticipated reduction of the dimer correlation time upon increasing the ionic strength. To see this figure in color, go online.

Figure 2

Localization of the assigned ¹H and ¹⁵N amide nuclear spins expected to experience chemical shift perturbation due to formation of the Ras dimer. Blue and white spheres indicate amide groups of the following residues: G48, E49, T50, S127, R128, R135, S136, Y137, I139, Y141, I142, E143, G151, D154, A155, Y157, T158, R161, and E162 (some are labeled for a visual guidance). One G domain is shown as a cartoon; another—with the van der Waals surface. Switch I, magenta; switch II, yellow; GDP, green sticks; and active site magnesium ion, a red sphere. To see this figure in color, go online.

Figure 3

Lack of significant chemical shift differences between amide resonances of the G domain in Ras181 and Ras-2-Ras. (A) Overlay of ¹⁵N-¹H HSQC NMR spectra for Ras-2-Ras (red) onto the Ras181 (blue) at low ionic strength at 20°C. Peak assignments are shown for resonances of the G domain; labels in crowded regions were removed for clarity. Signals from the C-terminal peptide affected by the conjugation reaction are indicated by black ovals. (B) Averaged chemical shift differences, Δ_av, plotted versus the residue number in the G domain. The C-terminal extensions were not included in analysis. Residues at the dimer interface (their NH groups indicated by spheres in Fig. 2) are indicated as shaded areas. Intervals without black bars correspond to gaps in the assignment or unresolved spectral overlap. (C) Enlarged spectral views of the two peaks indicated with an asterisk in (B).To see this figure in color, go online.