Rotational and Translational Dynamics of Ras Proteins upon Binding to Model Membrane Systems

Abstract

Plasma-membrane-associated Ras proteins typically control signal transduction processes. As nanoclustering and membrane viscosity sensing provide plausible signaling mechanisms, determination of the rotational and translational dynamics of membrane-bound Ras isoforms can help to link their dynamic mobility to their function. Herein, by using time-resolved fluorescence anisotropy and correlation spectroscopic measurements, we obtain the rotational-correlation time and the translational diffusion coefficient of lipidated boron-dipyrromethene-labeled Ras, both in bulk Ras and upon membrane binding. The results show that the second lipidation motif of N-Ras triggers dimer formation in bulk solution, whereas K-Ras4B is monomeric. Upon membrane binding, an essentially free rotation of the G-domain is observed, along with a high lateral mobility; the latter is essentially limited by the viscosity of the membrane and by lipid-mediated electrostatic interactions. This high diffusional mobility warrants rapid recognition-binding sequences in the membrane-bound state, thereby facilitating efficient interactions between the Ras proteins and scaffolding or effector proteins. The lipid-like rapid lateral diffusion observed here complies with in vivo data.

Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site

“…2C and Fig. S2B), consistent with orientational flexibility of the K-RAS4B GTPase domain (6); however, the relative population of each cluster was dependent on the activation state (Table S2). In the cluster that predominates in the GDP-bound form, most of the K-RAS4B helices are oriented parallel to the membrane, and a surface comprised of the N terminus of β1 (N-β1), α4, β6, α5, and the loop connecting β2 and β3 interfaces with the membrane (Fig.…”

“…Although the significance of membrane tethering of K-RAS4B is well appreciated, a high-resolution map of how K-RAS4B interacts with the membrane is lacking. Because membrane-anchored RAS presents a major challenge to crystallization, current structural insights into the behavior of membrane-anchored RAS have come from a variety of lower-resolution techniques including in vivo FRET-based studies (3), fluorescence and infrared spectroscopic studies (4)(5)(6), and in silico models (3). These pioneering studies suggested that the K-RAS4B GTPase domain adopts certain preferred orientations on the anionic membrane and that these orientations could be influenced by the bound nucleotide.…”

Computational Equilibrium Thermodynamic and Kinetic Analysis of K-Ras Dimerization through an Effector Binding Surface Suggests Limited Functional Role

“…Plugging these values into eq 7 yielded δω = 0.08 rad 5 , which measures the orientational freedom of the monomer. Using this value and integrating the PMF profile shown in Figure 6A over the entire range of r , we obtained K d = 870 μ M. This indicates a weak PPI 42 (e.g., a dimer fraction of 2% in a 10 μ M solution), and is consistent with the failure of various experimental studies to detect K-Ras dimers in solution 13 except at a relatively high concentration or upon membrane binding. 12 …”

“…Moreover, using gel filtration, Dementiev 12 showed that K-Ras dimerizes upon binding to a synthetic membrane via a meleimide-linked C-terminally farnesylated motif. On the other hand, Werkmüller et al 13 found that K-Ras is monomeric in bulk solution at concentrations as high as 2.0 μ M based on rotational-correlation times from time-resolved fluorescence anisotropy (TRFA) and lateral diffusion coefficients from fluorescence correlation spectroscopy (FCS) experiments. Another recent report by Nussinov and co-workers 14 combined microscale thermophoresis and dynamic light scattering experiments with computational modeling to propose that the soluble catalytic domain of GTP-loaded K-Ras forms dimers in solution at concentrations in the tens of micromolar range.…”

Computational and biochemical characterization of two partially overlapping interfaces and multiple weak-affinity K-Ras dimers

“…The evidence for Ras dimerization in solution is mixed. For example, N-Ras was shown to dimerize in bulk solution at concentrations as low as 0.34 μM while K-Ras is a monomer at 2.0 μM12. Yet the soluble catalytic domain of K-Ras was reported to dimerize with an apparent dissociation constant (K d ) of ~1 μM13, while a C-terminally cross-linked H-Ras does not14.…”