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

Abstract

Recent studies found that membrane-bound K-Ras dimers are important for biological function. However, the structure and thermodynamic stability of these complexes remained unknown because they are hard to probe by conventional approaches. Combining data from a wide range of computational and experimental approaches, here we describe the structure, dynamics, energetics and mechanism of assembly of multiple K-Ras dimers. Utilizing a range of techniques for the detection of reactive surfaces, protein-protein docking and molecular simulations, we found that two largely polar and partially overlapping surfaces underlie the formation of multiple K-Ras dimers. For validation we used mutagenesis, electron microscopy and biochemical assays under non-denaturing conditions. We show that partial disruption of a predicted interface through charge reversal mutation of apposed residues reduces oligomerization while introduction of cysteines at these positions enhanced dimerization likely through the formation of an intermolecular disulfide bond. Free energy calculations indicated that K-Ras dimerization involves direct but weak protein-protein interactions in solution, consistent with the notion that dimerization is facilitated by membrane binding. Taken together, our atomically detailed analyses provide unique mechanistic insights into K-Ras dimer formation and membrane organization as well as the conformational fluctuations and equilibrium thermodynamics underlying these processes.

Analysis of K-Ras Interactions by Biotin Ligase Tagging

“…As shown, our chimeric BirA*Ras proteins were expressed as expected, localized as predicted, and their localization patterns matched the patterns of the proteins they biotinylated (Figures 1-4). K-Ras proteins also have been reported to oligomerize (17,20,(89)(90)(91), and we observed that WT, G12D, and S17N BirA*Ras proteins biotinylated WT pAMCherry1-KRas proteins in trans ( Figure 5). These results are of interest in the context of data that show both GDPbound and GTP-bound K-Ras proteins oligomerize in cells (20).…”

Spatiotemporal Analysis of K-Ras Plasma Membrane Interactions Reveals Multiple High Order Homo-oligomeric Complexes

“…Diffusion of Ras is dominated by lateral mobility on the PM, 36,37 and we previously showed using EM and FLIM/FRET that clustering is decreased in the KE mutant and increased in CC relative to WT. 9 Together, these results suggest an altered molecular organization of KE and CC compared with WT. Consistent with this interpretation, the mobile fraction of molecular species is larger in KE (~89%) than WT and CC (73–76%).…”

“…9 To model larger K-Ras oligomers, we first required that each monomer within a cluster should bind a planar membrane. This eliminates oligomers of the D-symmetry group except D 2 (dimer of dimer).…”

“…As an initial step toward addressing these questions, we recently described two partially overlapping protein–protein interaction interfaces (PPIs) that underlie K-Ras self-interactions. 9 …”

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

“…(B) The proposed dimer interface with coordinating residues as obtained by MD simulations. A similar interface was proposed for K-Ras (Spencer-Smith et al, 2016;Prakash et al, 2017). Stephen et al, 2014;Chen et al, 2016;Lu et al, 2016), and elucidating the dimer interface at atomic detail will facilitate therapeutic approaches that modulate this interaction.…”