Augmentation of Interactions between Dimers Stabilizes Higher Order Oligomers of Membrane-Bound K-Ras: A Single Molecule Perspective

Abstract

Ras proteins are small GTPases that control cell growth, differentiation and proliferation. They primarily reside on the inner leaflet of the plasma membrane and are crucial anticancer drug targets. The structural organization of Ras proteins on the plasma membrane has attracted major interest in recent years. Computational work by our group predicted three interfaces that are variously involved in the oligomerization of K-Ras, the most frequently mutated isoform of Ras proteins. In the current study, we examined a charge reversal K-Ras mutant K101E and a double cysteine mutant K101C/E107C that represent constructs with predicted weak and strong potential for oligomerization, respectively relative to the oncogenic G12V control (WT). We used fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and image correlation spectroscopy (ICS) to monitor the distribution of dimers and higher-order oligomers of these proteins. Our data show that the distribution of monomers, dimers and higher-order oligomers is such that the K101E mutant is mostly monomeric, K-Ras G12V exists as both dimer and higher-order oligomer whereas the percentage of dimers and larger oligomers is much greater in K101C/E107C, consistent with our prediction and data from signaling assays. These results establish that K101 and E107 play a key role in K-Ras oligomerization and establish the co-existence of diverse K-Ras oligomeric states.