K-Ras G-domain binding with signaling lipid phosphoinositides: PIP2 association, orientation, function

Abstract

Ras genes are potent drivers of human cancers, with mutated K-Ras4B being the most abundant isoform. Targeted inhibition of oncogenic gene products is considered the holy grail of present-day cancer therapy, and recent discoveries of small molecule inhibitors for K-Ras4B greatly benefited from a deeper understanding of the protein structure and dynamics of the GTPase. Since interactions with biological membranes are key for Ras function, the details of Ras - lipid interactions have become a major focus of study, especially since it is becoming clear that such interactions not only involve the Ras C-terminus for lipid anchoring, but also the G-protein domain. Here we investigated the interaction between K-Ras4B with the signaling lipid phosphatidyl inositol (4,5) phosphate (PIP2) using NMR spectroscopy and molecular dynamics simulations, complemented by biophysical and cell biology assays. We discovered that the {beta}2 and {beta}3 strands as well as helices 4 and 5 of the GTPase G-domain bind to PIP2, and that these secondary structural elements employ specific residues for these interactions. These likely occur in two orientation states of the protein relative to the membrane. Importantly, we found that some of these residues, which are known to be oncogenic when mutated (D47K, D92N, K104M and D126N), are critical for K-Ras-mediated transformation of fibroblast cells, while not substantially affecting basal and assisted nucleotide hydrolysis and exchange. We further showed that mutation K104M can indeed abolish localization of mutant K-Ras to the plasma membrane. These findings suggest that specific G-domain residues play an important, previously-unknown role in regulating Ras function by mediating interactions with membrane PIP2 lipids. Thus, a detailed description of the novel K-Ras-PIP2 binding surfaces is likely to inform the future design of therapeutic reagents.