K-Ras G-domain binding with signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2): membrane association, protein orientation, and function

Abstract

Edited by Henrik G. DohlmanRas genes potently drive human cancers, with mutated protooncogene GTPase KRAS4B (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 KRas4B inhibitors were made thanks to a deeper understanding of the structure and dynamics of this GTPase. Because interactions with biological membranes are key for Ras function, Ras-lipid interactions have become a major focus, especially because such interactions evidently involve both the Ras C terminus for lipid anchoring and its G-protein domain. Here, using NMR spectroscopy and molecular dynamics simulations complemented by biophysicaland cell-biology assays, we investigated the interaction between K-Ras4B with the signaling lipid phosphatidylinositol (4,5)phosphate (PIP2). We discovered that the ␤2 and ␤3 strands as well as helices 4 and 5 of the GTPase G-domain bind to PIP2 and identified the specific residues in these structural elements employed in these interactions, likely occurring in two K-Ras4B orientation states relative to the membrane. Importantly, we found that some of these residues known to be oncogenic when mutated (D47K, D92N, K104M, and D126N) are critical for K-Ras-mediated transformation of fibroblast cells, but do not substantially affect basal and assisted nucleotide hydrolysis and exchange. Moreover, the K104M substitution abolished localization of K-Ras to the plasma membrane. The findings suggest that specific G-domain residues can critically regulate Ras function by mediating interactions with membrane-associated PIP2 lipids; these insights that may inform the future design of therapeutic reagents targeting Ras activity.

Machine Learning-driven Multiscale Modeling Reveals Lipid-Dependent Dynamics of RAS Signaling Proteins

“…In particular, the HRC is enriched in PIP2 and depleted in cholesterol and polyunsaturated lipids, whereas the LRC exhibits opposite trends. These ndings on our in silico arti cial membrane corroborate RAS' preferential association with PIP2 on experimental arti cial membranes (32,33). However, discrepancy still exists between reconstituted systems and the observed enrichment of PS and phosphatidic acids in RAS nanoclusters on intact-cell PM sheets (11).…”

Comparative effects of oncogenic mutations G12C, G12V, G13D, and Q61H on local conformations and dynamics of K-Ras

“…These studies have used MD simulations with the HVR attached to the lipid membrane to understand the membrane orientation of the catalytic domain of an oncogenic K-Ras (i.e. G12V-KRAS 46,47 ), but have not compared different mutant proteins or nucleotide states. Therefore, MD simulations of different membrane-bound oncogenic K-Ras proteins, as well as their comparative analyses are useful for further understanding the effects of oncogenic mutations on K-Ras dynamics.…”

Mechanisms of Ras Membrane Organization and Signaling: Ras Rocks Again

“…Based on an elegant application of NMR to K-Ras bound on lipid nanodiscs, Mazhab-Jafari MT, et al found that disease-causing RASopathy mutations such as K5N and D153V modulate the membrane orientation of K-Ras [65]. Combining MD and spectroscopic approaches, additional studies by the Sligar and Buck groups have established that membrane reorientation is modulated by the lipid composition of the host membrane [63,68]. Helix α4 and the HVR were proposed to constitute two structural elements that were used to balance the reorientation equilibrium, thus modulating the preferred orientation and probably also the ease of the reorientation [17,21].…”