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 KRas4B 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 β2 and β3 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.

Dynamics of Membrane-Bound G12V-KRAS from Simulations and Single-Molecule FRET in Native Nanodiscs

“…This result directly links internal conformational fluctuation to membrane reorientation. We would like to emphasize that, although the different orientations are sampled spontaneously, they may be stabilized by protein-lipid electrostatic interactions as suggested by several previous reports (16,18,23). That intrinsic flexibility underlies the membrane orientation dynamics of G12V-KRAS is further confirmed by performing another 20 ms MD simulation using the CHARMM36 force field (24).…”

“…OS 1 and OS 2 differ in the accessibility of functionally critical switch loops to partner proteins, suggesting that membrane reorientation in the cell may modulate KRAS signaling. However, our current understanding of this phenomenon is limited to inferences from structural models from molecular dynamics (MD) simulations or indirect spectroscopic techniques in simple model membranes (15)(16)(17)(18). While MD simulations typically access only relatively short length and timescales, the timescale of RAS reorientation in the complex cell membranes is likely too fast to resolve by experimental techniques alone.…”

Membrane curvature sensing of the lipid-anchored K-Ras small GTPase

“…Specifically, the basic residues in the helix α4 on the GTP-bound H-Ras associate with anionic lipids in the membranes (78,79). K-Ras G-domain residues, such as Arg73 and Arg102, associate with PS in the membranes (80), whereas several polar residues, such as Lys16, Asp47, and Glu49, of K-Ras G-domain associate with PIP 2 ((81) Preprint ). AFM experiments also show that the globular K-Ras G-domain is partially embedded in the supported bilayers (82).…”

Computational Design of Myristoylated Cell Penetrating Peptides Targeting Oncogenic K-Ras.G12D at the Effector Binding Membrane Interface

“…K-Ras4A and -4B have been found to bind to such a membrane relatively strongly but still with multiple distinct orientations. [17][18][19][20][21][22] Addition of such anionic lipid molecules may neutralize the charge of some of the arginines and thus may increase the binding of myr_R9 peptide residues, while possibly decreasing K-Ras -POPS interactions. However, in the simulations where 20% of POPC was replaced with POPS, the binding of the K-Ras4B core domain toward the membrane is also very strong ( Fig.…”

“…12 Both lobes bind to the membrane, based on the results of computational modeling, NMR and FRET. [17][18][19][20][21][22] For functional activity via the ERK or the MAPK pathway, the effector lobe needs to associate with down-stream effector proteins, such as Raf and PI3K. 23,24 Therefore, membrane binding of the effector lobe will occlude the association of an effector protein with this region of the GTPase, and thus attenuate the activity of K-Ras.…”