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

Abstract

Self-assembly of plasma membrane-associated Ras GTPases has major implications to the regulation of cell signaling. However, the structural basis of homo-oligomerization and the fractional distribution of oligomeric states remained undetermined. We have addressed these issues by deciphering the distribution of dimers and higher-order oligomers of K-Ras4B, the most frequently mutated Ras isoform in human cancers. We focused on the constitutively active G12V K-Ras and two of its variants, K101E and K101C/E107C, which respectively destabilize and stabilize oligomers. Using raster image correlation spectroscopy and number and brightness analysis combined with fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and electron microscopy in live cells, we show that G12V K-Ras exists as a mixture of monomers, dimers and larger oligomers, while the K101E mutant is predominantly monomeric and K101C/E107C is dominated by oligomers. This observation demonstrates the ability of K-Ras to exist in multiple oligomeric states whose population can be altered by interfacial mutations. Using molecular modeling and simulations we further show that K-Ras uses two partially overlapping interfaces to form compositionally and topologically diverse oligomers. Our results thus provide the first detailed insight into the multiplicity, structure, and membrane organization of K-Ras homomers.

Deciphering lipid codes: K‐Ras as a paradigm

“…[25][26][27][28][29][30][31] EM spatial analysis has been complemented by live cell imaging, including single-particle tracking, raster image correlation spectroscopy, fluorescence correlation spectroscopy, fluorescence recovery after photobleaching and photoactivated localization microscopy. [32][33][34] Coarse-grained molecular dynamic simulations also predict similar clustering behavior of Ras anchors on lipid bilayers in silico. 35,36 Together these studies suggest that~56% of Ras molecules exist as freely diffusing monomers or dimers and~44% of Ras molecules exist in higher order oligomers and nanoclusters.…”

Dissociation of the Signaling Protein K‐Ras4B from Lipid Membranes Induced by a Molecular Tweezer

“…Specifically,w ed emonstrate, how the lysine binder CLR01i sc apable of dissociating K-Ras4B from the lipid membrane by targeting the polybasic domain of K-Ras4B. [3,15] When K-Ras4Bb inds to the lipid membrane, simulations and biophysical studies have shownt hat the lysines of the polybasic domain are located outsidet he lipid bilayer,w hereas the farnesylated lipid anchor is inserted in the lipid membrane, [16][17][18] so that the polybasic domain shoulds till be largely accessible for the tweezer CLR01. Figure 1B depicts the exposed lysine residues in the largely disorderedp olybasic domain of the hypervariable regiono fK -Ras4B (in blue) and the argininer esidues (in violet)-further potentialb inding partners of the tweezer-on the surfaceo ftheK -Ras4Bp rotein.…”

“…Figure 1B depicts the exposed lysine residues in the largely disorderedp olybasic domain of the hypervariable regiono fK -Ras4B (in blue) and the argininer esidues (in violet)-further potentialb inding partners of the tweezer-on the surfaceo ftheK -Ras4Bp rotein. [3,15] When K-Ras4Bb inds to the lipid membrane, simulations and biophysical studies have shownt hat the lysines of the polybasic domain are located outsidet he lipid bilayer,w hereas the farnesylated lipid anchor is inserted in the lipid membrane, [16][17][18] so that the polybasic domain shoulds till be largely accessible for the tweezer CLR01.…”

The Role of Intestinal Fatty Acid Binding Proteins in Protecting Cells from Fatty Acid Induced Impairment of Mitochondrial Dynamics and Apoptosis

“…Acid Binding Protein present in the protein helps it to fold and attain native structure properly [15,71]. There are reports that suggest the roles of the hydrophobic patches of β-sheet proteins (66-69) [72][73][74][75] as also the alpha helices [76] to modulate the process of oligomerization. Hence, we hypothesized that during the lack of FA ligand in the cytosol, IFABP tends to form clusters by self-association (probably by forming non-covalent bonds between the hydrophobic patches of interacting proteins) and appear as bright dynamic species around the perinuclear region and within the cytosol.…”