Recapitulation of cell-like KRAS4b membrane dynamics on complex biomimetic membranes

Abstract

Summary Understanding the spatiotemporal distribution and dynamics of RAS on the plasma membrane (PM) is the key for elucidating the molecular mechanisms of the RAS signaling pathway. Single particle tracking (SPT) experiments show that in cells, KRAS diffuses in at least three interchanging states on the cellular PM; however, KRAS remains monomeric and always shows homogeneous diffusion on artificial membranes. Here, we show for the first time on a supported lipid bilayer composed of heterogeneous lipid components that we can recapitulate the three-state diffusion of KRAS seen in cells. The use of a biologically relevant eight-lipid system opens a new frontier in the biophysical studies of RAS and other membrane associated proteins on a biomimetic system that recapitulates the complexity of a cellular PM.

Asynchronous Reciprocal Coupling of Martini 2.2 Coarse-Grained and CHARMM36 All-Atom Simulations in an Automated Multiscale Framework

“…Systems contain fluorescently labeled RAS, 8:2 mixtures of DOPC and DOPS, and a small fraction of fluorescently labeled DOPE. Results show that the lateral diffusion of RAS is approximately 1.5 times faster than that of DOPE (Figure ), consistent with our recent work . Replacement of half the DOPC with ent-DOPC leads to a small increase in the diffusion rates of both Atto550 DOPE and RAS (Figure ).…”

“…Both proteins are loaded with nonhydrolyzable GTP analogue (GppNHp) prior to the experiment. Details on the expression, purification, exchange, and labeling of the protein can be found in our prior publication . Proteins are incubated for at least an hour and washed with imaging buffer (20 mM Hepes, 300 mM NaCl, 5 mM MgCl 2 , and 5 mM 2-mercaptoethanol) prior to imaging.…”

“…Results show that the lateral diffusion of RAS is approximately 1.5 times faster than that of DOPE (Figure 4), consistent with our recent work. 110 Replacement of half the DOPC with ent-DOPC leads to a small increase in the diffusion rates of both Atto550 DOPE and RAS (Figure 4). However, replacement of half or all of the DOPC with Δ9-trans DOPC has no statistically significant effect on either of these diffusion rates (Figure 4).…”

Membrane lipids drive formation of KRAS4b-RAF1 RBDCRD nanoclusters on the membrane

“…Here, we collected single molecule tracks of membrane-bound, Alexa647 labeled KRAS C118S/S106C before and after addition of unlabeled RBDCRD on a supported 8-lipid bilayer. Our 8-lipid system provides a PM-like lipid environment that is a key regulator of KRAS membrane organization otherwise not attainable in simpler lipid bilayers 17 , 18 . The use of recombinant proteins, along with photostable dyes, results in longer tracks and the capacity for prolonged monitoring of protein kinetics.…”

Direct Modulators of K-Ras–Membrane Interactions

“…K-Ras PMIs are mediated by the C-terminal membrane anchor that consists of a farnesylated hexa-lysine polybasic domain. This anchor selectively associates with defined species of phosphatidylserine to form nanoclusters, comprising 4–6 K-Ras proteins. − In addition, K-Ras diffusion is distinctive when compared to other paralogs, indicating that the lipid–protein environment that K-Ras explores is unique. ,, Importantly, the specific lipid environment within K-Ras nanoclusters facilitates effector recruitment and activation. − However, the precise mechanism underlying this PMI dependence in effector recruitment is currently unknown. New chemical tools that enable a precise modulation of these PMIs could therefore meet a critical need.…”