The KRAS and other prenylated polybasic domain membrane anchors recognize phosphatidylserine acyl chain structure

Abstract

KRAS interacts with the inner leaflet of the plasma membrane (PM) using a hybrid anchor that comprises a lysine-rich polybasic domain (PBD) and a C-terminal farnesyl chain. Electrostatic interactions have been envisaged as the primary determinant of interactions between KRAS and membranes. Here, we integrated molecular dynamics (MD) simulations and superresolution spatial analysis in mammalian cells and systematically compared four equally charged KRAS anchors: the wild-type farnesyl hexa-lysine and engineered mutants comprising farnesyl hexa-arginine, geranylgeranyl hexa-lysine, and geranylgeranyl hexa-arginine. MD simulations show that these equally charged KRAS mutant anchors exhibit distinct interactions and packing patterns with different phosphatidylserine (PtdSer) species, indicating that prenylated PBD–bilayer interactions extend beyond electrostatics. Similar observations were apparent in intact cells, where each anchor exhibited binding specificities for PtdSer species with distinct acyl chain compositions. Acyl chain composition determined responsiveness of the spatial organization of different PtdSer species to diverse PM perturbations, including transmembrane potential, cholesterol depletion, and PM curvature. In consequence, the spatial organization and PM binding of each KRAS anchor precisely reflected the behavior of its preferred PtdSer ligand to these same PM perturbations. Taken together these results show that small GTPase PBD-prenyl anchors, such as that of KRAS, have the capacity to encode binding specificity for specific acyl chains as well as lipid headgroups, which allow differential responses to biophysical perturbations that may have biological and signaling consequences for the anchored GTPase.

RAS Nanoclusters Selectively Sort Distinct Lipid Headgroups and Acyl Chains

“…As the sample Ripley's K-function curve in Figure 2C illustrates, peak clustering of GFP-RAS occurs at the radial length r of ∼20 nm, suggesting that the most probable radius of GFP-RAS nanoclusters is approximately 20 nm Plowman et al, 2005;Zhou et al, 2014;Liang and et al, 2019;Zhou et al, 2021). The K-function analysis further showed that RAS nanoclusters contain approximately 6-7 RAS molecules, and suggests that nearly half of GFP-RAS molecules exist as monomers, ∼30% as dimers, >10% as trimers, and <10% of GFP-RAS form higher order multimers .…”

“…The enrichment of specific lipids within different RAS nanoclusters has been investigated using EM-bivariate coclustering analysis of GFP-tagged lipid-binding domains that bind specific lipids (some examples listed in Figure 2H) and RFP-tagged RAS proteins on intact PM sheets (Zhou et al, 2014;Liang et al, 2019;Zhou et al, 2021). These experiments were complemented by FLIM-FRET in live cells expressing RFP-tagged RAS isoforms and spike-labeled TopFluor-tagged fluorescent lipids exogenously supplemented to these cells (Zhou et al, 2014;Liang et al, 2019;Zhou et al, 2021). The EM co-clustering analysis showed that RFP-KRAS4BG12V co-localized extensively with the PS probe GFP-LactC2 and the PA probe GFP-PASS, but not with the PIP 2 probe GFP-PH-PLCδ, the PIP 3 probe GFP-PH-Akt or the cholesterol probe GFP-D4H (Zhou et al, 2014;.…”

“…To manipulate PS content, PSS1 in Chinese hamster ovarian (CHO) cells was knocked down to generate a mutant line, termed as PSA3 cells (Lee et al, 2012). When grown in dialyzed fetal bovine serum (DFBS), PSA3 cells generate 35% less total PS and markedly lower PS levels in the PM inner leaflet (Lee et al, 2012;Zhou et al, 2014;Liang et al, 2019;Zhou et al, 2021). In the DFBS-treated PSA3 cells, supplementation of ethanolamine (Etn), which is a ligand upstream of PSS2, stimulates PSS2 and dose-dependently (0-10 μM for 72 h) elevates PS in the PM (Lee et al, 2012;Zhou et al, 2014;Liang and et al, 2019;Zhou et al, 2021).…”

“…When grown in dialyzed fetal bovine serum (DFBS), PSA3 cells generate 35% less total PS and markedly lower PS levels in the PM inner leaflet (Lee et al, 2012;Zhou et al, 2014;Liang et al, 2019;Zhou et al, 2021). In the DFBS-treated PSA3 cells, supplementation of ethanolamine (Etn), which is a ligand upstream of PSS2, stimulates PSS2 and dose-dependently (0-10 μM for 72 h) elevates PS in the PM (Lee et al, 2012;Zhou et al, 2014;Liang and et al, 2019;Zhou et al, 2021). Then, different extracts of mouse brain lipids were acutely added back (1-hour incubation) to the PSdepleted PSA3 cells (Zhou et al, 2014;Liang et al, 2019;Zhou et al, 2021).…”

“…In the DFBS-treated PSA3 cells, supplementation of ethanolamine (Etn), which is a ligand upstream of PSS2, stimulates PSS2 and dose-dependently (0-10 μM for 72 h) elevates PS in the PM (Lee et al, 2012;Zhou et al, 2014;Liang and et al, 2019;Zhou et al, 2021). Then, different extracts of mouse brain lipids were acutely added back (1-hour incubation) to the PSdepleted PSA3 cells (Zhou et al, 2014;Liang et al, 2019;Zhou et al, 2021). EM-univariate nanoclustering analysis of these cells showed that PS depletion effectively disrupted the nanoclustering and PM localization of GFP-KRAS4BG12V as well as the GFP-tK but had no effect on GFP-HRAS (Zhou et al, 2014;.…”