Organization, dynamics, and segregation of Ras nanoclusters in membrane domains

Abstract

Recent experiments have shown that membrane-bound Ras proteins form transient, nanoscale signaling platforms that play a crucial role in high-fidelity signal transmission. However, a detailed characterization of these dynamic proteolipid substructures by high-resolution experimental techniques remains elusive. Here we use extensive semiatomic simulations to reveal the molecular basis for the formation and domain-specific distribution of Ras nanoclusters. As model systems, we chose the triply lipidated membrane targeting motif of H-ras (tH) and a large bilayer made up of di160-PC (DPPC), di182-PC (DLiPC), and cholesterol. We found that 4-10 tH molecules assemble into clusters that undergo molecular exchange in the sub-μs to μs time scale, depending on the simulation temperature and hence the stability of lipid domains. Driven by the opposite preference of tH palmitoyls and farnesyl for ordered and disordered membrane domains, clustered tH molecules segregate to the boundary of lipid domains. Additionally, a systematic analysis of depalmitoylated and defarnesylated tH variants allowed us to decipher the role of individual lipid modifications in domain-specific nanocluster localization and thereby explain why homologous Ras isoforms form nonoverlapping nanoclusters. Moreover, the localization of tH nanoclusters at domain boundaries resulted in a significantly lower line tension and increased membrane curvature. Taken together, these results provide a unique mechanistic insight into how protein assembly promoted by lipid-modification modulates bilayer shape to generate functional signaling platforms.

Fig. 1.

Molecular models. (A) CG models for (from left to right): DPPC, DLiPC, CHOL, and tH. For the lipids the beads represent NCH₃ (blue), PO₄ (red), and glycerol (gray) groups. Cyan and pink represent the saturated and unsaturated portions of the lipid tails, respectively. For CHOL, the ring is shown in yellow, the tail in cyan and the OH group in red. The heptapeptide tH is comprised of Gly180, Pa181, Met182, Ser183, Pa184, Lys185, and Fa186; Pa and Fa represent palmitoylation and farnesylation, respectively. Note that, despite the different color schemes used here, the hydrocarbon tail of Pa is the same as that of DPPC. (B) Top view of the initial structure (left) and after 24 μs MD (right) of the 5∶3∶2 DPPC∶DLiPC∶CHOL ternary bilayer in the absence of tH. The initial structure shown here is after minimization and preequilibration (heating) of the system. DPPC, DLiPC and CHOL are shown as tan, blue and white dots, respectively. (C) Top view of the initial structure (left) and after 24 μs (right) of a system with 64 tH molecules bound to one side of the ternary bilayer. The 64 tH molecules were initially distributed on a grid with random orientations. Similarly to the free bilayer, the initial configuration is after minimization and heating of the system with the tH restrained to their original grid positions. The tH beads are represented in red balls, with palmitoyl and farnesyl tails colored in green and yellow, respectively. Images rendered with VMD (44).

Fig. 2.

Size and localization of tH nanoclusters. (A) Comparison of cluster size distributions derived from EM (gray bars) and MD simulations at 18 °C (turquoise), 28 °C (black) and 38 °C (red). Insets: left, results from single-linkage (empty squares) and Ripley’s K-function (filled circles) analysis of the EM data; right, results from a simulated 2D Poisson distribution of 64 noninteracting particles in the same box size as our system. (B) Lipid composition across the bilayer with the origin set to be the middle of the L_d domain. Dotted lines demarcate the center of the L_o/L_d boundary, defined as the location at which the average DPPC and DLiPC compositions are equal. (C) Probability distribution of tH across the bilayer (black line) and its decomposition into clusters of different sizes (s) shown in purple (monomers), cyan (dimers and trimers), and red (four or larger). All data in this and subsequent figures are from simulations performed at 28 °C.

Fig. 3.

Size and localization of mutant tH nanoclusters. (A) Comparison of wild-type tH cluster sizes (gray bars) with those of de-palmitoylated (de-Pa181: cyan, de-Pa184: blue, de-Pa181/184: green) and de-farnesylated (de-Fa186: magenta) tH variants. (B) and (C) The probability distribution of the four tH variants across the bilayer. The wild-type tH distribution is shown for reference (dashed line).

Fig. 4.

Nanocluster-induced bilayer shape change. (A) Average lipid tail tilt angle from the bilayer normal in the tH-free bilayer. (B) Cross-section of a snapshot from the tH-free simulation showing the opposite curvature of the two monolayers. (C) Same as in (A) but for the tH-bound bilayer, with the tH-containing monolayer in solid line and the tH-free monolayer in a dashed line. (D) Same as in (B) but for the tH-bound bilayer, showing the same overall curvature of the two monolayers and a larger nanocluster-induced curvature at the L_o/L_d interface.

Fig. 5.

Organization of tH components in the bilayer. (A) Average tilt angle of tH lipids from the bilayer normal. (B) DPPC/(DPPC + DLiPC) fraction around the backbone of each tH residue. A 0.7 nm cutoff was used. Since CHOL represents only approximately 0.5 % of the molecules at the interface, the interfacial line is assumed to be located at a DPPC/(DPPC + DLiPC) ratio of 0.5.

Fig. 6.

Schematic representation of the tH anchor configuration in the curved bilayer with the L_o domain in tan and L_d in blue. Backbone and phosphate beads in ball representation and lipid tails in lines, with straighter lines representing more ordered tails. tH backbone color-coding is the same as in Fig. 1A. Cholesterol is shown as gray ellipsoid within the L_o domain. For clarity, the distance between different tH backbone beads is exaggerated. The figure highlights the parallel alignment of the protein backbone, the L_o- and L_d-oriented palmitoyl and farnesyl tails, and membrane curvature (especially at the tH-containing monolayer).