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. 2024 Jul 11;128(27):6518-6528.
doi: 10.1021/acs.jpcb.4c01876. Epub 2024 Jun 28.

Intrinsically Disordered Membrane Anchors of Rheb, RhoA, and DiRas3 Small GTPases: Molecular Dynamics, Membrane Organization, and Interactions

Chase M Hutchins  1   2 , Alemayehu A Gorfe  1   2
Affiliations

Intrinsically Disordered Membrane Anchors of Rheb, RhoA, and DiRas3 Small GTPases: Molecular Dynamics, Membrane Organization, and Interactions

Chase M Hutchins et al. J Phys Chem B. .

Abstract

Protein structure has been well established to play a key role in determining function; however, intrinsically disordered proteins and regions (IDPs and IDRs) defy this paradigm. IDPs and IDRs exist as an ensemble of structures rather than a stable 3D structure yet play essential roles in many cell-signaling processes. Nearly all Ras superfamily GTPases are tethered to membranes by a lipid tail at the end of a flexible IDR. The sequence of the IDR is a key determinant of membrane localization, and interaction between the IDR and the membrane has been shown to affect signaling in RAS proteins through the modulation of dynamic membrane organization. Here, we utilized atomistic molecular dynamics simulations to study the membrane interaction, conformational dynamics, and lipid sorting of three IDRs from small GTPases Rheb, RhoA, and DiRas3 in model membranes representing their physiological target membranes. We found that complementarity between the lipidated IDR sequence and target membrane lipid composition is a determinant of conformational plasticity. We also show that electrostatic interactions between anionic lipids and basic residues on IDRs are correlated with sampling of semistable conformational substates, and lack of these interactions is associated with greater conformational diversity. Finally, we show that small GTPase IDRs with a polybasic domain alter local lipid composition by segregating anionic lipids and, in some cases, excluding other lipids from their immediate vicinity in favor of anionic lipids.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
Sequence (A) and initial structure (B–D) of prenylated C-terminal membrane anchors of Rheb and RhoA and myristoylated N-terminal extension of DiRas3 (DNTE). Color code as indicated.
Figure 2.
Figure 2.
(A) Time averaged area per lipid (APL) and (B) lateral diffusion coefficient (D) of each lipid type in each simulation. APL was calculated using the last 1.5 μs of Rheb and RhoA simulations and the entire trajectory of DNTE. D was obtained from mean square displacements (MSD) calculated using 200 ns time blocks of the last 1 μs data, with linear regression performed on the most linear portion of the MSD plots, as described previously.
Figure 3.
Figure 3.
Snapshot at the end of Rheb and RhoA simulations illustrating the organization of the lipidated IDR in bilayer, highlighting basic residues in blue, acidic in red, lipidated cysteines in magenta, and remaining backbone atoms in atom-colored licorice. Protein backbone is shown in cartoon and side chains in licorice. Lipid acyl chains are shown as surface and head groups as sphere colored as indicated. Lipid atoms including those of POPS within 10 Å of protein heavy atoms as well as water and ions are omitted for a better visualization of the peptide.
Figure 4.
Figure 4.
(A) Time evolution of the number of hydrogen bonds (NHB) between all protein hydrogen bond donors and phosphate oxygen atoms of each lipid type in Rheb (top) and RhoA (bottom) simulations. (B) Radial pair distribution of phosphate oxygen atoms around the NZ atom of lysine and NH1 atom of Arg side chains on the Rheb and RhoA anchors.
Figure 5.
Figure 5.
(A) Peak-normalized 2D probability number density distribution of the Rheb and RhoA simulated conformers along a pseudo-dihedral angle and end-end distance reaction coordinates. The dihedral angles were calculated using four consecutive Cα atoms and the distance was between Cα atoms of C and N-terminal residues. Representative structures for the highest peaks are shown in purple with residues used for pseudo-dihedral angle calculation marked in gold. (B) Waterfall plots of the average number of hydrogen bonds between RhoA and lipids comparing all semistable conformations (combining conformers in all of the high-density contours in Figure 2) and the rest of the conformers.
Figure 6.
Figure 6.
(A) Snapshot at the end of the DNTE simulation illustrating the organization of the peptide on the host monolayer with residues colored as in Figure 1D, except for the myristoylated Gly which is now in magenta. Lipid atoms including those of POPS within 10 Å of protein heavy atoms as well as water and ions are omitted for clarity. (B) Close-up view of the bilayer insertion depth and lipid interaction of DNTE. Lipids are in gray lines with phosphorus atoms shown as orange balls. DNTE is shown in cartoon representation with side chains colored in red (acidic), blue (basic), white (nonpolar), and green (polar). (C) Distribution of simulated conformers along the top two principal components, PC1 and PC2, based on pairwise Cα atom distances excluding adjacent residues. Example structures for the high-density regions labeled 1–4 are shown (arrows), highlighting the shape and insertion depth of DNTE relative to the phosphate groups of the host monolayer (red spheres). Side chains are shown in licorice with residues colored as in Figure 1. (D) Frequency of DNTE-PS hydrogen bonds (NHB, left) and van der Waals contacts (NvdW) of DNTE nonpolar side chains and lipid acyl chains. Calculations were done using conformers from the regions of interest (ROI) marked with black squares in panel (C).
Figure 7.
Figure 7.
2D particle density of lipids around each lipidated IDR during the three simulations (see the Methods section). Normalized Gaussian kernel density values are shaded according to a global color scale where highest and lowest densities are the maximum and minimum densities out of all data sets. This allows one to compare the relative probability among lipid types and across simulations.
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