Flexibility of Ras Lipid Modifications Studied by 2H Solid-State NMR and Molecular Dynamics Simulations

Abstract

Human posttranslationally modified N-ras oncogenes are known to be implicated in numerous human cancers. Here, we applied a combination of experimental and computational techniques to determine structural and dynamical details of the lipid chain modifications of an N-ras heptapeptide in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes. Experimentally, 2H NMR spectroscopy was used to study oriented membranes that incorporated ras heptapeptides with two covalently attached perdeuterated hexadecyl chains. Atomistic molecular dynamics simulations of the same system were carried out over 100 ns including 60 DMPC and 4 ras molecules. Several structural and dynamical experimental parameters could be directly compared to the simulation. Experimental and simulated 2H NMR order parameters for the methylene groups of the ras lipid chains exhibited a systematic difference attributable to the absence of collective motions in the simulation and to geometrical effects. In contrast, experimental 2H NMR spin-lattice relaxation rates for Zeeman order were well reproduced in the simulation. The lack of slower collective motions in the simulation did not appreciably influence the relaxation rates at a Larmor frequency of 115.1 MHz. The experimental angular dependence of the 2H NMR relaxation rates with respect to the external magnetic field was also relatively well simulated. These relaxation rates showed a weak angular dependence, suggesting that the lipid modifications of ras are very flexible and highly mobile in agreement with the low order parameters. To quantify these results, the angular dependence of the 2H relaxation rates was calculated by an analytical model considering both molecular and collective motions. Peptide dynamics in the membrane could be modeled by an anisotropic diffusion tensor with principal values of Dparallel=2.1x10(9) s(-1) and Dperpendicular=4.5x10(5) s(-1). A viscoelastic fitting parameter describing the membrane elasticity, viscosity, and temperature was found to be relatively similar for the ras peptide and the DMPC host matrix. Large motional amplitudes and relatively short correlation times facilitate mixing and dispersal with the lipid bilayer matrix, with implications for the role of the full-length ras protein in signal transduction and oncogenesis.

Structural Thermodynamics of myr-Src(2–19) Binding to Phospholipid Membranes

“…However, as pointed out in that study, polar and charged residues near the C-terminus may be located closer to the membrane than indicated by the spin labels, and distances from the membrane >5 Å are not determined unambiguously. Hence, it is very likely that all basic amino acids of myr-Src are indeed localized close to the headgroup region of the phospholipid membrane, supporting the above conclusion that additional short-range electrostatic interactions with anionic lipid headgroups contribute to membrane association of myrSrc (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). …”

“…We have shown that myr-Src (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) interacts with zwitterionic membranes by partial insertion of its N-terminal myristoyl chain, where insertion depth and chain extent are dictated by a compromise between the hydrophobic effect and optimization of membrane packing on the one hand and Born repulsion and dehydration effects on the other. The interplay of these contributions results in a higher affinity of the Src peptide for DLPC than for DMPC, although the latter is expected to enable better hydrophobic matching.…”

“…In particular, nuclear magnetic resonance (NMR) spectroscopy of the human N-Ras protein has provided a structural view of the membraneinserted lipid modifications of the protein (10)(11)(12), the conformation of the membrane-associated protein moiety (13,14), and the structural dynamics of the amino-acid residues interacting with the membrane surface (15,16). Interestingly, the Ras palmitoyl chain has been shown to adjust its length to the hydrophobic thickness of the host membrane to maximize favorable hydrophobic interactions (17).…”

“…For nonmyristoylated Src (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), a free-energy minimum has been postulated at a distance of~3 Å between the van der Waals surfaces of the peptide and an anionic membrane (9), corresponding to one layer of water molecules (24). In the case of myristoylated Src (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), 2 H NMR spectroscopy has revealed that the acyl chain of the peptide is much more extended than the phospholipid acyl chains in a zwitterionic DMPC bilayer, such that insertion of onlỹ 11.5 carbons of the myristoyl chain suffices to match the hydrophobic thickness of the host membrane. In the presence of negatively charged lipids, the length difference between the myristoyl chain of Src and the phospholipid acyl chains is somewhat diminished (26) because the latter are slightly longer than in a zwitterionic membrane.…”

Palmitoylation and localisation of RAS isoforms are modulated by the hypervariable linker domain

“…These studies revealed that the linker domain is highly flexible and enables basic residues within the HVR and the G-domain of H-RAS to interact with membranes (Gorfe et al, 2007). A separate observation from the spectroscopy and modelling studies was that, in the context of the N-RAS targeting domain, the farnesyl group forms a stable anchor, whereas the palmitoyl group can flip in and out of the membrane (Vogel et al, 2007). Both our data and previous studies of H-RAS reveal that the N-and H-RAS Gdomains partially oppose the membrane interaction forces supplied by the HVR (Fig.…”