Backbone conformational flexibility of the lipid modified membrane anchor of the human N-Ras protein investigated by solid-state NMR and molecular dynamics simulation

Abstract

The lipid modified human N-Ras protein, implicated in human cancer development, is of particular interest due to its membrane anchor that determines the activity and subcellular location of the protein. Previous solid-state NMR investigations indicated that this membrane anchor is highly dynamic, which may be indicative of backbone conformational flexibility. This article aims to address if a dynamic exchange between three structural models exist that had been determined previously. We applied a combination of solid-state nuclear magnetic resonance (NMR) methods and replica exchange molecular dynamics (MD) simulations using a Ras peptide that represents the terminal seven amino acids of the human N-Ras protein. Analysis of correlations between the conformations of individual amino acids revealed that Cys 181 and Met 182 undergo collective conformational exchange. Two major structures constituting about 60% of all conformations could be identified. The two conformations found in the simulation are in rapid exchange, which gives rise to low backbone order parameters and nuclear spin relaxation as measured by experimental NMR methods. These parameters were also determined from two 300 ns conventional MD simulations, providing very good agreement with the experimental data.

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). …”

“…However, insertion into thinner DLPC bilayers does not require the myristoyl chain to stretch to avoid unfavorably close contact between and, thus, extensive dehydration of the peptide and lipid headgroups. Furthermore, we could show that Coulombic and intrinsic contributions to membrane binding of myr-Src (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) are not simply additive, as short-range electrostatic effects in the presence of anionic lipids enhance membrane binding more strongly than would be expected on the basis of Coulombic attraction alone. …”

“…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.…”