The Lipidated Membrane Anchor of Full Length N-Ras Protein Shows an Extensive Dynamics as Revealed by Solid-State NMR Spectroscopy

Abstract

Many proteins involved in signal transduction are equipped with covalently attached lipid chains providing a hydrophobic anchor targeting these molecules to membranes. Despite the considerable biological significance of this membrane binding mechanism for 5-10% of all cellular proteins, to date very little is known about structural and dynamical features of lipidated membrane binding domains. Here we report the first comprehensive study of the molecular dynamics of the C-terminus of membrane-associated full-length lipidated Ras protein determined by solid-state NMR. Fully functional lipid-modified N-Ras protein was obtained by chemical-biological synthesis ligating the expressed water soluble N-terminus with a chemically synthesized (2)H or (13)C labeled lipidated heptapeptide. Dynamical parameters for the lipid chain modification at Cys 181 were determined from static (2)H NMR order parameter and relaxation measurements. Order parameters describing the amplitude of motion in the protein backbone and the side chain were determined from site-specific measurements of (1)H-(13)C dipolar couplings for all seven amino acids in the membrane anchor of Ras. Finally, the correlation times of motion were determined from temperature dependent relaxation time measurements and analyzed using a modified Lipari Szabo approach. Overall, the C-terminus of Ras shows a versatile dynamics with segmental fluctuations and axially symmetric overall motions on the membrane surface. In particular, the lipid chain modifications are highly flexible in the membrane.

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

“…Chemically synthesised, lipid-modified tN and full-length H-and N-RAS have been analysed spectroscopically and modelled using Molecular Dynamics software to determine how the HVR orientates when inserted into membranes. The majority of the HVR is embedded at the lipid-water interface, whereas the lipid modifications insert into the hydrophobic core of the membrane (Gorfe et al, 2004;Huster et al, 2003;Reuther et al, 2006). 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).…”

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

“…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).…”

“…Accordingly, the peptide moiety of Src is thought to be localized above the membrane surface, with one or more water layers in between the peptide and the membrane surface, thus preventing full insertion of the myristoyl chain (25). 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.…”

The Lipid Modifications of Ras that Sense Membrane Environments and Induce Local Enrichment

“…[4] Although inserted into the membrane, the lipid modifications experience a high degree of motional freedom that is also transmitted to the adjacent polypeptide chain. [5,6] Although the highly homologous Ras proteins interact with the same effectors in vitro, they produce distinctly different output signals in vivo, which suggests that these differences are imparted by the lipid-modified C termini of the proteins, where the homology is very low. [7] Moreover, depending on the nucleotide binding state, the localization of Ras in liquid-crystalline or raft domains [8] of the membrane appears to be regulated.…”