Revealing conformational substates of lipidated N-Ras protein by pressure modulation

Abstract

Regulation of protein function is often linked to a conformational switch triggered by chemical or physical signals. To evaluate such conformational changes and to elucidate the underlying molecular mechanisms of subsequent protein function, experimental identification of conformational substates and characterization of conformational equilibria are mandatory. We apply pressure modulation in combination with FTIR spectroscopy to reveal equilibria between spectroscopically resolved substates of the lipidated signaling protein N-Ras. Pressure has the advantage that its thermodynamic conjugate is volume, a parameter that is directly related to structure. The conformational dynamics of N-Ras in its different nucleotide binding states in the absence and presence of a model biomembrane was probed by pressure perturbation. We show that not only nucleotide binding but also the presence of the membrane has a drastic effect on the conformational dynamics and selection of conformational substates of the protein, and a new substate appearing upon membrane binding could be uncovered. Population of this new substate is accompanied by structural reorientations of the G domain, as also indicated by complementary ATR-FTIR and IRRAS measurements. These findings thus illustrate that the membrane controls signaling conformations by acting as an effective interaction partner, which has consequences for the G-domain orientation of membrane-associated N-Ras, which in turn is known to be critical for its effector and modulator interactions. Finally, these results provide insights into the influence of pressure on Ras-controlled signaling events in organisms living under extreme environmental conditions as they are encountered in the deep sea where pressures reach the kbar range.

H-Ras forms dimers on membrane surfaces via a protein–protein interface

“…Membrane-induced conformational changes have been reported for both H-and N-Ras (15,17), and membrane-specific conformations of the HVR in full-length H-Ras have been predicted by MD simulations (18). Our analysis of membrane surface dimerization energetics indicates that membrane localization alone is insufficient to drive dimerization; a different protein configuration or significant rotational constraints are required.…”

“…In addition to biochemical evidence for communication between the C-terminal membrane binding region and the nucleotide binding pocket, NMR and IR spectroscopic observations suggest that the HVR and lipid anchor membrane insertion affects Ras structure and orientation (15)(16)(17). Molecular dynamics (MD) modeling of bilayer-induced H-Ras conformations has identified two nucleotide-dependent states, which differ in HVR conformation, membrane contacts, and G-domain orientation (18).…”

Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin

“…Despite their functional relevance, excited states are sparsely populated and may escape detection by standard spectroscopic techniques. Hydrostatic pressure apparently offers a solution to this problem by reversibly populating excited states of proteins, allowing for spectroscopic characterization (9)(10)(11)(12)(13)(14). Pressure application is particularly useful because it shifts the relative population of preexisting conformational states while minimally affecting the conformational landscape itself (15).…”

The Ras–Membrane Interface: Isoform-Specific Differences in the Catalytic Domain

“…Because active N-Ras resides in lipid rafts, helix 4 residues in this isoform will encounter a higher concentration of cholesterol and sphingomyelin. Activated N-RasGTP is stabilized mainly by helix 4 and 5 residues, whereas N-RasGDP predominantly resides in an orientation stabilized by the HVR (30,57). Additional basic residues in the linker region help prevent rapid reorientation of the monopalmitolyated isoform (30).…”