Solid-state ² H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin

Abstract

Rhodopsin is a canonical member of the family of G proteincoupled receptors, which transmit signals across cellular membranes and are linked to many drug interventions in humans. Here we show that solid-state 2 H NMR relaxation allows investigation of light-induced changes in local ps-ns time scale motions of retinal bound to rhodopsin. Site-specific 2 H labels were introduced into methyl groups of the retinal ligand that are essential to the activation process. We conducted solid-state 2 H NMR relaxation (spinlattice, T 1Z , and quadrupolar-order, T 1Q ) experiments in the dark, Meta I, and Meta II states of the photoreceptor. Surprisingly, we find the retinylidene methyl groups exhibit site-specific differences in dynamics that change upon light excitation-even more striking, the C9-methyl group is a dynamical hotspot that corresponds to a crucial functional hotspot of rhodopsin. Following 11-cis to trans isomerization, the 2 H NMR data suggest the β-ionone ring remains in its hydrophobic binding pocket in all three states of the protein.We propose a multiscale activation mechanism with a complex energy landscape, whereby the photonic energy is directed against the E2 loop by the C13-methyl group, and toward helices H3 and H5 by the C5-methyl of the β-ionone ring. Changes in retinal structure and dynamics initiate activating fluctuations of transmembrane helices H5 and H6 in the Meta I-Meta II equilibrium of rhodopsin. Our proposals challenge the Standard Model whereby a single light-activated receptor conformation yields the visual response -rather an ensemble of substates is present, due to the entropy gain produced by photolysis of the inhibitory retinal lock.GPCR | solid-state NMR | generalized model-free analysis G protein-coupled receptors (GPCRs) (1-3) are the largest family of integral membrane proteins in the human genome, and they are the targets of about 30% of all human pharmaceuticals. At present the 3D structures of several GPCRs-including rhodopsin (4-8), the β 1 -and β 2 -adrenergic receptors (9, 10), and the adenosine A 2A receptor (11)-have been established in various functional states (2). Yet the mechanisms of GPCR activation remain elusive, as they are membrane-embedded molecules that are often recalcitrant to crystallization, and push the size limits of solution NMR spectroscopy. Rhodopsin is the quintessential prototype for Family A GPCRs, because previous X-ray (5,6) and electron diffraction (12) studies have yielded crystal structures of its dark state (4-6) and several photointermediates (13,14). Notably, the retinal ligand is buried deeply within a 7-transmembrane (TM) helical bundle, where it locks the receptor in the inactive state, with negligible basal (constitutive) activity (4). Upon light absorption, rhodopsin catalyzes the rapid and highly selective 11-cis to trans isomerization of retinal (15, 16) switching it from an inverse agonist to an agonist. In the Metarhodopsin I-Metarhodopsin II equilibrium, recognition sites are exposed for a heterotrimeric G protein (transducin or...

Chemistry and Biology of Vision

“…An enhanced insight into the dynamics of rhodopsin activation and interaction with ligand and G protein has been obtained more recently by NMR techniques that show confor- mational flexibility of this receptor and the G protein upon activation (45)(46)(47)(48). Several methods demonstrated that membrane proteins (49), including rhodopsin (50), contain integral ordered water molecules that play important roles in both structure and function.…”

Retinal Conformation Changes Rhodopsin’s Dynamic Ensemble

“…Previous efforts by several labs have shown that retinal makes state-specific protein-ligand interactions (68)(69)(70)(71)(72). We monitored retinal using three regions of the ligand equivalent to those identified by Brown and coworkers, the orientations of the C5-, C9-, and C13-methyl groups (46,47,(73)(74)(75)(76). In this analysis, the vectors denoted by the three arrows in Fig.…”

Membrane Protein Structure and Dynamics from NMR Spectroscopy

“…Transitioning to the Meta I state changes the C6-C7 torsion angle to 32° or 57° and makes planes B and C roughly parallel (94). Analysis of 2 H T 1Z and T 1Q relaxation times of the methyl groups as a function of temperature yielded the activation energies (E a ) and order parameters of methyl rotations for the dark, Meta I, and Meta II states (95, 96). The methyl group attached to C5 of the β-ionone ring has the highest E a , which is insensitive to photoactivation (95), consistent with confinement of the ring in the hydrophobic pocket of the protein.…”

“…Analysis of 2 H T 1Z and T 1Q relaxation times of the methyl groups as a function of temperature yielded the activation energies (E a ) and order parameters of methyl rotations for the dark, Meta I, and Meta II states (95, 96). The methyl group attached to C5 of the β-ionone ring has the highest E a , which is insensitive to photoactivation (95), consistent with confinement of the ring in the hydrophobic pocket of the protein. The methyl groups associated with C9 and C13 have energy barriers that change with photoactivation: Retinal isomerization increases the C9 E a but decreases the C13 E a .…”

“…The methyl groups associated with C9 and C13 have energy barriers that change with photoactivation: Retinal isomerization increases the C9 E a but decreases the C13 E a . These changes were attributed to the rearrangement of the TM helices and the polyene chain protons near the methyl groups (95). Movement of the β-ionone ring was proposed to affect the H3-H5 interface and hydrogen-bonding network, whereas the C13 methyl rotation was proposed to impact EL2 and the H4-H6 hydrogen-bonding network (96).…”