Retinal dynamics during light activation of rhodopsin revealed by solid-state NMR spectroscopy

Abstract

Rhodopsin is a canonical member of class A of the G protein-coupled receptors (GPCR) that are implicated in many of the drug interventions in humans and are of great pharmaceutical interest. The molecular mechanism of rhodopsin activation remains unknown as atomistic structural information for the active metarhodopsin II state is currently lacking. Solid-state 2H NMR constitutes a powerful aproarch to study atomic-level dynamics of membrane proteins. In the present application we describe how information is obtained about interactions of the retinal cofactor with rhodopsin that change with light activation of the photoreceptor. The retinal methyl groups play an important role in rhodopsin function by directing conformational changes upon transition into the active state. Site-specific 2H labels have been introduced into the methyl groups of retinal and solid-state 2H NMR methods applied to obtain order parameters and correlation times that quantify the mobility of the cofactor in the inactive dark state, as well as the cryo-trapped metarhodopsin I and metarhodopsin II states. Analysis of the angular-dependent 2H NMR lineshapes for selectively deuterated methyl groups of rhodopsin in aligned membranes enables determination of the average ligand conformation within the binding pocket. The relaxation data suggest that the β-ionone ring is not expelled from its hydrophobic pocket in the transition from the pre-activated metarhodopsin I to the active metarhodopsin II state. Rather, the major structural changes of the retinal cofactor occur already at the metarhodopsin I state in the activation process. The metarhodopsin I to metarhodopsin II transition involves mainly conformational changes of the protein within the membrane lipid bilayer rather than the ligand. The dynamics of the retinylidene methyl groups upon isomerization are explained by an activation mechanism involving cooperative rearrangments of extracellular loop E2 together with transmembrane helices H5 and H6. These activating movements are triggered by steric clashes of the isomerized all-trans retinal with the β4 strand of the E2 loop and the side chains of Glu122 and Trp265 within the binding pocket. The solid-state 2H NMR data are discussed with regard to the pathway of the energy flow in the receptor activation mechanism.

Conformational Selection and Equilibrium Governs the Ability of Retinals to Bind Opsin

“…Additionally, we recapitulate the extended ternary complex model originally established to explain ligand binding to CAM ␤ 2 -adrenergic receptor for rhodopsin, greatly implying that the dim light receptor behaves like a ligand-binding GPCR rather than being a special case (68). Interestingly, activation of rhodopsin has been proposed to be an extreme example of an induced fit mechanism as the inverse agonist isomerizes to the agonist while inside the binding pocket and appears to actively push the inactive conformation to active MII (69). Taken with the data presented here, it appears that rhodopsin compartmentally exhibits both classical models of ligand-receptor interactions.…”

Combined Use of Optical Spectroscopy and Computational Methods to Study the Binding and the Photoinduced Conformational Modification of Proteins When NMR and X-Ray Structural Determinations Are Not an Option

“…Such structural change can prompt the opening of the transmembrane channel, thus triggering a depolarization of the cellular membrane and in turn stimulate the optical nerve, or it promotes the binding to G-proteins that trigger a signaling cascade that depolarizes the neuronal membrane and sends the stimulus to the optical nerve. Once again, a key step in the conversion of photon energy into a biological response is caused by a conformational change of a protein as a response to the photoinduced event (Brown, Salgado, & Struts, 2010).…”

24S-hydroxycholesterol and cholesterol-24S-hydroxylase (CYP46A1) in the retina: from cholesterol homeostasis to pathophysiology of glaucoma

“…Absorption of photon by rhodopsin yields conformational movements of rhodopsin that result in activation of the G-protein and biological response. The lipid environment of rhodopsin is a key effector of these changes (Brown et al, 2010). Docosahexaenoic acid (DHA) is a long chain polyunsaturated fatty acid from the omega 3 series.…”