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 (GPCRs) 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 (2)H NMR constitutes a powerful approach 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 (2)H labels have been introduced into the methyl groups of retinal and solid-state (2)H 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 cryotrapped metarhodopsin I and metarhodopsin II states. Analysis of the angular-dependent (2)H NMR line shapes 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 beta-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 rearrangements 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 beta4 strand of the E2 loop and the side chains of Glu(122) and Trp(265) within the binding pocket. The solid-state (2)H NMR data are discussed with regard to the pathway of the energy flow in the receptor activation mechanism.

Figure 1

Structure and dynamics of retinal ligand of rhodopsin are probed by solid-state ²H NMR spectroscopy in a native-like membrane environment. Geometry of the NMR experiment is depicted for aligned bilayers. (a) 11-cis-retinylidene chromophore of rhodopsin in the dark state. Angle of C–C²H³ bond axis to the local membrane normal n is designated as θ_B, with static rotational symmetry described by azimuthal angle ϕ. Alignment disorder is characterized by angle θ′ of n relative to the average membrane normal n₀, and is uniaxially distributed as given by ϕ′. The tilt angle of n₀ to the main magnetic field B₀ is denoted by θ about which there is also cylindrical symmetry. Finally, θ″ and ϕ″ are the angles for overall transformation from n to B₀. (b) Membrane-bound rhodopsin including the N-retinylidene cofactor within its binding cavity. The van der Waals surface of rhodopsin is depicted in light grey, with the seven transmembrane helices indicated by rods. Note that the extracellular side is at top and the cytoplasmic side at bottom. (c) Schematic view of stack of aligned membranes containing rhodopsin within the radiofrequency coil of the NMR spectrometer, showing geometry relative to the B₀ magnetic field. Adapted with permission from Ref. [24].

Figure 2

Solid-state ²H NMR allows determination of structure and orientation of 11-cis-retinal in the dark state of rhodopsin. The retinal conformation is described by three planes of unsaturation (designated A, B, C). (a) ²H NMR spectra for 11-cis-retinal in the rhodopsin dark state depend on the methyl bond orientation and mosaic spread of aligned membranes. Representative ²H NMR spectra are shown for 11-Z-[5–C²H₃]-retinylidene rhodopsin (blue), 11-Z-[9–C²H₃]-retinylidene rhodopsin (magenta), and 11-Z-[13–C²H₃]-retinylidene rhodopsin (green), respectively. Results are included for rhodopsin/POPC bilayers (1:50) at θ = 0° orientation of membrane normal to the static magnetic field B₀ at pH = 7 and T = −150 °C. (b) Simple three-plane model having dihedral twisting only about C6–C7 and C12–C13 bonds. (c) Extended three-plane model with additional pre-twisting about C11=C12 double bond. Note that the extracellular side of rhodopsin is up and the cytoplasmic side is down (cf. text). Adapted with permission from Ref. [24].

Figure 3

Retinal structure in metarhodopsin I is established by ²H NMR spectroscopy. Three planes of unsaturation (A, B, C) are assumed. (a) Simulations of solid-state ²H NMR spectra provide orientations of the methyl groups relative to membrane normal. Spectra are indicated for θ = 0° orientation of the average membrane normal to the static magnetic field B₀. (b) Orientations of methyl groups and electronic transition dipole moment define orientations of the molecular fragments; two possible solutions for the planes are indicated in each case. (c) Different ²H NMR structures for retinylidene ligand are eliminated using rotational-resonance ¹³C NMR carbon-carbon distances [71, 73] and molecular simulations (cf. text). Figure reprinted with permission from Ref. [53].

Figure 4

Analysis of solid-state ²H NMR data gives structure of retinal cofactor in the dark and meta I states of rhodopsin. Membrane normal corresponds to vertical direction; note that the extracellular side is up and cytoplasmic side is down. (a) NMR structure (green) of 11-cis-retinal in the dark state compared to retinal structure (red) from X-ray crystallography (PDB accession code 1U19) [4]. Inset: NMR structure calculated with polyene dihedral twisting about C12–C13 bond only (simple three-plane model; red) compared to structure with dihedral twisting about both the C11=C12 and C12–C13 bonds (extended three-plane model; green). (b) NMR structure for 11-cis-retinal in the rhodopsin dark state (green) versus NMR structure of trans-retinal in the meta I state (red). Figure produced with PyMOL [174]. Adapted with permission from Ref. [24].

Figure 5

Solid-state ²H NMR relaxation of retinal cofactor within rhodopsin binding pocket manifests steric hindrance of spinning methyl groups. Rotational dynamics of methyl groups of retinal either as axial 3-fold jumps (rate constant k) or alternatively diffusion within a potential of mean torque (diffusion coefficients D_∥ and D_⊥ for axial and off-axial rotations). Note that the correlation times for 3-fold jumps and continuous axial diffusion have the same activation energy; only the pre-exponential factors differ. Retinal geometry is characterized by Euler angles Ω_PM for transformation of principal axis system (PAS) of the C–²H bond (electric field gradient) to the methyl rotor axis (M); Ω_MD for rotation of methyl axis to membrane normal n₀ (director, D); Ω_ML for rotation of methyl axis to laboratory frame (L); and lastly Ω_DL for rotation of the membrane normal to the laboratory frame defined by the external magnetic field B₀.

Figure 6

Analysis of NMR relaxation data reveals site-specific dynamics of retinal underlying the rhodopsin activation mechanism. (a)–(c) Summary of results for the dark, meta I, and meta II states respectively. Methyl rotation is treated as axial 3-fold jumps (with rate constant k) or alternatively continuous diffusion (with coefficients D_∥ and D_⊥). The pre-exponential factor is either k₀ for 3-fold axial jumps or D₀ for continuous diffusion; and E_a is the barrier height (activation energy). For the diffusion model results are included for η_D≡D_∥/D_⊥=1 (in the front) and D_⊥= 0 (in the back) except for the C5-methyl of the β-ionone ring in meta I, where both axial (∥) and off-axial (⊥) motions are included with η_D≠1, or alternatively a two-conformer model with both positive (+) and negative (−) C5=C6–C7=C8 dihedral angles and D_⊥= 0.

Figure 7

Mechanism of rhodopsin activation involves conformational changes in the binding pocket and cytoplasmic side of the protein following chromophore photoisomerization. (a) Concerted rearrangement of the extracellular E2 loop and helices H5, H6, and H3. (b) Due to geometry of the binding pocket and the ligand pretwist around the C11=C12 bond in the dark state, retinal isomerization occurs in the direction indicated. The C9-methyl group prevents rotation of the chromophore about its long axis; hence the C13-methyl group rotates upwards towards the β4 strand of the extracellular loop E2. The C13-methyl pushes E2 and the retinal polyene chain away from each other. Concomitantly the retinal β-ionone ring moves towards Glu¹²² and Met²⁰⁷ due to the retinal straightening, together with the steric hindrance between the polyene chain and Trp²⁶⁵. Disruption of the hydrogen-bonding networks around Glu¹²² and the E2 loop as well as the ionic lock between the protonated Schiff base and counterions Glu¹⁸¹ and/or Glu¹¹³ (see text) destabilizes the inactive rhodopsin conformation. (c) Rearrangements of helices H5 and H6 in the cytoplasmic region [39] renders a second ionic lock involving Glu¹³⁴, Arg¹³⁵, and Glu²⁴⁷ in the inactive rhodopsin state [26] less favorable than interactions between Tyr²²³, Arg¹³⁵, and Met²⁵⁷. The later are assumed to be maintained in the activated meta II conformation [26, 40]. The activation mechanism is highly cooperative suggesting that large-scale conformational fluctuations of the protein at lower frequencies are involved.