Retinal dynamics during light activation of rhodopsin revealed by solid-state NMR spectroscopy - PubMed (original) (raw)

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

Michael F Brown et al. Biochim Biophys Acta. 2010 Feb.

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.

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Figures

Figure 1

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

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

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

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

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

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 _k_0 for 3-fold axial jumps or _D_0 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

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.

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