Retinal dynamics underlie its switch from inverse agonist to agonist during rhodopsin activation

Abstract

X-ray and magnetic resonance approaches, though central to studies of G protein-coupled receptor (GPCR)-mediated signaling, cannot address GPCR protein dynamics or plasticity. Here we show that solid-state (2)H NMR relaxation elucidates picosecond-to-nanosecond-timescale motions of the retinal ligand that influence larger-scale functional dynamics of rhodopsin in membranes. We propose a multiscale activation mechanism whereby retinal initiates collective helix fluctuations in the meta I-meta II equilibrium on the microsecond-to-millisecond timescale.

Figure 1

Site-specific ²H NMR relaxation illuminates functional dynamics of retinylidene methyl groups within binding pocket of rhodopsin. (a) Light absorption yields 11-cis to trans isomerization, converting retinal from an inverse agonist to an agonist by a series of intermediates with different time scales. (b–c) Solid-state ²H NMR spectra for dark-state rhodopsin with 11-cis retinal deuterated at C5-, C9-, or C13–C²H₃ groups in POPC bilayers (1:50 molar ratio). The ²H NMR lineshapes indicate rapid axial spinning of C–C²H₃ groups down to at least −160 °C. (e,f) Partially relaxed ²H NMR spectra for retinylidene C9- and C13–C²H₃ groups of rhodopsin in aligned POPC membranes (θ=0°) at −150 °C. (g) Inversion-recovery plots showing site-specific variations in spin-lattice (T_1Z) relaxation times for C9- and C13-Me groups at −150 °C.

Figure 2

Solid-state ²H NMR of captures site-specific changes in retinal mobility during light activation of rhodopsin. Spin-lattice (T_1Z) relaxation times (±s.d.) of retinylidene methyl groups are shown versus reciprocal temperature in (a) the dark, (b) Meta I, and (c) Meta II states (−30 to −160 °C). Methyl dynamics are described by an axial 3-fold jump model or a continuous diffusion model with coefficients D_‖ and D_⊥. In panels a–c rotation about the methyl 3-fold (C₃) axis corresponds to solid lines with D_⊥=0; the dashed lines include restricted off-axial diffusion (D_⊥=D_‖). Fits for the C5-Me in Meta I in panel b assume unlike rotational diffusion constants (D_‖≠D_⊥) (dashed line), or two conformers with different bond orientations and axial diffusion coefficients (solid line).

Figure 3

²H NMR relaxation of retinal sheds new light on activation mechanism of rhodopsin. (a) Summary of analysis of solid-state ²H NMR measurements. Order parameters of rapidly spinning methyl groups are designated by $S_{{\mathrm{C}}_3}$; the pre-exponential factor is k₀ for 3-fold axial jumps or D₀ for continuous diffusion; and E_a indicates the activation energy. (The diffusion model assumes either D_⊥=0 (right) or η_D ≡ D_‖/D_⊥=1 (left) except for the C5-Me in Meta I, where η_D≠1.) (b–d) Proposed activation mechanism for rhodopsin in membranes based on X-ray, FTIR, and ²H NMR data (see text). Isomerization of retinal displaces the E2 loop towards the extracellular (e) side with fluctuations of TM helices H5 and H6 exposing transducin (G_t) recognition sites on the opposing cytoplasmic (c) surface. Figure produced (PDB code 1U19) using PyMOL [http://pymol.sourceforge.net/]