Dynamic Structure of Retinylidene Ligand of Rhodopsin Probed by Molecular Simulations (original) (raw)
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Dynamic Structure of Retinyl Probed by Molecular Simulat
2015
Rhodopsin is currently the only available atomic-resolution template for understanding biological functions of the G protein-coupled receptor (GPCR) family. The structural basis for the phenomenal dark state stability of 11-cis-retinal bound to rhodopsin and its ultrafast photoreaction are active topics of research. In particular, the β-ionone ring of the retinylidene inverse agonist is crucial for the activation mechanism. We analyzed a total of 23 independent, 100 ns all-atom molecular dynamics simulations of rhodopsin embedded in a lipid bilayer in the microcanonical (N,V,E) ensemble. Analysis of intramolecular fluctuations predicts hydrogen-out-of-plane (HOOP) wagging modes of retinal consistent with those found in Raman vibrational spectroscopy. We show that sampling and ergodicity of the ensemble of simulations are crucial for determining the distribution of conformers of retinal bound to rhodopsin. The polyene chain is rigidly locked into a single, twisted conformation, consistent with the function of retinal as an inverse agonist in the dark state. Most surprisingly, the β-ionone ring is mobile within its binding pocket; interactions are non-specific and the cavity is sufficiently large to enable structural heterogeneity. We find that retinal occupies two distinct conformations in the dark state, contrary to most previous assumptions. The β-ionone ring can rotate relative to the polyene chain, thereby populating both positively and negatively twisted 6-s-cis enantiomers. This result, while unexpected, strongly agrees with experimental solid-state 2 H NMR spectra. Correlation analysis identifies the residues most critical to controlling mobility of retinal; we find that Trp265 moves away from the ionone ring prior to any conformational transition. Our findings reinforce how molecular dynamics simulations can challenge conventional assumptions for interpreting experimental data, especially where existing models neglect conformational fluctuations.
Photochemistry and Photobiology, 2009
Solid-state NMR spectroscopy gives a powerful avenue for investigating G protein-coupled receptors and other integral membrane proteins in a native-like environment. This article reviews the use of solid-state 2 H NMR to study the retinal cofactor of rhodopsin in the dark state as well as the meta I and meta II photointermediates. Site-specific 2 H NMR labels have been introduced into three regions (methyl groups) of retinal that are crucially important for the photochemical function of rhodopsin. Despite its phenomenal stability 2 H NMR spectroscopy indicates retinal undergoes rapid fluctuations within the protein binding cavity. The spectral lineshapes reveal the methyl groups spin rapidly about their three-fold (C 3 ) axes with an order parameter for the off-axial motion of S C3 % 0:9: For the dark state, the 2 H NMR structure of 11-cis-retinal manifests torsional twisting of both the polyene chain and the b-ionone ring due to steric interactions of the ligand and the protein. Retinal is accommodated within the rhodopsin binding pocket with a negative pretwist about the C11=C12 double bond. Conformational distortion explains its rapid photochemistry and reveals the trajectory of the 11-cis to trans isomerization. In addition, 2 H NMR has been applied to study the retinylidene dynamics in the dark and light-activated states. Upon isomerization there are drastic changes in the mobility of all three methyl groups. The relaxation data support an activation mechanism whereby the b-ionone ring of retinal stays in nearly the same environment, without a large displacement of the ligand. Interactions of the b-ionone ring and the retinylidene Schiff base with the protein transmit the force of the retinal isomerization. Solid-state 2 H NMR thus provides information about the flow of energy that triggers changes in hydrogen-bonding networks and helix movements in the activation mechanism of the photoreceptor. †This paper is part of the
Biophysical Journal, 2008
As one of the best studied members of the pharmaceutically relevant family of G-protein-coupled receptors, rhodopsin serves as a prototype for understanding the mechanism of G-protein-coupled receptor activation. Here, we aim at exploring functionally relevant conformational changes and signal transmission mechanisms involved in its photoactivation brought about through a cis-trans photoisomerization of retinal. For this exploration, we propose a molecular dynamics simulation protocol that utilizes normal modes derived from the anisotropic network model for proteins. Deformations along multiple lowfrequency modes of motion are used to efficiently sample collective conformational changes in the presence of explicit membrane and water environment, consistent with interresidue interactions. We identify two highly stable regions in rhodopsin, one clustered near the chromophore, the other near the cytoplasmic ends of transmembrane helices H1, H2, and H7. Due to redistribution of interactions in the neighborhood of retinal upon stabilization of the trans form, local structural rearrangements in the adjoining H3-H6 residues are efficiently propagated to the cytoplasmic end of these particular helices. In the structures obtained by our simulations, all-trans retinal interacts with Cys 167 on H4 and Phe 203 on H5, which were not accessible in the dark state, and exhibits stronger interactions with H5, while some of the contacts made (in the cis form) with H6 are lost.
Journal of Molecular Biology, 2007
Rhodopsin is a prototype for G protein-coupled receptors (GPCRs) that are implicated in many biological responses in humans. A site-directed 2 H NMR approach was used for structural analysis of retinal within its binding cavity in the dark and pre-activated meta I states. Retinal was labeled with 2 H at the C5, C9, or C13 methyl groups by total synthesis, and was used to regenerate the opsin apoprotein. Solid-state 2 H NMR spectra were acquired for aligned membranes in the low-temperature lipid gel phase versus the tilt angle to the magnetic field. Data reduction assumed a static uniaxial distribution, and gave the retinylidene methyl bond orientations plus the alignment disorder (mosaic spread). The darkstate 2 H NMR structure of 11-cis-retinal shows torsional twisting of the polyene chain and the β-ionone ring. The ligand undergoes restricted motion, as evinced by order parameters of ≈ 0.9 for the spinning C-C 2 H 3 groups, with off-axial fluctuations of ≈ 15°. Retinal is accommodated within the rhodopsin binding pocket with a negative pre-twist about the C11 = C12 double bond that explains its rapid photochemistry and the trajectory of 11-cis to trans isomerization. In the cryo-trapped meta I state, the 2 H NMR structure shows a reduction of the polyene strain, while torsional twisting of the β-ionone ring is maintained. Distortion of the retinal conformation is interpreted through substituent control of receptor activation. Steric hindrance between trans retinal and Trp265 can trigger formation of the subsequent activated meta II state. Our results are pertinent to quantum and molecular mechanics simulations of ligands bound to GPCRs, and illustrate how 2 H NMR can be applied to study their biological mechanisms of action.
Retinal dynamics during light activation of rhodopsin revealed by solid-state NMR spectroscopy
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2010
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 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 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 β4 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.
Retinal dynamics underlie its switch from inverse agonist to agonist during rhodopsin activation
Nature Structural & Molecular Biology, 2011
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.
Proceedings of the National Academy of Sciences of the United States of America, 2011
Rhodopsin is a canonical member of the family of G protein-coupled 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 (spin-lattice, 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 ...
Deuterium NMR Structure of Retinal in the Ground State of Rhodopsin †
Biochemistry, 2004
The conformation of retinal bound to the G protein-coupled receptor rhodopsin is intimately linked to its photochemistry, which initiates the visual process. Site-directed deuterium ( 2 H) NMR spectroscopy was used to investigate the structure of retinal within the binding pocket of bovine rhodopsin. Aligned recombinant membranes were studied containing rhodopsin that was regenerated with retinal 2 H-labeled at the C 5 , C 9 , or C 13 methyl groups by total synthesis. Studies were conducted at temperatures below the gel to liquid-crystalline phase transition of the membrane lipid bilayer, where rotational and translational diffusion of rhodopsin is effectively quenched. The experimental tilt series of 2 H NMR spectra were fit to a theoretical line shape analysis [Nevzorov, A. A., Moltke, S., Heyn, M. P., and Brown, M. F. (1999) J. Am. Chem. Soc. 121,[7636][7637][7638][7639][7640][7641][7642][7643] giving the retinylidene bond orientations with respect to the membrane normal in the dark state. Moreover, the relative orientations of pairs of methyl groups were used to calculate effective torsional angles between different planes of unsaturation of the retinal chromophore. Our results are consistent with significant conformational distortion of retinal, and they have important implications for quantum mechanical calculations of its electronic spectral properties. In particular, we find that the -ionone ring has a twisted 6-s-cis conformation, whereas the polyene chain is twisted 12-s-trans. The conformational strain of retinal as revealed by solid-state 2 H NMR is significant for explaining the quantum yields and mechanism of its ultrafast photoisomerization in visual pigments. This work provides a consensus view of the retinal conformation in rhodopsin as seen by X-ray diffraction, solid-state NMR spectroscopy, and quantum chemical calculations. mean square deviation; MNDO, modified neglect of diatomic overlap; TD, transition dipole.
Biophysical Journal, 2021
Classical molecular dynamics (MD) and combined quantum mechanical/molecular mechanical (QM/MM) MD simulations have been performed to investigate the structural and dynamical properties of the Tl(III) ion in water. A six-coordinate hydration structure with a maximum probability of the Tl O distance at 2.21 Å was observed, which is in good agreement with X-ray data. The librational and vibrational spectra of water molecules in the first hydration shell are blue-shifted compared with those of pure liquid water, and the Tl O stretching force constant was evaluated as 148 N m −1. Both structural and dynamical properties show a distortion of the first solvation shell structure. The second shell ligands' mean residence time was determined as 12.8 ps. The Tl(III) ion can be classified as "structure forming" ion; the calculated hydration energy of −986 ± 9 kcal mol agrees well with the experimental value of −986 kcal mol.