Dynamic structure of retinylidene ligand of rhodopsin probed by molecular simulations

Abstract

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 beta-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 beta-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 beta-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.

Figure 1

Structure of dark-state rhodopsin and retinal in its binding cavity is illuminated by X-ray crystallography, resonance Raman, and solid-state ²H NMR spectroscopy. (a) Structural model of rhodopsin (PDB code 1U19) showing retinal (green) within its binding pocket. (b) Extended view of the retinal binding pocket showing the C6–C7 bond (yellow) and the key C5-, C9-, and C13-methyl groups. (c) HOOP modes for H11 and H12 are identified at 910 cm⁻¹ and are independent of both negative (cyan) and positive (green) 6-s-cis conformers. (d) Comparison of experimental solid-state ²H NMR spectra to theoretical spectra for C5-, C9-, and C13-methyl groups with membrane tilt angles of 0, 45, and 90° and bond orientation distributions from MD simulations.

Figure 2

Multiple conformations of β-ionone ring of the retinylidene inverse agonist are shown by molecular simulations. (a) Distribution of C6–C7 torsion angle in all 23 simulations of 100-ns duration. The two most prevalent conformations are shown, representing negatively- and positively-twisted 6-s-cis conformations of the β-ionone ring. (b) Plot of C8-to-C18 internuclear distance as a function of C6–C7 torsion angle showing the symmetry with respect to 0°. (c) Three representative C6–C7 torsion angle distributions from the 23 individual 100-ns trajectories comprising the distribution in part (a).

Figure 3

Polyene chain of retinylidene ligand is locked in a twisted conformation in the inverse agonist form. (a)–(h) Torsion angle distributions along the conjugated polyene chain from C7 to C15. Twisting of the polyene chain from planarity (torsion angle χ = 180°) is mainly confined to the C10–C11 and C12–C13 torsions to either side of the C11=C12 double bond.

Figure 4

Retinal ligand of rhodopsin has conformations of β-ionone ring whose ²H NMR spectra differ from experiment. (a) Predicted ²H NMR spectra considering only distribution of C6–C7 torsion angles < −70° (cyan) and (b) torsion angles > −70° (purple). Synthetic spectra in each case are superimposed on experimental ²H NMR data for membrane tilt angles of 0, 45, and 90°. (c) Probability distribution for all 23 simulations (cf. Fig. 2) showing range of C6–C7 torsional angles used to calculate the synthetic spectra.

Figure 5

Covariance analysis of individual 100-ns simulations shows variability of protein environment of retinylidene C5-, C9-, and C13-methyl groups. Elements of the covariance matrix are depicted by colors and range from −1 (completely anti-correlated; blue) to 1 (completely correlated; red). Panels (a)–(c) correspond to C5-, C9- and C13-methyl positions, respectively. Note that the matrix in panel (a) has three different blocks: (i) simulations 1– 11, (ii) simulations 12– 15, and (iii) simulations 16–23. Each of the blocks has a distinct C5-methyl environment. By contrast, in (b) and (c) the color map for the C9-, and C13-methyls has a consistent green color, indicating all 23 simulations yield similar methyl group environments. Panels (d)–(f) show contact matrices for the C5-, C9-, and C13-methyl groups, respectively. Columns list the simulation number and rows correspond to residues within a 4 Å-radius sphere centered on the methyl carbon. The color scale ranges from blue (low fraction value; indicating residue is infrequent within 4 Å-radius sphere) to red (high fraction showing sampling of different environments. For the C9- and C13-methyls in (b) and (c), bands of similar colors across all 23 simulations evince similar environments.

Figure 6

Conformation of the β-ionone ring is correlated with its distance to Trp²⁶⁵ side chain. (a) Distance between center of the β-ionone ring and the indole 6-carbon ring of Trp²⁶⁵ side chain used in the cross-correlation calculation is shown by the white line. (b) Average cross-correlation profile of the 19 simulations containing the 6-s-trans state shows a non-zero correlation value above the noise level at τ = −1 ns. Thus a correlation between the C6–C7 torsion angle and the distance between the β-ionone ring and Trp²⁶⁵ is revealed.

Figure 7

Dynamical structure of the retinal inverse agonist in its binding pocket is obtained for dark-state rhodopsin. Both positive (red) and negative (blue) 6-s-cis conformers are placed into the binding pocket of rhodopsin. The three planes used to describe the structure of retinal are designated by A, B, and C (cf. text).