Retinal ligand mobility explains internal hydration and reconciles active rhodopsin structures

Abstract

Rhodopsin, the mammalian dim-light receptor, is one of the best-characterized G-protein-coupled receptors, a pharmaceutically important class of membrane proteins that has garnered a great deal of attention because of the recent availability of structural information. Yet the mechanism of rhodopsin activation is not fully understood. Here, we use microsecond-scale all-atom molecular dynamics simulations, validated by solid-state (2)H nuclear magnetic resonance spectroscopy, to understand the transition between the dark and metarhodopsin I (Meta I) states. Our analysis of these simulations reveals striking differences in ligand flexibility between the two states. Retinal is much more dynamic in Meta I, adopting an elongated conformation similar to that seen in the recent activelike crystal structures. Surprisingly, this elongation corresponds to both a dramatic influx of bulk water into the hydrophobic core of the protein and a concerted transition in the highly conserved Trp265(6.48) residue. In addition, enhanced ligand flexibility upon light activation provides an explanation for the different retinal orientations observed in X-ray crystal structures of active rhodopsin.

Figure 1

Comparison of X-ray crystal structures of rhodopsin in the inactive and active states. Coordinates were obtained from the Protein Data Bank (entries 1U19, 2X72, and 3PXO). (a) Overall structure of rhodopsin (rainbow cartoon representation) with retinal in the binding pocket (black sticks). The figure is oriented with the extracellular side (intradiscal) at the top. The protein conformation is similar in both active structures. (b) Close-up of the binding pocket that reveals a ligand more elongated in the active structures (purple and tan) than in the inactive dark structure (cyan). (c) View after a 90° rotation in the plane of the membrane that shows the two retinal orientations in the active structures. Note that retinal differs by a long-axis flip of 180° between these two structures.

Figure 2

Solid-state ²H NMR spectra calculated from molecular dynamics (MD) simulations corroborate complex-counterion mechanism of rhodopsin activation. (a) Schematic of retinal (gray) in its binding pocket. Carbon atoms of retinal are colored as follows: (red) C5-methyl, (green) C9-methyl, and (purple) C13-methyl. This coloring is used in panels b–g for the simulation-based line shape calculations. (b–g) Solid-state ²H NMR spectra computed from MD simulations (color) plotted with experimental data (black). Each row of data in a panel indicates a different orientation of the bilayer to the magnetic field: 0° (first row), 45° (second row), and 90° (third row). The full series of tilt angles can be seen in Figures S2–S7 of the Supporting Information. Spectra were calculated from the dark-state (b and c), complex-counterion (d and e), and counterion-switch (f and g) simulations. These were compared to the dark-state (b, d, and f) and Meta I (c, e, and g) experimental ²H ssNMR spectra.

Figure 3

Retinal makes a concerted transition and is elongated early in rhodopsin’s activation. Results are shown from (left) the dark-state and (right) complex-counterion simulations. (a) Close-up of the ligand binding pocket that shows retinal, Lys296^7.43, and Trp265^6.48 motion in the dark-state simulation. Atomic coordinates were taken over the course of the trajectory at equal time intervals progressing from blue to white to red (time bar shown below). The position of the ligand can be seen with respect to rhodopsin transmembrane helices (the extracellular portions of H6 and H7 have been removed for the sake of clarity). Rhodopsin is colored as in Figure 1. (b) Expanded view of the retinal binding pocket for the complex-counterion trajectory. As in panel a, retinal, Lys296^7.43, and Trp265^6.48 are plotted as the simulation progressed. (c) 11-cis-retinal’s (cyan) most concerted motion is shown for the dark-state simulation. The principal component of motion is shown as yellow sticks superimposed on the average structure of retinal. (d) All-trans-retinal’s (purple) principal component of motion (yellow sticks) is shown for the complex-counterion simulation. (e) Displacement of 11-cis-retinal and Lys296^7.43 along the first principal component for the dark-state simulation. This displacement is calculated using the average structure. In the top panel, the displacement along the principal component of motion in panel c is illustrated as a function of simulation time. The coloring matches the progression of time shown in panel a. The middle panel shows the time course of displacement where the principal component analysis has been expanded to include the entire binding pocket (see Results). The bottom panel shows the time series of the χ₁ torsion of Trp265^6.48. (f) Displacement of all-trans-retinal and Lys296^7.43 along the first principal component of motion during the complex-counterion simulation. The top panel shows the displacement along the principal component (as shown in panel d). The middle panel shows the displacement along the principal component where the PCA is expanded to include the entire binding pocket (see Results). The bottom panel shows the time series of the χ₁ torsion of Trp265^6.48.

Figure 4

Complex-counterion simulation that shows the influx of bulk water into the hydrophobic core of the protein upon activation. (a) The number of water molecules within 7 Å of transmembrane residues was plotted for both the dark-state (cyan) and complex-counterion (purple) simulations. (b and c) Water density in the complex-counterion simulation averaged (b) from 100 to 250 ns and (c) from 450 to 1470 ns.

Figure 5

Retinal mobility is increased in the complex-counterion simulation. (a) Illustration of retinal and its Schiff base-linked lysine (Lys296^7.43) (carbons colored yellow and nitrogens blue). The β-ionone ring is located at the bottom of the image. The breakouts show each torsion angle plotted as a function of trajectory time for both the dark-state (cyan) and complex-counterion (purple) simulations. Each torsion is labeled by the two atoms involved, with a single dash (—) representing a single bond and a double dash (=) representing a double bond. (b) Retinal elongation is shown for both the dark-state and complex-counterion simulations. The distance between the Lys296^7.43 α-carbon and C3 in retinal’s β-ionone ring is plotted for both simulations.