Location of the Retinal Chromophore in the Activated State of Rhodopsin^*

Abstract

Rhodopsin is a highly specialized G protein-coupled receptor (GPCR) that is activated by the rapid photochemical isomerization of its covalently bound 11-cis-retinal chromophore. Using two-dimensional solid-state NMR spectroscopy, we defined the position of the retinal in the active metarhodopsin II intermediate. Distance constraints were obtained between amino acids in the retinal binding site and specific ¹³C-labeled sites located on the β-ionone ring, polyene chain, and Schiff base end of the retinal. We show that the retinal C20 methyl group rotates toward the second extracellular loop (EL2), which forms a cap on the retinal binding site in the inactive receptor. Despite the trajectory of the methyl group, we observed an increase in the C20-Gly¹⁸⁸ (EL2) distance consistent with an increase in separation between the retinal and EL2 upon activation. NMR distance constraints showed that the β-ionone ring moves to a position between Met²⁰⁷ and Phe²⁰⁸ on transmembrane helix H5. Movement of the ring toward H5 was also reflected in increased separation between the Cε carbons of Lys²⁹⁶ (H7) and Met⁴⁴ (H1) and between Gly¹²¹ (H3) and the retinal C18 methyl group. Helix-helix interactions involving the H3-H5 and H4-H5 interfaces were also found to change in the formation of metarhodopsin II reflecting increased retinal-protein interactions in the region of Glu¹²² (H3) and His²¹¹ (H5). We discuss the location of the retinal in metarhodopsin II and its interaction with sequence motifs, which are highly conserved across the pharmaceutically important class A GPCR family, with respect to the mechanism of receptor activation.

The first step in the activation mechanism of most G protein-coupled receptors (GPCRs)3 is the binding of a signaling ligand. Ligand binding to the extracellular loops or within the transmembrane helical bundle of these receptors leads to an allosteric conformational change that causes G protein activation. The detailed location of the activating ligand and the conformational changes triggered by ligand binding are still not well established for any GPCR. The visual photoreceptor rhodopsin is a member of the large family of class A GPCRs (1). These receptors have a common architecture consisting of seven-transmembrane α-helices. Activation of rhodopsin begins with the absorption of light by the photoreactive 11-cis-retinal chromophore. The retinal is covalently bound as a protonated Schiff base to Lys²⁹⁶ and tightly packed within the bundle of helices (Fig. 1, B and C). The retinal binding site is restricted along the axis of the retinal polyene chain by transmembrane helices H5 and H7 and is enclosed on the extracellular side of the dark, inactive state of rhodopsin by the second extracellular (or intradiscal) loop (EL2). The retinal binding site in the inactive state of rhodopsin can accommodate the 7-cis,9-cis, and 11-cis isomers of retinal but not the longer all-trans or 13-cis isomers (2). Within this restricted space, the rapid 11-cis-to-all-trans isomerization of the retinal upon absorption of light triggers receptor activation. The question of how the absorbed light energy leads to structural changes within the rhodopsin protein has not been fully answered since Hubbard and Wald (3) first established that the retinal undergoes a large conformational change upon light absorption.

FIGURE 1.

Structure of the retinal binding site in rhodopsin. A, structures of the 11-cis- and all-trans-retinal chromophores. B, view of the rhodopsin crystal structure (Protein Data Bank code: 1U19) from the extracellular surface showing transmembrane helices H3-H7 and the position of key amino acids discussed in the text. C, view along the axis of the retinal from the perspective of H5.

Calorimetric studies have shown that 35 kcal/mol of the absorbed light energy is stored in bathorhodopsin, the first relatively long-lived intermediate (4). Resonance Raman spectra of bathorhodopsin exhibit intense hydrogen out-of-plane modes indicating that the retinal is conformationally distorted (5) due to the rapid change in chromophore structure from 11-cis to all-trans within a highly constrained binding site. There have been many detailed proposals for how the energy stored in bathorhodopsin is released during the thermal decay to metarhodopsin II (Meta II).4 Volumetric measurements show that there is an increase in the overall volume of the protein upon activation (6, 7), implying that retinal isomerization leads to rearrangement of the transmembrane helices. Site-directed spin labeling and EPR measurements show that the cytoplasmic end of H6 undergoes an outward rotation upon activation (8, 9). These studies are consistent with models in which the retinal moves toward and contacts H6, placing the β-ionone ring in the H5-H6 interface. Analysis of helix-helix packing in rhodopsin indicates that the H5-H6 interface is the most loosely packed in rhodopsin (10) and may provide a low energy path for the retinal to enter/exit the retinal binding site. In fact, a recent crystal structure of opsin lacking the retinal chromophore reveals an opening in the H5-H6 interface (11).

In contrast to the idea that the retinal moves toward H6 following isomerization, studies using retinal analogs that can cross-link to the protein in the dark or following photoreaction place the ionone ring in the H4-H5 interface in Meta II. Specifically, Nakanishi and colleagues (12) found a cross-link between the β-ionone ring of retinal and Ala¹⁶⁹ on helix H4 after illumination. Because Ala¹⁶⁹ is on the lipid-facing surface of H4, it has been noted that a straight line drawn between Lys²⁹⁶ and Ala¹⁶⁹ passes through helix H3, suggesting that this helix must consequently move upon activation (13). Additionally, a comparison of the recent 2.6 Å crystal structure of lumirhodopsin with the structure of rhodopsin reveals differences in the middle of H3 (14).

There are also proposals that the β-ionone ring does not change position upon formation of Meta II. Watts and co-workers (15) argued that the β-ionone ring anchors the retinal in position and that retinal isomerization leads to motion of H6 by inducing changes in the dihedral angles of Tyr²⁶⁸ and Ala²⁶⁹. Also, in a low-resolution crystal structure of a rhodopsin photointermediate having an unprotonated Schiff base (16), only minimal structural changes were observed relative to rhodopsin on the extracellular side of the receptor. Both models suggest that the energy stored in bathorhodopsin is largely dissipated by structural changes in helix H6 and the cytoplasmic loops.

We have previously presented solid-state NMR measurements of rhodopsin showing that there is movement of the retinal toward H5 in Meta II (17). In this study (17), the retinal was labeled with ¹³C at the C19 and C20 methyl groups and at the C14 and C15 carbons of the polyene chain close to the retinal protonated Schiff base linkage with Lys²⁹⁶. These measurements reported only on the position of the retinal methyl groups and the region of the retinal near the Schiff base and, consequently, provided only weak constraints on the final position of the β-ionone ring relative to helix H5. As a result, it was not possible to distinguish between different models that had previously been proposed for the location of the retinal in Meta II. Here, we have extended our solid-state NMR studies on rhodopsin4 and Meta II using retinals isotopically ¹³C-labeled on the retinal chain (C7, C12, C19, C20) and the β-ionone ring (C5, C6, C16, C17, and C18). These retinals are incorporated into rhodopsin containing ¹³C labels on different amino acids (methionine, histidine, glycine, cysteine, lysine, tyrosine, tryptophan, threonine, and phenylalanine) to define the location of the retinal in the activated Meta II intermediate. High-resolution structural constraints on rhodopsin and Meta II were established using the two-dimensional (2D) dipolar-assisted rotational resonance (DARR) NMR experiment. This approach enabled us to obtain multiple constraints by measurement of long-range ¹³C... ¹³C through-space correlations between unique ¹³C labels. On the basis of the structural changes observed upon Meta II formation, a mechanism for the activation of rhodopsin, as well as other class A GPCRs, is proposed that involves the concerted motion of helices H5, H6, and H7 through the coupling between conserved microdomains (18, 19).

MATERIALS AND METHODS

Expression and Purification of ¹³C-Labeled Rhodopsin—Rhodopsin was expressed in stable tetracycline-inducible HEK293S cells (20) containing the wild-type opsin gene or mutant derivatives (21). The cells were grown in Dulbecco's modified Eagle's medium formulation (22) prepared from cell culture-tested components (Sigma). Fetal bovine serum was heat-inactivated before its addition to the growth medium. For suspension growth, the medium was supplemented with specific ¹³C-labeled amino acids (Cambridge Isotope Laboratories, Andover, MA), dialyzed fetal bovine serum (10%), Pluronic F-68 (0.1%), dextran sulfate (300 mg/liter), penicillin (100 units/ml), and streptomycin (100 μg/ml) (23, 24). The opsin gene expression was induced by addition of 2 mg/liter tetracycline and 5 mm sodium butyrate on day 5 after inoculation (20), and cells were harvested on day 7.

The HEK293S cell pellets were resuspended in phosphate-buffered saline buffer (40 ml/liter cell culture plus protease inhibitors) (21), and unlabeled 11-cis-retinal was added in two steps to a final concentration of 15 μm. The cells (40 ml/liter of cell culture) were solubilized in phosphate-buffered saline buffer plus 1% n-dodecyl-β-d-maltoside (DDM) at room temperature for 4 h. Subsequent immunoaffinity chromatography purification was carried out using the rho-1D4 antibody (National Cell Culture Center, Minneapolis, MN) as described previously (21). Column fractions containing rhodopsin were concentrated (∼0.4-0.6 mm) to a final volume of ∼400 μl using Centricon cones (Amicon, Bedford, MA) with a 10-kDa cutoff.

Synthesis of ¹³C-Labeled Retinals and Regeneration into Rhodopsin—Synthetic retinals with specific ¹³C labels were produced by standard methods (25) and purified as described previously (23). Rhodopsin pigments in DDM micelles were regenerated by illuminating concentrated samples of a 2:1 molar ratio of ¹³C-labeled retinal-to-protein as discussed previously (17) and concentrated to a volume of 60 μl or less by evaporation using a stream of argon gas.

Solid-state NMR Spectroscopy—Solid-state NMR experiments on 7-10 mg of ¹³C-labeled rhodopsin were performed at 14.1 teslas (600 MHz ¹H frequency) on a Bruker Avance spectrometer using 4-mm magic angle spinning (MAS) probes as described previously (26). One-dimensional ¹³C NMR spectra were collected at MAS spinning rates between 8 and 12 kHz using ramped amplitude cross-polarization (27) with contact times of 2 ms in all experiments.

2D ¹³C DARR NMR measurements (28) were performed with 1024 points in the direct (f2) dimension and 64 points in the indirect (f1) dimension as described previously (26). A DARR mixing time of 600 ms was used to maximize homonuclear recoupling (29). The ¹H radiofrequency field strength during the mixing period was matched to the MAS speed for each sample, satisfying the n = 1 matching condition. Two-pulse phase-modulated proton decoupling (30) or SPINAL64 proton decoupling (31) was used during the acquisition and evolution periods. The decoupling field strengths were typically 80-90 kHz. To obtain the observed signal-to-noise ratios for each data set discussed below, a 2D spectrum typically required a week of signal averaging, which is approximately equal to 5120 scans for each of the 64 rows in the f1 dimension of the 2D spectrum. The carbonyl resonance of powdered glycine at 176.46 ppm relative to tetramethylsilane was used as an external ¹³C reference. All experiments were conducted at -80 °C.

Trapping of the Metarhodopsin II Intermediate—Upon light activation of rhodopsin, the active Meta II intermediate is formed within milliseconds in rod outer segment (ROS) membranes and decays with a half-time of ∼10 min at pH 6.0 and 20 °C (32). Lowering the temperature to -80 °C blocks the decay of the Meta II intermediate; this is essential for the long periods of data acquisition required for the 2D NMR experiments. However, in ROS or model membranes one typically obtains a mixture of Meta I and Meta II after photoreaction of the retinal and low temperature trapping. To quantitatively (>85%) trap the sample in the Meta II intermediate, we solubilized rhodopsin in DDM detergent.

Samples were irradiated for 45-60 s at room temperature in the NMR rotor using a 400-watt lamp equipped with a >495 nm long pass filter and immediately put in the NMR probe with the probe stator warmed to 5 °C. Under slow spinning (∼2 kHz) the sample was frozen within 3 min of illumination using N₂ gas cooled to -80 °C (17).

To confirm that we had trapped Meta II and completely converted the sample from rhodopsin and Meta I, we monitored the chemical shift changes of the ¹³C labels of the retinal that are sensitive to the protonation state of the Schiff base nitrogen and the configuration of the C11=C12 double bond. Meta II is the only intermediate with an all-trans-retinal and an unprotonated Schiff base. We observed no residual rhodopsin or Meta I after illumination and typically were able to trap >85% of our original sample in the Meta II state based on integration of the retinal ¹³C resonances in rhodopsin and Meta II (17).

The line widths of the resolved protein and retinal NMR resonances are generally between 1 and 2 ppm in both rhodopsin and Meta II. The absence of line broadening or resonance splitting indicated that we had trapped a spectroscopically well defined Meta II state. Molecular dynamics (MD) simulations and ²H NMR measurements on rhodopsin have suggested that there are two major conformations of the β-ionone ring, one with a positively twisted C6—C7 single bond and one with a negatively twisted C6—C7 single bond (33, 34). As described below, ¹³C chemical shift and DARR NMR measurements suggest that, predominantly, a single conformation exists in rhodopsin and in Meta II.

Meta II in DDM has been found to be functionally equivalent to Meta II in ROS membranes as it can activate transducin (35, 36). Also, it has been shown that the vibrational frequencies observed in the Fourier transform infrared difference spectrum of Meta II minus rhodopsin are identical for rhodopsin in DDM and ROS membranes (37, 38). The mean half-time for proton uptake of Meta II in DDM is 25 ms (39). As mentioned above, the time between illumination and freezing of the sample is ∼3 min, indicating that the proton uptake in our sample is complete and that we were observing the form of Meta II that activates transducin.

RESULTS

Location of the β-Ionone Ring Relative to H5 in Metarhodopsin II—Our previous solid-state NMR studies concluded that the retinal moves toward H5 in the rhodopsin-to-Meta II transition (17). We sought to establish whether the β-ionone ring increases its contact with H5 by targeting the terminal ε-methyl group of Met²⁰⁷, which is on the face of H5 oriented toward the β-ionone ring of the retinal. For these experiments, rhodopsin containing ¹³Cε-labeled methionine was regenerated with 11-cis-retinal labeled with ¹³C at the C6 and C7 carbons (Fig. 1A).

Fig. 2A presents the 2D DARR NMR spectra of rhodopsin showing only the region containing cross-peaks between the ¹³C6 and ¹³C7 retinal resonances and the ¹³Cε resonance of methionine. The rhodopsin spectrum (Fig. 2A, black) exhibits a weak cross-peak between the ¹³C7 resonance of the retinal chromophore and Met²⁰⁷ (14.7 ppm). The weak intensity of the Met²⁰⁷-C7 cross-peak (Fig. 2A) is consistent with the separation of 4.9 Å observed in the rhodopsin crystal structure (40). No cross-peaks are observed between the ¹³Cε resonance of Met²⁰⁷ and the ¹³C6 resonance of the retinal; the 5.4 Å distance between these ¹³C labels is near the upper limit of the distance range of the DARR experiment (29). Upon conversion to Meta II (Fig. 2A, red) both the ¹³C6 and ¹³C7 resonances exhibit strong cross-peaks with the ¹³Cε-labeled Met²⁰⁷ resonance at 13.8 ppm. The intensity of the Meta II cross-peaks indicates that the C6 and C7 carbons are in van der Waals contact with Met²⁰⁷.

FIGURE 2.

2D DARR NMR of rhodopsin and Meta II labeled with [¹³Cε]methionine and regenerated with ¹³C6,¹³C7-labeled retinal or with ¹³C5,¹³C18-labeled retinal. A, regions of the 2D DARR NMR spectra are shown for rhodopsin (black) and Meta II (red) labeled with [¹³Cε]Met and regenerated with ¹³C6,¹³C7-labeled retinal. A weak cross-peak is observed between the ¹³C7 resonance of the retinal (132.8 ppm) and the ¹³Cε resonance of Met²⁰⁷ (14.7 ppm) on H5 in rhodopsin. In Meta II, the cross-peak between the ¹³C7 (127.6 ppm) retinal resonance and Met²⁰⁷ (13.8 ppm) gains intensity, and a new cross-peak is observed between the ¹³C6 (139.5 ppm) retinal resonance and Met²⁰⁷. B, rows through the [¹³Cε]Met diagonal resonance from the 2D DARR NMR spectra of rhodopsin (black) and Meta II (red) labeled with ¹³Cε-Met and regenerated with 11-cis-retinal labeled at ¹³C5 and ¹³C18 on the β-ionone ring. In rhodopsin, there were no cross-peaks observed between Met²⁰⁷ (14.7 ppm) and the ¹³C5 (131.0 ppm) or ¹³C18 (21.6 ppm) resonances of the retinal. On conversion to Meta II, strong contacts are observed between the ¹³Cε methyl resonance of Met²⁰⁷ (13.8 ppm) and both the ¹³C5 (126.0 ppm) and ¹³C18 (20.9 ppm) resonances of the retinal.

NMR measurements were also made on rhodopsin containing [¹³Cε]methionine and regenerated with ¹³C5,¹³C18-labeled retinal. In the DARR NMR spectra of rhodopsin, cross-peaks were not observed between Met²⁰⁷ and either the ¹³C5 or ¹³C18 resonances of the retinal β-ionone ring. In the rhodopsin crystal structure (40), the Met²⁰⁷(Cε)-retinal C5 (6.7 Å) and the Met²⁰⁷(Cε)-retinal C18 (7.7 Å) distances are outside the range of the DARR NMR experiment. However, upon conversion to Meta II, we observed strong cross-peaks between Met²⁰⁷ and both the ¹³C5 and ¹³C18 resonances on the β-ionone ring (Fig. 2B, red).

The Met²⁰⁷-C5/C6/C7/C18 contacts are consistent with our previous observation of movement of the retinal toward H5 (17). However, the data do not tightly constrain the location of the ionone ring relative to H5 because of the flexibility of the long methionine side chain. To better establish the position of the ionone ring relative to H5 in Meta II, additional ¹³C-¹³C recoupling experiments involving the retinal and several amino acids in the H4-H5 and H5-H6 interfaces were undertaken.

To address the trajectory of the retinal suggested by retinal cross-linking to Ala¹⁶⁹ on helix H4 (12), we obtained 2D DARR NMR spectra of rhodopsin ¹³C-labeled at the Cβ carbon of Cys¹⁶⁷ and at the C5, C6, and C7 carbons of retinal. On the basis of molecular modeling of the retinal binding site, Cys¹⁶⁷ should be in close proximity to one of these carbons if the retinal adopts the position in the H4-H5 interface suggested by cross-linking to Ala¹⁶⁹. In rhodopsin, the β-carbon of Cys¹⁶⁷ and the C5—C7 carbons are separated by >7.8 Å. In the 2D DARR NMR spectrum of rhodopsin, no cysteine-retinal cross-peaks were observed (data not shown). Similarly, in the 2D DARR NMR spectrum of Meta II we were unable to detect cysteine-retinal cross-peaks, arguing that the retinal C5—C7 carbons are more than 6 Å from the β carbon of Cys¹⁶⁷ (data not shown).

To observe contacts between the retinal chromophore and phenylalanine in the H5-H6 interface, we ¹³C-labeled the C16 and C17 methyl groups on the retinal β-ionone ring, as well as the ring carbons of phenylalanine. The ¹³C resonances of the aromatic ring of phenylalanine at 120-145 ppm are well resolved from the ¹³C resonances of the C16 and C17 retinal methyl groups between 25 and 33 ppm. Fig. 3A presents the one-dimensional MAS difference spectrum between rhodopsin (positive) and Meta II (negative) in the region of the retinal methyl resonances. The positive peaks at 30.6 and 26.1 ppm have previously been assigned to the ¹³C16 and ¹³C17 resonances in rhodopsin (41), respectively. On the basis of IUPAC nomenclature, the C1—C16 bond is oriented into the plane of page in Fig. 1A, whereas the C1—C17 bond is oriented out of the plane of the page. The difference between the ¹³C chemical shifts (4.5 ppm) of the C16 and C17 resonances is attributed to one methyl group being in an equatorial orientation and the other methyl group being in an axial orientation. A steric contact between the methyl group in the axial orientation and the proton on C3 of the β-ionone ring results in an upfield chemical shift (41, 42). The negative peaks at 28.6 and 33.4 ppm correspond to the chemical shifts of the C16 and C17 methyl groups in Meta II, respectively (data not shown). The difference in chemical shift (4.8 ppm) between the ¹³C16 and ¹³C17 resonances is again consistent with a difference in the relative orientation of the methyl groups. However, the shift of C16 from 30.6 to 28.6 ppm and of C17 from 26.1 to 33.4 ppm indicates that C16 has now moved to the axial position in Meta II.

FIGURE 3.

Retinal ¹³C16 and ¹³C17 contacts with phenylalanine, histidine, and methionine. A, NMR difference spectrum of rhodopsin (positive) minus Meta II (negative), showing only the region of the ¹³C16 and ¹³C17 resonances of the retinal. B, row taken from the 2D DARR NMR spectrum of rhodopsin containing ¹³C-ring-labeled phenylalanine. The row passes through the diagonal ¹³C-ring resonances of phenylalanine, although only the region of the cross-peaks to the retinal ¹³C16 and ¹³C17 resonances is shown. C, row taken from the 2D DARR NMR spectrum of Meta II of the same sample as in B. Vertical lines are drawn to indicate the positions of phenylalanine-retinal cross-peaks. D, 2D DARR NMR spectra of rhodopsin (black) and Meta II (red) using rhodopsin labeled at [¹³C1]histidine and regenerated with ¹³C16,¹³C17 11-cis-retinal. E, 2D DARR NMR spectra of rhodopsin (black) and Meta II (red) using rhodopsin labeled at [¹³C1]methionine and regenerated with ¹³C16,¹³C17 11-cis-retinal. Boxes outline the regions of interest.

Fig. 3, B and C, presents rows extracted from the 2D DARR NMR spectra of rhodopsin containing ¹³C-ring-labeled phenylalanine and regenerated with 11-cis-retinal ¹³C-labeled at the C16 and C17 positions. The rows are taken through the ¹³C resonance of the aromatic phenylalanine ring on the diagonal of the 2D spectrum. Vertical lines drawn from the MAS difference spectrum indicate the positions of the retinal-phenylalanine cross-peaks. There are 31 phenylalanines in rhodopsin. In the rhodopsin crystal structure, the closest ring carbons of Phe²⁰⁸ are 4.2 Å from C17 and 4.7 Å from C16. Phe²¹² is the only other phenylalanine within 7 Å of the C16 and C17 methyl groups. Therefore, the contacts detected between phenylalanine and the ¹³C16 and ¹³C17 resonances at 30.6 and 26.1 ppm, respectively, in Fig. 3B are assigned to Phe²⁰⁸ and/or Phe²¹². Although the 4.2 and 4.7 Å distances are within the range of the DARR experiment, the low signal intensity (compared with an isolated pair of ¹³C spins separated by the same distance) is due to dipolar truncation (43), which makes it difficult to measure a weak dipolar coupling in the presence of a strongly coupled network of spins.

Upon conversion to Meta II, the cross-peaks assigned to retinal-phenylalanine contacts in rhodopsin disappear. New cross-peaks of similar intensity appear at the chemical shifts assigned to the ¹³C16 and ¹³C17 resonances in Meta II (Fig. 3C). These contacts are also assigned to Phe²⁰⁸ and/or Phe²¹² based on the proximity of the retinal to Met²⁰⁷ discussed above. The similar intensities of the C16,C17-phenylalanine cross-peaks in the inactive and active states are consistent with the β-ionone ring maintaining an approximate distance of 4-5 Å from Phe²⁰⁸ and/or Phe²¹² in Meta II.

To further characterize the position of the retinal, DARR NMR measurements were made between the retinal ¹³C16- and ¹³C17-labeled methyl groups and the backbone carbonyls of Met²⁰⁷ (¹³C1) and His²¹¹ (¹³C1). Fig. 3D presents the 2D DARR NMR spectra of rhodopsin (black) and Meta II (red) containing [¹³C1]histidine and ¹³C16,¹³C17-labeled retinal. His²¹¹ is the only histidine within 15 Å of the C16 and C17 methyl groups in rhodopsin. The [¹³C1]histidine chemical shifts for His²¹¹ in rhodopsin (172.4 ppm) and Meta II (170.2 ppm) agree with those assigned previously to His²¹¹ (44). Fig. 3E presents the 2D DARR NMR spectra of rhodopsin (black) and Meta II (red) containing methionine ¹³C-labeled at the carbonyl position and regenerated with ¹³C16-,¹³C17-labeled retinal. The Met-retinal cross-peaks in these spectra are assigned to Met²⁰⁷ based on the close proximity of these groups in rhodopsin. Although [¹³C1]His²¹¹-retinal and [¹³C1]Met²⁰⁷-retinal cross-peaks are observed in both rhodopsin and Meta II, an increase of intensity upon conversion to Meta II is consistent with tighter packing of the β-ionone ring of retinal with the backbone of H5.

Location of the β-Ionone Ring Relative to H3 and H6 in Metarhodopsin II—In the crystal structure of rhodopsin, the 11-cis-retinal chromophore is tightly packed in the protein interior (Fig. 1, B and C). As noted in the introduction, the all-trans isomer of retinal cannot fit in the binding site of the inactive state of rhodopsin, indicating that the shape of the binding cavity must change upon activation. The results described above and in our previous studies (17) are consistent with motion of the retinal chromophore toward H5. In this section, ¹³C DARR NMR measurements are described for rhodopsin ¹³C-labeled at Thr¹¹⁸ (H3), Gly¹²¹ (H3), and Trp²⁶⁵ (H6) and regenerated with 11-cis-retinal ¹³C-labeled at the C18 and C19 methyl groups to further address the motion of the retinal β-ionone ring relative to H3 and H6.

Fig. 4A shows the positions of Gly¹²¹ and Trp²⁶⁵ and the C18 methyl group on the retinal β-ionone ring. In the rhodopsin crystal structure (40), the retinal C18 methyl group is at 3.7 Å from the Cα carbon of Gly¹²¹. In the 2D DARR NMR spectrum, we observe a strong cross-peak between these groups (Fig. 4B, black). Upon conversion to Meta II, the Gly¹²¹-retinal C18 contact is lost (Fig. 4B, red) consistent with an increase in their separation. On the basis of T₁ relaxation measurements of the C18 methyl group, we can show that the retinal C6—C7 single bond is in the s-cis conformation in both rhodopsin and Meta II (data not shown), indicating that the change in distance is not due to a change in the conformation of the β-ionone ring from 6-s-cis to 6-s-trans. Furthermore, on the basis of an NMR contact between Met⁸⁶ on H2 and Gly¹²⁰ on H3 that does not change upon formation of Meta II, we have argued that the position of Gly¹²¹, the adjacent residue on H3, does not change (at least relative to H2) (26). As a result, we attribute the increase in the Gly¹²¹-retinal C18 distance to the motion of the retinal β-ionone ring toward H5 by at least 1.5 Å. Finally, on the basis of the C18-Gly¹²¹ contact in rhodopsin, we conclude that the 6-s-cis conformation in rhodopsin has a negative twist (i.e. the C18 methyl group is oriented toward H3). In MD simulations on rhodopsin, Lau et al. (34) observed two distinct conformations, one conformation with a negative twist about the C6—C7 single bond, placing the C18 methyl group near Gly¹²¹ (as observed) and one with a positive twist placing the C18 methyl group near Tyr²⁶⁸. DARR NMR experiments on [¹³Cζ]tyrosine-labeled rhodopsin containing ¹³C18-labeled retinal failed to exhibit a C18-Tyr cross-peak (data not shown), indicating that if present the positively twisted conformer is only a minor component.

FIGURE 4.

Position of the β-ionone ring with respect to helices H3 and H6 in rhodopsin and Meta II. A, view of the retinal-binding pocket highlighting the interaction of the C18 methyl group on the β-ionone ring of the retinal with Gly¹²¹ (H3) and Trp²⁶⁵ (H6) and the interaction of the C19 methyl group on the retinal polyene chain with Thr¹¹⁸ (H3). Panels B-D present 2D DARR NMR spectra of rhodopsin (black) and Meta II (red). B, region of Gly¹²¹-retinal C18 contacts. In rhodopsin, a cross-peak (highlighted by a green box) is observed between the ¹³Cα resonance of Gly¹²¹ on H3 and the retinal ¹³C18 methyl group, consistent with a separation of 3.7 Å (Protein Data Bank code: 1U19). This contact is lost upon conversion to Meta II. C, region of Trp²⁶⁵-retinal C18 contacts. In rhodopsin, a weak contact is observed between the ring carbons of [U-¹³C]Trp²⁶⁵ and the retinal ¹³C18 methyl group. This contact is lost in Meta II. D, region of Thr¹¹⁸-retinal C19 contacts. In rhodopsin, a cross-peak between [U-¹³C]Thr¹¹⁸ on H3 and the retinal ¹³C19 methyl group overlaps with the intense intra-residue cross-peaks of [U-¹³C]Thr¹¹⁸. The cross-peak is more clearly resolved in Meta II (red) where the ¹³C19 resonance has shifted to a lower frequency. The observation in Meta II of a retinal C19-Thr¹¹⁸ cross-peak of roughly the same intensity as in rhodopsin indicates that the retinal-Thr¹¹⁸ distance does not change considerably upon activation.

In the rhodopsin crystal structure (40), the side chain of the highly conserved Trp²⁶⁵ (H6) is tightly packed against the β-ionone ring of the retinal. The retinal C18 methyl group is at 3.7 Å from the nearest indole ring carbon. To test whether the retinal C18 methyl group remains in contact with Trp²⁶⁵ after conversion to Meta II, we obtained 2D DARR NMR spectra of rhodopsin and Meta II containing [U-¹³C]tryptophan, [¹³Cα]glycine, and [¹³C5,¹³C18]retinal. We observed a weak cross-peak between the aromatic ring carbons of Trp²⁶⁵ and the C18 methyl group in rhodopsin (Fig. 4C). The cross-peak was not observed in Meta II, consistent with an increase in the distance between the β-ionone ring and Trp²⁶⁵. We attribute the weak intensity of the C18-Trp²⁶⁵ cross-peak in rhodopsin to dipolar truncation (43), a problem encountered in the measurement of long internuclear distances (i.e. weak dipolar couplings) using uniformly ¹³C-labeled amino acids (i.e. in the presence of strongly ¹³C-coupled networks).

To assess the contribution of dipolar truncation to the observed intensities in rhodopsin containing [U-¹³C]tryptophan, [¹³Cα]glycine, and [¹³C5,¹³C18]retinal, DARR NMR experiments were run on a parallel rhodopsin sample containing only ¹³Cα-glycine and [¹³C5,¹³C18]retinal. Fig. 5, A and B, corresponds to rows through the retinal C5 diagonal at 131.0 ppm of these two rhodopsin samples. The cross-peak at 21.6 ppm arising from the directly bonded C5—C18 carbons increases in intensity when Trp²⁶⁵ is not ¹³C-labeled, consistent with dipolar truncation being the cause of the intensity loss in our measurement of the retinal C18-Trp²⁶⁵ contact. In contrast, on conversion to Meta II the intensity of the ¹³C5-¹³C18 cross-peak (measured through the C5 diagonal at 126.0 ppm) in the sample labeled with [U-¹³C]tryptophan (Fig. 5C) is comparable with the intensity of the ¹³C5-¹³C18 cross-peak in the sample containing unlabeled tryptophan (Fig. 5D), suggesting that Trp²⁶⁵ (H6) and C5—C18 have moved away (>6 Å) from each other in Meta II. As a result, the difference in intensity of the ¹³C5-¹³C18 cross-peak in Fig. 5, A and C, is consistent with an increase in separation between the retinal C18 methyl group and Trp²⁶⁵ in Meta II.

FIGURE 5.

Effect of dipolar truncation on the intensity of DARR cross-peaks. Rows are shown through the ¹³C5 diagonal from the 2D DARR NMR spectra of wild-type rhodopsin (black) and Meta II (red) regenerated with ¹³C5,¹³C18-labeled retinal and containing either [U-¹³C]Trp and [¹³Cα]Gly (A and C) or only [¹³Cα]Gly (B and D). In all four spectra the region containing the cross-peak between the directly bonded ¹³C5 and ¹³C18 carbons of the retinal is highlighted. In A, a relatively weak cross-peak is observed between the retinal ¹³C5 (131.0 ppm) and ¹³C18 (21.6 ppm) resonances as compared with B. The loss of intensity in the ¹³C5-¹³C18 cross-peak between A and B has been attributed to dipolar truncation due to the presence of [U-¹³C]Trp²⁶⁵ in the vicinity of the directly bonded ¹³C5-¹³C18 pair in A. On conversion to Meta II, the cross-peak observed in C between the retinal ¹³C5 (126.0 ppm) and ¹³C18 (20.9 ppm) resonances is almost the same in intensity as the one observed in D. This observation suggests that the presence of [U-¹³C]Trp²⁶⁵ no longer modulates the intensity of the cross-peak between directly bonded ¹³C5 and ¹³C18 carbons and provides indirect evidence for the increase in separation between the conserved Trp²⁶⁵ (H6) and the β-ionone ring of the retinal in Meta II. Asterisks indicate MAS side bands.

The retinal C18-Gly¹²¹ and retinal C18-Trp²⁶⁵ distance constraints described above complement previous Gly¹²¹-Trp²⁶⁵ and retinal C19-Trp²⁶⁵ distance measurements in which we observed the loss of a Gly¹²¹-Trp²⁶⁵ contact and the gain of a retinal C19-Trp²⁶⁵ contact in Meta II (26). These earlier results were interpreted in terms of rotation or translation of the Trp²⁶⁵ side chain toward the extracellular side of the retinal binding site upon formation of Meta II.

The cross-peak between the retinal C19 methyl group and Trp²⁶⁵ provides a point of contact between the retinal chromophore and H6 in Meta II. To obtain a corresponding point of contact with H3, we undertook DARR NMR measurements of rhodopsin containing ¹³C19-labeled retinal and [U-¹³C]threonine (Fig. 4A). In the rhodopsin crystal structure (40), the retinal is packed against Ala¹¹⁷, Thr¹¹⁸, and Glu¹²² on H3. Thr¹¹⁸ is adjacent to the middle of the retinal polyene chain and is the only amino acid among these three that can be ¹³C-labeled in our HEK293S expression system. The retinal C19 methyl group is one of the closest points of contact with H3 and is within 5 Å of Thr¹¹⁸. The next closest threonine is over 10 Å away. In Meta II, the cross-peak between the ¹³C19 methyl group and the unresolved ¹³Cα and ¹³Cβ carbons of Thr¹¹⁸ does not change appreciably in intensity, indicating that the retinal C19 methyl group maintains contact with H3 at this position (Fig. 4D).

Location of the Polyene Chain in Metarhodopsin II—In our earlier work (17), the conclusion that the retinal moves toward H5 was based largely on the loss of cross-peaks between the retinal C19 methyl group and the Cζ carbons of Tyr¹⁹¹ (EL2) and Tyr²⁶⁸ (H6), as well as on the gain of a tyrosine contact with the retinal C20 methyl group that we had tentatively assigned to Tyr¹⁷⁸ (EL2). To conclusively assign the C20-Tyr contact in Meta II, we obtained DARR NMR spectra of the Y178F rhodopsin mutant labeled with [¹³Cζ]tyrosine and regenerated with [¹³C12,¹³C20]retinal.

Fig. 6 presents the DARR NMR spectra of wild-type rhodopsin (left) and the Y178F mutant of rhodopsin (right). Fig. 6 presents rows through the diagonal resonance of tyrosine at ∼156 ppm in both the dark state of rhodopsin (black) and the Meta II intermediate (red); the upper panels (A and C) show the region containing retinal C12-tyrosine cross-peaks, and the lower panels (B and D) show the region containing the C20-Tyr cross-peaks. In rhodopsin (Fig. 6, black spectra), we observe cross-peaks between Tyr²⁶⁸ and both the C12 and C20 resonances. The ¹³Cζ-carbon of Tyr²⁶⁸ is close to both C12 (4.9 Å) and C20 (4.2 Å) in the inactive state of rhodopsin (40).

FIGURE 6.

2D DARR NMR of wild-type rhodopsin and the Y178F rhodopsin mutant labeled with [¹³Cζ]tyrosine and regenerated with [¹³C12, ¹³C20]retinal. A and B, rows are shown taken through the [¹³Cζ]Tyr diagonal from the DARR NMR spectra of wild-type rhodopsin (black) and Meta II (red). The region containing cross-peaks to the retinal ¹³C12 carbon is shown in A and to the retinal ¹³C20 carbon in B. C and D, the same regions of the DARR NMR spectra of the Y178F mutant of rhodopsin (black) and Meta II (red) are taken through the [¹³Cζ]Tyr diagonal. Asterisks indicate MAS side bands.

In Fig. 6, B and D, we observe a single retinal-tyrosine cross-peak in both wild-type Meta II and the Y178F mutant of Meta II (red spectra). The observation of a retinal-tyrosine cross-peak in the Y178F mutant rules out Tyr¹⁷⁸ as the tyrosine having a direct contact with the C20 methyl group in Meta II. According to the crystal structure, there are two other tyrosines in relatively close proximity to the C20 methyl group, Tyr²⁶⁸ on H6 and Tyr¹⁹¹ on EL2. We assigned the C20-tyrosine cross-peak in Meta II to Tyr²⁶⁸ on H6 (see below).

To assign the C20-tyrosine cross-peak to either Tyr¹⁹¹ (EL2) or Tyr²⁶⁸ (H6), DARR NMR measurements were made on rhodopsin and the Y178F mutant of rhodopsin ¹³C-labeled at [¹³Cα]glycine and regenerated with [¹³C12,¹³C20]retinal. In the crystal structure of rhodopsin, the two closest glycines to the C20 methyl group are Gly¹⁸⁸ on EL2 (6.2 Å) and Gly¹¹⁴ on H3 (7.2 Å) (Fig. 7A). We used the assignment of a single C20-glycine contact in Meta II to either Gly¹⁸⁸ or Gly¹¹⁴ to aid with the C20-tyrosine assignment. Fig. 7B presents rows through the C20 diagonal resonance showing contacts with glycines at 42.0 and 45.5 ppm, which are assigned to Gly¹⁸⁸ and Gly¹¹⁴, respectively, in rhodopsin, (Fig. 7B, top). The chemical shift of the 45.5 ppm resonance is consistent with the location of Gly¹¹⁴ in α-helical secondary structure. In Meta II, both C20-glycine contacts are lost, and a single new contact is observed at 46.5 ppm (Fig. 7B, bottom) in both the Y178F mutant and wild-type rhodopsin. We could assign the new 46.5 ppm resonance to Gly¹¹⁴ (H3) rather than Gly¹⁸⁸ (EL2), as the same cross-peak is observed in the G188A mutant of rhodopsin (data not shown).

FIGURE 7.

2D DARR NMR of rhodopsin and Meta II labeled with [¹³Cα]glycine and regenerated with [¹³C12,¹³C20]retinal. A, view of the retinal-binding pocket highlighting the interaction of retinal with Gly¹⁸⁸ on EL2 and Gly¹¹⁴ on H3. B, rows through the [¹³Cα]glycine diagonal of rhodopsin (black) and Meta II (red). The cross-peaks between C20 and Gly¹⁸⁸ on EL2 observed at 42.0 ppm and Gly¹¹⁴ on H3 at 45.5 ppm in rhodopsin are lost, and a new cross-peak is observed between C20 and Gly¹¹⁴ on H3 at 46.5 ppm in Meta II. (The rhodopsin spectrum in B is of the Y178F mutant obtained with a high signal-to-noise ratio; in wild-type rhodopsin we observed a single cross-peak at 42.0 ppm, which we assigned to the closer C20-Gly¹⁸⁸ contact.)

The assignment of the C20-glycine contact to Gly¹¹⁴ in Meta II has several implications. First, the additional distance constraints describe above on Thr¹¹⁸ and Gly¹¹⁴ agree with our previous conclusion (17) that the C20 methyl group undergoes a large rotation (>90°) upon cis-trans isomerization of the retinal, whereas the C19 methyl group remains roughly in the same position. Moreover, we find that in rhodopsin the C12 carbon exhibits cross-peaks to both Gly¹¹⁴ and Gly¹⁸⁸ (data not shown), consistent with the crystal structure of rhodopsin (40). However, these cross-peaks are lost in Meta II, indicating that the C12-C13-C20 plane is oriented such that the C20 methyl group is closer to Gly¹¹⁴ on the extracellular side of the binding site, and C12 is pointing away from Gly¹¹⁴ (H3) toward the cytoplasmic side of the binding site. MD simulations (see supplemental material), using the C20-Gly¹¹⁴ assignment, produce a model of the all-trans-retinal in Meta II with the C13-C20 bond oriented toward H3 at an angle of ∼60° to the membrane normal. Independently, site-directed deuterium NMR studies on Meta I by Brown and co-workers (45) have shown that the C13-C20 bond is oriented at an angle of 59 ± 3°. Although the deuterium NMR studies are on Meta I, they support the assignment of the C20-Gly¹¹⁴ contact in Meta II.

Second, the assignment suggests that EL2 moves away from the retinal chromophore upon activation. The large rotation of the C20 methyl group agrees with the crystal structure of bathorhodopsin (46, 47), which reveals a clockwise rotation of the C20 methyl group (viewed from the Schiff base end of the retinal) and a number of computational (46, 48) and biophysical (45) studies. Such a rotation would place the C20 methyl group close to Gly¹⁸⁸ on EL2 in the absence of EL2 motion. We had previously assumed that the position of EL2 did not change in the formation of Meta II (17) because of the network of hydrogen bonding interactions involving EL2 and the extracellular ends of the transmembrane helices. These new data challenge this assumption. Moreover, recent DARR NMR distance measurements (49) show that contacts between the retinal chromophore and the β4 strand of EL2 are lost in Meta II.

Third, the assignment of the C20-Gly contact to Gly¹¹⁴ in Meta II suggests that the C20-tyrosine contact is with Tyr²⁶⁸ rather than with Tyr¹⁹¹. In rhodopsin, the C20 methyl group has contacts with Tyr²⁶⁸ and Trp²⁶⁵, whereas the C19 methyl group has contacts with Tyr¹⁹¹ and Tyr²⁶⁸ (17). In Meta II, there are no tyrosine cross-peaks associated with C19, and as noted, we observed only a single tyrosine cross-peak to C20. We had previously argued for a 4-5 Å translation of the retinal toward H5 in order to move the C19 methyl group away from Tyr¹⁹¹ and Tyr²⁶⁸. The distance constraints presented here and in a manuscript on the motion of EL2 published elsewhere (49) indicate that the translation of the retinal is more modest (∼2 Å) and that EL2 moves away from the retinal upon activation. Motion of EL2 away from the retinal chromophore would increase both the C19-Tyr¹⁹¹ distance and the C20-Tyr¹⁹¹ distance, leaving only Tyr²⁶⁸ in relatively close proximity to the C20 methyl group. We have not been able to confirm this assignment by mutational studies because mutation of Tyr²⁶⁸ to phenylalanine appears to alter its interactions with EL2 (49).

Location of the Retinal Schiff Base in Metarhodopsin II—The DARR NMR measurements described above between the β-ionone ring and amino acids on H5 and the retinal C18 methyl group and Gly¹²¹ (H3) argue for a small (∼2 Å) shift of the retinal toward H5. To obtain further support for motion of the retinal toward H5, we also measured the distance between the ε-carbon of Lys²⁹⁶ on H7 and the ε-CH₃ carbon of Met⁴⁴ on H1. The [¹³Cε]Met⁴⁴-to-[¹³Cε]Lys²⁹⁶ distance in rhodopsin is 4.7 Å. Fig. 8A presents rows through the ¹³Cε diagonal resonance of methionine in the DARR NMR spectrum of rhodopsin (black) and Meta II (red). The Met⁴⁴-Lys²⁹⁶ cross-peak intensity decreases in the conversion to Meta II consistent with an increase in the internuclear distance.

FIGURE 8.

2D DARR NMR of rhodopsin and Meta II labeled with [¹³Cε]methionine and [¹³Cε]lysine. A, rows through the 2D DARR NMR spectra of rhodopsin (black) and Meta II (red) taken through the Met⁴⁴ (H1) diagonal resonance at 10.5 ppm showing the cross-peak with [¹³Cε]Lys296 (H7) at 49.1 ppm. On conversion to Meta II (red) the cross-peak becomes weaker consistent with an increase in separation between Met⁴⁴ (19.4 ppm) and Lys²⁹⁶ (60.8 ppm). B, view of the retinal binding site in rhodopsin (Protein Data Bank code: 1U19) from the extracellular side of the protein highlighting the orientation of the retinal with respect to specific residues on H1 (Met⁴⁴), H3 (Thr¹¹⁸), H5 (Met²⁰⁷), H6 (Trp²⁶⁵), and H7 (Lys²⁹⁶).

Rearrangement of H3-H4-H5 Contacts in Metarhodopsin II—In this section, we describe additional solid-state NMR measurements to address the impact of retinal movement on the position of helix H5. Solvent accessibility studies (50) and recent double electron-electron resonance EPR measurements (9) suggest that the cytoplasmic end of H5 does not undergo large, rigid body motion as observed for H6. However, we have recently shown that a hydrogen bond between His²¹¹ on H5 and Glu¹²² on H3 is broken in Meta II (44), suggesting a change in helix-helix packing and H5 motion. Below, we take advantage of the known chemical shifts of His²¹¹ and Met²⁰⁷ to probe how the H4-H5 interface changes in Meta II.

Fig. 9 shows the 2D DARR NMR spectra of rhodopsin (black) and of Meta II (red) labeled with [¹³Cβ]cysteine, [¹³Cε]methionine, and [¹³Cε1]histidine in the region of the Met-Cys, His-Cys, and His-Met cross-peaks. There are 10 cysteines, 16 methionines, and 6 histidines in rhodopsin. Fig. 9A shows the Cys-Met region of the full 2D NMR spectrum. In rhodopsin, there were no Cys-Met contacts observed, as the closest Cys-Met pair is separated by more than 7.6 Å. In Meta II, we observed a contact between Cys¹⁶⁷ at 25.3 ppm and Met²⁰⁷ at 13.8 ppm, indicating that they are in close proximity (< 5.0 Å). The Met²⁰⁷ chemical shift in Meta II is identical to that defined by the methionine-retinal (¹³C6, ¹³C7) cross-peaks (Fig. 2A), whereas the Cys¹⁶⁷ chemical shift is identical to that defined by the His²¹¹-Cys¹⁶⁷ cross-peak (Fig. 9B).

FIGURE 9.

2D DARR NMR spectra of rhodopsin and Meta II labeled with [¹³Cβ]cysteine, [¹³Cε]methionine, and [¹³Cε1]histidine. A, Cys-Met contacts in rhodopsin (black) and Meta II (red). Above the 2D spectrum are rows taken through the Met²⁰⁷ diagonal. A cross-peak with Cys¹⁶⁷ at 25.3 ppm appears in Meta II. B, His-Cys and His-Met contacts in rhodopsin and Meta II. Above the 2D spectrum are rows taken through the His²¹¹ diagonal in rhodopsin (136.9 ppm) and Meta II (137.5 ppm). In rhodopsin, cross-peaks are observed with Cys¹⁶⁷ at 23.7 ppm and Met¹⁶³ at 13.1 ppm. In Meta II, cross-peaks are observed with Cys¹⁶⁷ at 25.3 ppm, Met²⁰⁷ at 13.8 ppm, and Met¹⁶³ at 11.2 ppm.

A mutation of His²¹¹ to alanine allows us to unambiguously assign the resonance at 136.9 ppm in rhodopsin to His²¹¹ (44). The ¹³Cε1 resonances of the other 5 histidines in rhodopsin are observed as a broad line between 130 and 140 ppm. Above the 2D spectrum in Fig. 9B, we show rows taken through the diagonal of His²¹¹ in rhodopsin (black) and Meta II (red). There is only one cysteine within 17 Å of His²¹¹, i.e. Cys¹⁶⁷ at a distance of 3.9 Å. The His²¹¹-Cys¹⁶⁷ cross-peak is observed at 23.7 ppm in rhodopsin. Similarly, the only methionine in close proximity to His²¹¹ is Met¹⁶³ at a distance of 4.1 Å in rhodopsin. The next closest methionine in rhodopsin is Met²⁰⁷, which is 9.7 Å away. The broad His²¹¹-Met cross-peak at 13.1 ppm is consequently assigned to Met¹⁶³.

In Meta II (Fig. 9B, red), the His²¹¹-Cys¹⁶⁷ cross-peak loses intensity and shifts to 25.3 ppm. The assignment to Cys¹⁶⁷ can be made because it is the only cysteine within 17 Å of His²¹¹. The decrease in intensity of the histidine-cysteine cross-peak indicates that there is an increase in the His²¹¹-Cys¹⁶⁷ distance.

The His-Met region of the 2D DARR NMR spectrum in Fig. 9B also places constraints on the position of H5 in Meta II; two cross-peaks (red) are observed between His²¹¹ and methionine in Meta II. There are only two methionines within 16 Å of His²¹¹ in rhodopsin. The cross-peak at 13.8 ppm is assigned to Met²⁰⁷ based on the chemical shift of the cross-peak observed between Met²⁰⁷ and the retinal ¹³C6,¹³C7 carbons (Fig. 2A). The cross-peak at 11.2 ppm is assigned to Met¹⁶³. These data indicate that the terminal methyl groups of both Met¹⁶³ and Met²⁰⁷ are within 6.0 Å of His²¹¹.

The multiple contacts observed for Met²⁰⁷ in Meta II provide strong constraints on its location. The Met²⁰⁷ side chain must be positioned between Cys¹⁶⁷ and the retinal because we did not observe a retinal-Cys¹⁶⁷ cross-peak. The chemical shift of Met²⁰⁷ undergoes a small (0.9 ppm) shift in frequency upon conversion from rhodopsin to Meta II. The unique shift and narrow line width indicate that the Met²⁰⁷ side chain is not disordered. The decrease in intensity of the His²¹¹-Cys¹⁶⁷ cross-peak and the appearance of a new Met²⁰⁷-Cys¹⁶⁷ contact in Meta II are consistent with motion of the extracellular end of the H5 helix.

Recently, Watts and co-workers (15) have argued that the position of the β-ionone ring does not change upon the formation of Meta II on the basis of an analysis of the ¹³C chemical shifts of the C16 and C17 methyl groups. This conclusion is essentially correct relative to the large translation proposed by Nakanishi and co-workers (12). However, the small, but significant, changes we observed, which are in disagreement with the detailed model proposed on the basis of an immobile ionone ring (15), are critical for the motions of H5 and H6, as discussed below. Changes in the chemical shifts observed due to sample illumination in the previous study (15) (∼0.7 ppm for C17) are smaller than those observed here (2-3 ppm for both C16 and C17). The differences can arise from incomplete trapping of Meta II or differences in the Meta II intermediate trapped in lipid and detergent environments. We (51, 52) and others (37, 53) find that illumination of rhodopsin in DDM detergent results in nearly quantitative conversion (> 85%) to Meta II, whereas illumination of rhodopsin in unsaturated lipids and ROS membranes results in a mixture of intermediates containing both protonated and unprotonated retinal Schiff bases at pH 7.0 (54).

DISCUSSION

We have previously presented solid-state NMR measurements of rhodopsin showing that there is movement of the retinal toward H5 in Meta II (17). These measurements only probed the position of the retinal C19 and C20 methyl groups and the region of the retinal near the Schiff base and, consequently, provided only weak constraints on the final position of the β-ionone ring relative to helix H5. As a result, it was not possible to distinguish between different models that had previously been proposed for the location of the retinal in Meta II (12, 15). In this manuscript, we provide additional structural constraints for the location of the retinal in the activated Meta II intermediate and consider their implications on the structural changes that occur upon conversion of rhodopsin to Meta II.

Retinal-H3 and Retinal-H5 Interactions—The cross-peaks observed between the ¹³C labels on the retinal and histidine, phenylalanine, and methionine on H5 tightly constrain the position of the β-ionone ring between Met²⁰⁷ and Phe²⁰⁸ in Meta II. Moreover, the observation of a new Met²⁰⁷-Cys¹⁶⁷ contact and the lack of a contact between the retinal and Cys¹⁶⁷ in Meta II rule out the possibility that the β-ionone ring is located in the H4-H5 interface. These data argue that the position of the retinal derivative used in the cross-linking studies of Nakanishi and co-workers (12) is different than in wild-type Meta II.

A structural model resulting from MD simulations guided by our NMR data (see supplemental material) suggests an explanation for the role of the β-ionone ring in the stability of Meta II (Fig. 10). The crystal structure of rhodopsin shows a hydrogen bonding contact between the side chain of Glu¹²² on H3 and the backbone carbonyl of His²¹¹ on H5. The His²¹¹ carbonyl does not participate in main chain hydrogen bonding because of the highly conserved Pro²¹⁵ at the i+4 position. In the Meta II model derived from guided MD simulations, the β-ionone ring contacts the side chain of Glu¹²² on H3 as well as the backbone of H5 near His²¹¹. The hydrogen bond between the main chain carbonyl of His²¹¹ and the side chain of Glu¹²² is disrupted as observed previously by solid-state NMR (44). The position of H5 allows the formation of a new hydrogen bond between Glu¹²² and the His²¹¹ δ-nitrogen, which stabilizes the Meta II structure.

FIGURE 10.

Influence of retinal motion on H3-H5 interactions. A, stereo view of the rhodopsin crystal structure (Protein Data Bank code: 1U19) from the extracellular surface showing the 11-cis-retinal and key amino acids on H3, H4, and H5. B, structure of Meta II obtained from a restrained molecular dynamics simulation (see supplemental material) showing the relative positions of all-trans-retinal (orange) and helices H3-H5 after retinal isomerization. The position of the 11-cis-retinal chromophore in rhodopsin is shown in red. The β-ionone ring of retinal contacts the side chain of Glu¹²² on H3 as the retinal shifts position upon formation of Meta II. This motion of the retinal disrupts the hydrogen bonding interaction between the backbone carbonyl of His²¹¹ (H5) and the side chain of Glu¹²² (H3) and leads to a rearrangement of other bonding and non-bonding interactions between pairs of residues (such as Cys¹⁶⁷-Met²⁰⁷, Trp¹²⁶-Glu¹²², and His²¹¹-Tyr²⁰⁶) in the H3-H4-H5 interface. This rearrangement is essential for H5 to adopt an active conformation. Displacement of the β-ionone ring of the retinal allows the side chain of Trp²⁶⁵ to rotate toward the extracellular side of the receptor triggering a shift in the positions of helices H6 and H7. The retinal C6-C7 bond is shown as 6-s-cis with a slight negative twist on the basis of the observed retinal C18-Gly¹²¹ (H3) contact. The retinal C6-C7 bond remains negatively twisted 6-s-cis in Meta II on the basis of the observed retinal C18-Met²⁰⁷ contact. No evidence was found for a positively twisted 6-s-cis conformation in rhodopsin or in Meta II.

The role of Glu¹²² and His²¹¹ in stabilizing the Meta II state is supported by comparative studies between rhodopsin and the cone pigments, which lack these residues. Shichida and co-workers (55) found that substitution of Glu¹²² in rhodopsin with the corresponding amino acid in the green- or red-sensitive cone pigments converts the rate of retinal regeneration and Meta II decay into those characteristic of the respective cone pigments. In contrast, when glutamate is substituted into the green-sensitive cone pigment, the rates of retinal regeneration and Meta II decay are similar to those of rhodopsin.

H5 in many other class A GPCRs has been shown to interact with receptor-specific ligands. For instance, in the amine receptors (such as the β₂-adrenergic receptors), the amino acids corresponding to Met207, Phe208 and His211 in rhodopsin are conserved as serines. These serines hydrogen bond to hydroxyl groups on the catechol ring of the amine agonist to stabilize an active receptor conformation (56-58). In the dopamine (59) and serotonin (60) receptors, the amino acids at the positions equivalent to His²¹¹ and Phe²¹² have been shown to interact with their receptor-specific ligands.

The recent crystal structure of opsin (11), which is formed following the release of all-trans-retinal in Meta II, shows that the position of the extracellular end of helix H5 is the same as in rhodopsin, although some of the side chain interactions predicted for Meta II are retained. For example, the backbone carbonyl of His²¹¹ (H5) is no longer hydrogen bonded to the Glu¹²² (H3) side chain. Instead a new interaction is observed directly between the Glu¹²² (H3) and His²¹¹ (H5) side chains consistent with our Meta II data. Also, Trp¹²⁶ (H3) has lost its hydrogen bonding interaction with Glu¹²² (H3) in opsin and appears to be hydrogen bonded only to His²¹¹ on H5. A weakening of the hydrogen bonding interaction for Trp¹²⁶ was observed in Meta II by NMR (26) and UV-visible absorption spectroscopy (61). However, the hydrogen bonding interactions for Tyr²⁰⁶ in opsin are not consistent with recent NMR results showing that Tyr²⁰⁶ undergoes a significant weakening of hydrogen bonding in Meta II (49). Also, the distance constraints obtained from our NMR experiments for pairs of amino acids in the H3-H4-H5 interface in Meta II, such as His²¹¹(H5)-Cys¹⁶⁷(H4), His²¹¹(H5)-Met¹⁶³(H4), His²¹¹(H5)-Met²⁰⁷(H5), and Met²⁰⁷(H5)-Cys¹⁶⁷(H4), are not consistent with the arrangement of residues in the opsin crystal structure. As a result, it appears that the all-trans-retinal chromophore holds helix H5 in an active conformation and that loss of the retinal allows the receptor to shift (at least partially) back to an inactive conformation. These changes are in agreement with retinal analog studies showing the importance of the β-ionone ring in receptor activation (62).

In contrast to the inactive conformation of the extracellular end of H5, the cytoplasmic end of H5 in the opsin crystal structure (11) appears to adopt the “active” conformation of the receptor. The cytoplasmic end of H5 has moved inward and rotated to place conserved Tyr²²³ in contact with Arg¹³⁵, breaking the ionic lock between Arg¹³⁵ on H3 and Glu²⁴⁷ on H6. The NMR results showing a movement of H5 due to steric interactions with the ionone ring and coupling with EL2 (49) are consistent with the key role of H5 motion in activation.

Retinal-H6 Interactions—An aromatic cluster of amino acids at the extracellular end of H6 represents a functional microdomain that is key to receptor activation in the class A GPCRs. In rhodopsin, Phe²⁶¹, Trp²⁶⁵, and Tyr²⁶⁸ on H6 form this cluster. The aromatic side chain of Trp²⁶⁵ lies in the arc created by the 11-cis-retinal and the side chain of Lys²⁹⁶. We have previously proposed that preventing motion of Trp²⁶⁵ locks the receptor off by inhibiting the motion of H6 (26). Motion of the β-ionone ring toward H5 in the rhodopsin-to-Meta II transition allows motion of the Trp²⁶⁵ side chain or the H6 backbone toward EL2. Previously, we observed that Trp²⁶⁵ comes into close contact with the retinal C19 methyl group in Meta II (26) and loses its contact with Gly¹²¹ on H3. Here, we report that the C18 methyl group of the β-ionone ring moves away from Trp²⁶⁵ and Gly¹²¹. In the guided MD simulations, to satisfy the NMR constraints (see supplemental Table 1) the Trp²⁶⁵ side chain undergoes significant motion toward EL2 while remaining in van der Waals contact with the C19 methyl group on the retinal polyene chain. In the opsin crystal structure (11), the indole side chain of Trp²⁶⁵ has moved away from Gly¹²¹ and Ala²⁹⁵ consistent with the NMR data but has not undergone a change in rotameric state or shifted toward EL2 to any significant extent. We attribute the differences between the opsin structure and our model of Meta II to the lack of all-trans-retinal in the binding cavity.

Our conclusions regarding retinal-H6 interactions are in agreement with a wide range of previous studies on both rhodopsin and other GPCRs. For instance, UV absorbance and linear dichoism studies on rhodopsin show that Trp²⁶⁵ moves into a more hydrophilic environment and that the plane of the indole side chain of Trp²⁶⁵ changes from an orientation parallel to the bilayer normal to an orientation roughly perpendicular to the bilayer normal (61, 63). Javitch and co-workers (64) proposed a “rotamer toggle switch” for receptor activation based on studies of the β2 adrenergic receptor, in the subclass of biogenic amine receptors. In these receptors, ligand binding is thought to induce a change in the side chain conformation of a conserved phenylalanine, which is coupled to changes in the side chain conformations of conserved cysteine, tryptophan, and proline residues on H6. The location of Trp²⁶⁵ in opsin is consistent with the linear dichroism studies in which rotation of a tryptophan in the Meta I to Meta II transition was found to be reversed upon decay of Meta II (63).

Retinal-H7 and Retinal-EL2 Interactions—The extracellular and cytoplasmic ends of H7 are both important in the activation mechanism of rhodopsin (17). The extracellular end of H7 contains residues that are specific to the subfamily of class A GPCRs (17, 65). For example, in rhodopsin the retinal is attached to Lys²⁹⁶ (H7) as a positively charged protonated Schiff base that forms a salt bridge with the negatively charged side chain of Glu¹¹³ (H3). This interaction is important for keeping the receptor in the inactive state and is broken upon receptor activation (66).

The cytoplasmic end contains the highly conserved NPXXY sequence. The recent crystal structures of opsin shows that Tyr³⁰⁶ is reoriented toward Arg¹³⁵ of the ionic lock (11, 67), which is stabilized in an open conformation by elements from H5 (Tyr²²³), H6 (Met²⁵⁷), and H7 (Tyr³⁰⁶). Together, these observations indicate that retinal isomerization (or ligand binding in the ligand-activated GPCRs) results in structural changes involving the extracellular end of H7 that allow the cytoplasmic end to adopt an active conformation.

How does retinal isomerization and Schiff base deprotonation lead to motion of H7? There are two possibilities. The first possibility is that a change in the position of the retinal directly affects the position of H7 through its covalent linkage to Lys²⁹⁶. We previously proposed a “push-pull” mechanism for activation involving a push on H5 by the ionone ring and a pull on H7 via the covalently attached retinal (17). As mentioned above, the distance constraints presented here and in a manuscript on the motion of EL2 published elsewhere (49) indicate that translation of the retinal toward H5 is more modest (∼2 Å) and that EL2 moves away from the retinal binding site upon activation. The reduced motion of retinal toward H5 described above would suggest a more modest role for the direct covalent bond between the retinal and H7. In fact, Oprian and co-workers (68) have shown that a covalent bond between retinal and Lys²⁹⁶ is not required for activation.

The second and more likely possibility for how retinal isomerization and Schiff base deprotonation lead to a change in the position of the extracellular end of H7 is that the H7 helix responds to changes in other structural elements in the protein. The closest elements that change position in Meta II are EL2 and H6. The extracellular end of H7 between Met²⁸⁸ and Ala²⁹² packs against EL2 and will likely shift due to displacement of EL2 as a result of changes in both steric and electrostatic interactions. H6 packs against H7 along its entire length, and mutations along the H6-H7 interface modulate activity (69). For example, Oprian and co-workers have shown that mutants of Lys²⁹⁶ result in constitutive activity (70) and that the magnitude of the activity is inversely correlated with residue size (71). They concluded that the electrostatic Lys²⁹⁶-Glu¹¹³ interaction and a steric contact involving the Lys²⁹⁶ side chain both contribute to preventing receptor activation, presumably by preventing motion of H7. A similar loss of steric interactions between Trp²⁶⁵ and Ala²⁹⁵ (adjacent to Lys²⁹⁶) occurs in the activation of the wild-type receptor. Specifically, as described above, motion of the retinal allows for the rotation of Trp²⁶⁵ away from Ala²⁹⁵ on H7, which in turn may facilitate a shift of H7 into an active orientation.

Conclusions—Our structural studies on the location of the retinal and surrounding residues in Meta II described above and elsewhere (49) suggest that the all-trans-retinal Schiff base is holding EL2 and the extracellular ends of the receptor in an active conformation. It is known that mutation of a number of residues that line the retinal binding site can significantly influence the light dependent activation of transducin (55, 72-75), emphasizing the intricate interplay between the all-trans geometry of the retinal and amino acids on the extracellular side of the receptor in stabilizing the active state.

Together these results provide a new working model for how structural changes in the retinal are involved in receptor activation and are coupled to structural changes in several functional microdomains described previously (18, 19). Retinal isomerization leads to steric strain within the retinal binding site between the β-ionone ring and H5, and between the C19/C20 methyl groups and EL2. These interactions trigger the displacement of EL2, deprotonation of the Schiff base nitrogen and protonation of Glu¹¹³. The retinal C19 and C20 methyl groups are involved in rearrangement of EL2, whereas the β-ionone ring leads to rearrangement of the hydrogen bonding network centered on H5. We propose that EL2 motion and SB deprotonation are coupled to the motion of H5 and are needed for the retinal and H7 to shift into their active positions. Motion of the β-ionone ring is also coupled to the motion of Trp²⁶⁵, which likely happens in concert with motion of the cytoplasmic ends of H6 and H7 and the rearrangement of the hydrogen-bonding network centered on the conserved NPXXY sequence. Motion of helices H5, H6, and H7, in turn, is coupled to the rearrangement of electrostatic interactions involving the conserved ERY sequence at the cytoplasmic end of H3, exposing the G protein binding site on the cytoplasmic surface of the protein. The location of the retinal and reorganization of the protein upon receptor activation provide a structural basis for understanding the action of agonists and antagonists in the large family of class A GPCRs.