6-s-cis conformation and polar binding pocket of the retinal chromophore in the photoactivated state of rhodopsin

Article abstract of DOI:10.1021/ja9034768

The visual pigment rhodopsin is unique among the G protein-coupled receptors in having an 11-cis retinal chromophore covalently bound to the protein through a protonated Schiff base linkage. The chromophore locks the visual receptor in an inactive conformation through specific steric and electrostatic interactions. This efficient inverse agonist is rapidly converted to an agonist, the unprotonated Schiff base of all-trans retinal, upon light activation. Here, we use magic angle spinning NMR spectroscopy to obtain the ¹³C chemical shifts (C5-C20) of the all-trans retinylidene chromophore and the ¹⁵N chemical shift of the Schiff base nitrogen in the active metarhodopsin II intermediate. The retinal chemical shifts are sensitive to the conformation of the chromophore and its molecular interactions within the protein-binding site. Comparison of the retinal chemical shifts in metarhodopsin II with those of retinal model compounds reveals that the Schiff base environment is polar. In particular, the ¹³C15 and ¹⁵Nε chemical shifts indicate that the CdN bond is highly polarized in a manner that would facilitate Schiff base hydrolysis. We show that a strong perturbation of the retinal ¹³C12 chemical shift observed in rhodopsin is reduced in wild-type metarhodopsin II and in the E181Q mutant of rhodopsin. On the basis of the T₁ relaxation time of the retinal ¹³C18 methyl group and the conjugated retinal ¹³C5 and ¹³C8 chemical shifts, we have determined that the conformation of the retinal C6-C7 single bond connecting the β-ionone ring and the retinylidene chain is 6-s-cis in both the inactive and the active states of rhodopsin. These results are discussed within the general framework of ligand-activated G protein-coupled receptors.