Location of the Retinal Chromophore in the Activated State of Rhodopsin

Shivani Ahuja,Evan Crocker,Markus Eilers,Viktor Hornak,Amiram Hirshfeld,Martine Ziliox,Natalie Syrett,Philip J Reeves,H.Gobind Khorana,Mordechai Sheves,Steven O Smith

doi:10.1074/jbc.m805725200

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 (13)C-labeled sites located on the beta-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-Gly(188) (EL2) distance consistent with an increase in separation between the retinal and EL2 upon activation. NMR distance constraints showed that the beta-ionone ring moves to a position between Met(207) and Phe(208) on transmembrane helix H5. Movement of the ring toward H5 was also reflected in increased separation between the Cepsilon carbons of Lys(296) (H7) and Met(44) (H1) and between Gly(121) (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 Glu(122) (H3) and His(211) (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.

Highlights

3 The abbreviations used are: GPCR, G protein-coupled receptor; dipolar-assisted rotational resonance (DARR), dipolar assisted rotational resonance; EL2, second extracellular loop; DDM, n-dodecyl-␤-D-maltoside; MAS, magic angle spinning; Molecular dynamics (MD), 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
We sought to establish whether the ␤-ionone ring increases its contact with H5 by targeting the terminal ⑀-methyl group of Met207, which is on the face of H5 oriented toward the ␤-ionone ring of the retinal
The retinal C19 and C20 methyl groups are involved in rearrangement of EL2, whereas the ␤ionone ring leads to rearrangement of the hydrogen bonding network centered on H5

Summary

Introduction

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. Retinal Location in Metarhodopsin II 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 ␤-ionone ring in the H5-H6 interface. 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

Methods

Results

Discussion

Conclusion