title: 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 (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.
authors.affiliation: Departments of Physics & Astronomy and Biochemistry & Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215.
authors.forename: Shivani
authors.initials: S
authors.lastname: Ahuja
authors.forename: Evan
authors.initials: E
authors.lastname: Crocker
authors.forename: Markus
authors.initials: M
authors.lastname: Eilers
authors.forename: Viktor
authors.initials: V
authors.lastname: Hornak
authors.forename: Amiram
authors.initials: A
authors.lastname: Hirshfeld
authors.forename: Martine
authors.initials: M
authors.lastname: Ziliox
authors.forename: Natalie
authors.initials: N
authors.lastname: Syrett
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Reeves
authors.forename: H Gobind
authors.initials: HG
authors.lastname: Khorana
authors.forename: Mordechai
authors.initials: M
authors.lastname: Sheves
authors.forename: Steven O
authors.initials: SO
authors.lastname: Smith
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D011090:Polyenes / Q000737:chemistry; D011487:Protein Conformation; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*; D012160:Retina / Q000378:metabolism*; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D012545:Schiff Bases / Q000737:chemistry
chemical_list: D011090:Polyenes; D043562:Receptors, G-Protein-Coupled; D012545:Schiff Bases; C013110:metarhodopsins; D012243:Rhodopsin