Rhodopsin activation exposes a key hydrophobic binding site for the transducin alpha-subunit C terminus.
Conformational changes enable the photoreceptor rhodopsin to couple with and activate the G-protein transducin. Here we demonstrate a key interaction between these proteins occurs between the C terminus of the transducin alpha-subunit (G(Talpha)) and a hydrophobic cleft in the rhodopsin cytoplasmic face exposed during receptor activation. We mapped this interaction by labeling rhodopsin mutants with the fluorescent probe bimane and then assessed how binding of a peptide analogue of the G(Talpha) C terminus (containing a tryptophan quenching group) affected their fluorescence. From these and other assays, we conclude that the G(Talpha) C-terminal tail binds to the inner face of helix 6 in a retinal-linked manner. Further, we find that a "hydrophobic patch" comprising key residues in the exposed cleft is required for transducin binding/activation because it enhances the binding affinity for the G(Talpha) C-terminal tail, contributing up to 3 kcal/mol for this interaction. We speculate the hydrophobic interactions identified here may be important in other GPCR signaling systems, and our Trp/bimane fluorescence methodology may be generally useful for mapping sites of protein-protein interaction.
Janz
Jay M
JM
Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, USA.
Farrens
David L
DL
D000818:Animals; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000737:chemistry / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D008027:Light; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D015293:Transducin / Q000737:chemistry* / Q000378:metabolism*
D019086:Bridged Bicyclo Compounds, Heterocyclic; D010455:Peptides; C021149:bimanes; D012243:Rhodopsin; D015293:Transducin