Rhodopsin activation exposes a key hydrophobic binding site for the transducin α-subunit C terminus

Abstract

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 α-subunit (GTα) 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 GTα. C terminus (containing a tryptophan quenching group) affected their fluorescence. From these and other assays, we conclude that the GTα 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 GTα 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.