Conformational Selection and Equilibrium Govern the Ability of Retinals to Bind Opsin.

Despite extensive study, how retinal enters and exits the visual G protein-coupled receptor (GPCR) rhodopsin remains unclear. One clue may lie in two openings between TM1/TM7 and TM5/TM6 in the active receptor structure. Recently, retinal has been proposed to enter the inactive apoprotein (ops) through these holes when the receptor transiently adopts the active (ops*) conformation. Here, we directly test this transient activation hypothesis (TAH) using a fluorescence-based approach to measure rates of retinal binding to samples containing differing relative fractions of ops and ops*. In contrast to what the TAH model would predict, we find binding for the inverse agonist, 11-cis retinal (11CR), slows when the sample contains more ops* (produced using M257Y, a constitutively activating mutation). Interestingly, the increased presence of ops* allows for binding of the agonist, all-trans retinal (ATR), whereas WT opsin shows none. Shifting the conformational equilibrium towards even more ops*, using a G protein peptide mimic (either free in solution or fused to the receptor), accelerates the rate of ATR binding and slows 11CR binding. An arrestin peptide mimic shows little effect on 11CR binding, yet stabilizes opsin-ATR complexes. The TM5/TM6 hole is apparently not involved in this conformational selection. Increasing its size by mutagenesis does not enable ATR binding, but instead, slows 11CR binding, suggesting it may play a role in trapping 11CR. In summary, our results indicate conformational selection dictates stable retinal binding, which we propose involves ATR and 11CR binding to different states, the latter a previously unidentified, open-but-inactive conformation.Copyright 2014, The American Society for Biochemistry and Molecular Biology.