Abstract
The G-protein-coupled receptor rhodopsin is activated by photoconversion of its covalently bound ligand 11-cis-retinal to the agonist all-trans-retinal. After light-induced isomerization and early photointermediates, the receptor reaches a G-protein-dependent equilibrium between active and inactive conformations distinguished by the protonation of key opsin residues. In this report, we study the role of the 9-methyl group of retinal, one of the crucial steric determinants of light activation. We find that when this group is removed, the protonation equilibrium is strongly shifted to the inactive conformation. The residually formed active species is very similar to the active form of normal rhodopsin, metarhodopsin II. It has a deprotonated Schiff base, binds to the retinal G-protein transducin, and is favored at acidic pH. Our data show that the normal proton transfer reactions are inhibited in 9-demethyl rhodopsin but are still mandatory for receptor activation. We propose that retinal and its 9-methyl group act as a scaffold for opsin to adjust key proton donor and acceptor side chains for the proton transfer reactions that stabilize the active conformation. The mechanism may also be applicable to related receptors and may thus explain the partial agonism of certain ligands.
Highlights
The G-protein-coupled receptor rhodopsin is activated by photoconversion of its covalently bound ligand 11-cis-retinal to the agonist all-trans-retinal
We find that when this group is removed, the protonation equilibrium is strongly shifted to the inactive conformation
The retinal photoreceptor rhodopsin is one of the archetypes of the G-protein-coupled receptor superfamily. It is composed of the apoprotein opsin comprising seven transmembrane helices (TMs)1 and the chromophore 11-cis-retinal, which is covalently bound to Lys296 in TM7 via a protonated Schiff base (SB), keeps the receptor in the inactive conformation, and acts as a highly effective inverse agonist
Summary
The 9-methyl group of the retinal, which has been shown to interact directly with the Gly121 residue of opsin in the middle of TM3 [21, 22], is known to be one of the crucial steric determinants of photoactivation Removal of this group (see Fig. 2) causes steric defects that reduce the ability of the pigment to progress to the meta II state and impair its catalytic efficiency [23]. Either a breakdown of the steric mechanisms prevents light-activated 9-demethyl (9-dm) rhodopsin from reaching the protonation equilibrium and instead causes the formation of a weakly active product (b) or the lack of retinal’s 9-methyl group allows the photoreaction to proceed to the protonation equilibrium, but shifts this equilibrium in favor of the inactive conformation analogous to meta I (c).
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