Effect of channel mutations on the uptake and release of the retinal ligand in opsin

Abstract

In the retinal binding pocket of rhodopsin, a Schiff base links the retinal ligand covalently to the Lys296 side chain. Light transforms the inverse agonist 11-cis-retinal into the agonist all-trans-retinal, leading to the active Meta II state. Crystal structures of Meta II and the active conformation of the opsin apoprotein revealed two openings of the 7-transmembrane (TM) bundle towards the hydrophobic core of the membrane, one between TM1/TM7 and one between TM5/TM6, respectively. Computational analysis revealed a putative ligand channel connecting the openings and traversing the binding pocket. Identified constrictions within the channel motivated this study of 35 rhodopsin mutants in which single amino acids lining the channel were replaced. 11-cis-retinal uptake and all-trans-retinal release were measured using UV/visible and fluorescence spectroscopy. Most mutations slow or accelerate both uptake and release, often with opposite effects. Mutations closer to the Lys296 active site show larger effects. The nucleophile hydroxylamine accelerates retinal release 80 times but the action profile of the mutants remains very similar. The data show that the mutations do not probe local channel permeability but rather affect global protein dynamics, with the focal point in the ligand pocket. We propose a model for retinal/receptor interaction in which the active receptor conformation sets the open state of the channel for 11-cis-retinal and all-trans-retinal, with positioning of the ligand at the active site as the kinetic bottleneck. Although other G protein-coupled receptors lack the covalent link to the protein, the access of ligands to their binding pocket may follow similar schemes.G protein-coupled-receptor | regeneration | signal transduction T he photoreceptor rhodopsin is a prototypical member of the superfamily of seven transmembrane (7 TM) helix or G protein-coupled receptors (GPCRs). Rhodopsin consists of the apoprotein opsin and the covalently bound chromophoric ligand 11-cis-retinal, which acts as a powerful inverse agonist and holds the receptor in its inactive conformation. Absorption of a photon isomerizes the chromophore to the agonist all-trans-retinal which in turn triggers conformational changes of the protein leading to the active, G protein-binding form metarhodopsin II (Meta II). In rod cells Meta II decays within minutes by hydrolysis of the Schiff base and release of all-trans-retinal. The regeneration of the rhodopsin dark state by uptake of new 11-cis-retinal effectively suppresses the basal activity of opsin and primes it at the same time for photoactivation (1, 2).In the rhodopsin dark state, 11-cis-retinal is buried in its binding pocket in the core of the 7 TM bundle. The side chain of Lys296 in TM7 protrudes into the pocket and provides the active site for the protonated Schiff base linkage between ligand and protein. The protonated Schiff base is stabilized by a salt-bridge with its counterion, Glu113, and by residues in the second extracellular loop, which is folded deeply into the...

Structural role of the T94I rhodopsin mutation in congenital stationary night blindness

“…Our 2.3 Å crystal structure of this mutant reveals the water network around retinal in an unprecedented detail: We observe four new water molecules that are probably not specific for this mutant, as the structure of the retinal binding pocket has not changed compared to previous structures of metarhodopsin-II [17,21]. However, one of the previously described water molecules in this region is missing in our structure, likely because the hydrophobic mutation T94I [26], as the smaller alanine side chain would not be able to affect retinal hydrolysis as described above.…”

“…According to a study of mutations in the retinal entry channel [26], T94I 2.61 may even reduce constitutive activity of opsin. The same study shows increased constitutive activity for 11 out of 15 mutations close to the retinal binding pocket, none of which is correlated with CSNB.…”

Insights into congenital stationary night blindness based on the structure of G90D rhodopsin

“…The question remains why only four mutations in rhodopsin cause CSNB, whereas many other known mutations increase constitutive activity of opsin [26], interfere with retinal binding [19], increase thermal isomerization [34] or preactivate dark-state rhodopsin [17,35]. Our structural data indicate that substitutions that specifically perturb the E113-K296 activation switch result in CSNB (Fig 4), likely by destabilizing the inactive conformation and selectively favouring a short-lived preactivated conformation at the same time.…”

“…The active conformation of opsin thus seems to preform a binding pocket into which several retinal isomers can bind. Once the right retinal isomer is in place, a SB is formed and snaps into the strong salt bridge with its counterion E113 to force the seven-helix bundle into the inactive rhodopsin dark state [2,19]. The G90D mutation prevents an efficient snapping by forming a salt bridge with K296, the site of SB formation.…”

Cone-like rhodopsin expressed in the all cone retina of the colubrid pine snake as a potential adaptation to diurnality

“…Cones have been optimized for fast regeneration, with cone opsin meta-intermediate states being short lived compared with those of rhodopsin (Imai et al, 2005), and a cone-specific Müller cell retinoid cycle (Das et al, 1992) providing a dedicated pool of 11-cis retinal. These faster kinetic properties are hypothesized to be facilitated in cone opsins via the relative 'openness' of the chromophore binding pocket, which allows water molecules, and therefore other small molecules such as hydroxylamine, to access the chromophore where they can participate in Schiff base hydrolysis (Chen et al, 2012;Piechnick et al, 2012;Wald et al, 1955). Rhodopsins, in contrast, are optimized for sensitivity and signal amplification; therefore, E122/I189 and a tighter overall structure contribute to a slower active state decay, allowing for the activation of multiple G proteins (Chen et al, 2012;Starace and Knox, 1997), increased thermal stability relative to cone opsins (Barlow, 1964) and a resistance to hydroxylamine (Dartnall, 1968).…”