Alkylated Hydroxylamine Derivatives Eliminate Peripheral Retinylidene Schiff Bases but Cannot Enter the Retinal Binding Pocket of Light-Activated Rhodopsin

Piechnick, Ronny; Heck, M.; Sommer, Monika

doi:10.1021/bi200675y

Cited by 13 publications

(22 citation statements)

References 61 publications

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“…In agreement with previous observations, tbHA has little effect on the release of ATR from WT rhodopsin (Fig. 2E) (16). However, for the CAM, tbHA completely abolished the initial incomplete release (Fig.…”

Section: After Rhodopsin Photoactivation An Equilibrium Of Atr Releasupporting

confidence: 93%

“…An alkylated derivative of HA, tbHA is too big to enter the receptor binding pocket and therefore can only react with retinals that have dissociated (16). As expected, tbHA did not affect WT rhodopsin (compare Fig.…”

Section: Resultssupporting

confidence: 59%

“…Like HA, tbHA reacts with ATR to form retinal-oxime that cannot rebind to the receptor. However, because tbHA is too large to enter the ligand-binding pocket (16), and thus can only modify ATR that has exited the protein, retinal release would only be affected if release and rebinding is occurring. In agreement with previous observations, tbHA has little effect on the release of ATR from WT rhodopsin (Fig.…”

Section: After Rhodopsin Photoactivation An Equilibrium Of Atr Releamentioning

confidence: 99%

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Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state

Schafer

Fay

Janz

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Here, we describe two insights into the role of receptor conformational dynamics during agonist release (all-trans retinal, ATR) from the visual G protein-coupled receptor (GPCR) rhodopsin. First, we show that, after light activation, ATR can continually release and rebind to any receptor remaining in an active-like conformation. As with other GPCRs, we observe that this equilibrium can be shifted by either promoting the active-like population or increasing the agonist concentration. Second, we find that during decay of the signaling state an active-like, yet empty, receptor conformation can transiently persist after retinal release, before the receptor ultimately collapses into an inactive conformation. The latter conclusion is based on timeresolved, site-directed fluorescence labeling experiments that show a small, but reproducible, lag between the retinal leaving the protein and return of transmembrane helix 6 (TM6) to the inactive conformation, as determined from tryptophan-induced quenching studies. Accelerating Schiff base hydrolysis and subsequent ATR dissociation, either by addition of hydroxylamine or introduction of mutations, further increased the time lag between ATR release and TM6 movement. These observations show that rhodopsin can bind its agonist in equilibrium like a traditional GPCR, provide evidence that an active GPCR conformation can persist even after agonist release, and raise the possibility of targeting this key photoreceptor protein by traditional pharmaceutical-based treatments.T he superfamily of G protein-coupled receptors (GPCRs) is one of the largest targets of pharmaceutical drugs in the human genome. Classically, GPCR signaling occurs when a diffusible ligand (such as a drug) binds to the receptor and stabilizes conformations that can couple with and activate intracellular proteins. Our understanding of this process has built on the classical "ternary complex" model of receptor-ligand-G protein interaction (1), a model that, with revisions, has continued to guide our knowledge of how this critical event occurs.However, this paradigm has faced problems when applied to rhodopsin, the dim-light visual receptor. Rhodopsin is kept in an "off" state by a covalently bound inverse agonist, 11-cis retinal (11CR). Light converts the 11CR to an agonist, all-trans retinal (ATR), which enables the receptor to activate its G protein, transducin (G t ) (2, 3). The active receptor, metarhodopsin II (MII), continues signaling until the Schiff base linking ATR to the receptor is hydrolyzed, resulting in the release of ATR and the decay of MII into an inactive apoprotein, opsin (4, 5). Binding of a new 11CR to opsin reforms the dark state (DS), enabling another round of photon detection (6).Due to this unusual light-activated, covalently bound ligand, rhodopsin has usually been considered "different" from the larger superfamily of diffusible ligand-binding GPCRs. However, we recently discovered that rhodopsin behaves more like a traditional ligand-binding GPCR than previously thought (7). Our expe...

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Section: After Rhodopsin Photoactivation An Equilibrium Of Atr Releasupporting

confidence: 93%

Section: Resultssupporting

confidence: 59%

Section: After Rhodopsin Photoactivation An Equilibrium Of Atr Releamentioning

confidence: 99%

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Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state

Schafer

Fay

Janz

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…3B). To confirm the interpretation of this phenomenon as a fast release of the ligand (22) we applied an additional test with alkylated hydroxylamine (18). UV-visible spectra of K296G/11-cis-PrSB taken immediately after illumination showed an absorption band at 440 nm which corresponds to protonated all-trans-PrSB (Fig.…”

Section: Resultsmentioning

confidence: 92%

“…This channel connects the open cytoplasmic crevice with the ligand binding pocket and is thought to allow the access of water to the retinal Schiff base site (6). Recent work suggests that this solvent channel is so narrow that water and hydroxylamine but not its alkylated derivatives can pass it (18). Our study starts from these two putative channel structures, the retinal channel which traverses the protein in parallel to the membrane plane and the water channel which runs perpendicular to it.…”

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confidence: 99%

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

Piechnick

Ritter

Hildebrand

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Quantification of Arrestin–Rhodopsin Binding Stoichiometry

Lally

Sommer

2015

Methods in Molecular Biology

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We have developed several methods to quantify arrestin-1 binding to rhodopsin in the native rod disk membrane. These methods can be applied to study arrestin interactions with all functional forms of rhodopsin, including dark-state rhodopsin, light-activated metarhodopsin II (Meta II), and the products of Meta II decay, opsin and all-trans-retinal. When used in parallel, these methods report both the actual amount of arrestin bound to the membrane surface and the functional aspects of arrestin binding, such as which arrestin loops are engaged and whether Meta II is stabilized. Most of these methods can also be applied to recombinant receptor reconstituted into liposomes, bicelles, and nanodisks.

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Alkylated Hydroxylamine Derivatives Eliminate Peripheral Retinylidene Schiff Bases but Cannot Enter the Retinal Binding Pocket of Light-Activated Rhodopsin

Cited by 13 publications

References 61 publications

Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state

Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state

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

Quantification of Arrestin–Rhodopsin Binding Stoichiometry

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