The Role of Glu181 in the Photoactivation of Rhodopsin

Lüdeke, Steffen; Beck, Mareike; Yan, Elsa C. Y.; Sakmar, Thomas P.; Siebert, Friedrich; Vogel, Reiner

doi:10.1016/j.jmb.2005.08.039

Cited by 102 publications

(165 citation statements)

References 60 publications

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“…S9D), we propose that the Rh6mr Schiff base remains protonated after light activation. This PSB must be stabilized by a nearby nucleophile such as Glu181, a counterion switch in Rh Meta-II precursor states (40), to render Rh6mr resistant to PSB hydrolysis. Indeed, our MM analyses show a direct interaction between the PSB and Glu181 in the active Rh6mr bound with the 11,13-dicis 6mr isomer (SI Appendix, Fig.…”

Section: Discussionmentioning

confidence: 99%

Photocyclic behavior of rhodopsin induced by an atypical isomerization mechanism

Gulati

Jastrzębska

Banerjee

et al. 2017

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

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Vertebrate rhodopsin (Rh) contains 11-cis-retinal as a chromophore to convert light energy into visual signals. On absorption of light, 11-cis-retinal is isomerized to all-trans-retinal, constituting a oneway reaction that activates transducin (G t ) followed by chromophore release. Here we report that bovine Rh, regenerated instead with a six-carbon-ring retinal chromophore featuring a C 11 =C 12 double bond locked in its cis conformation (Rh6mr), employs an atypical isomerization mechanism by converting 11-cis to an 11,13-dicis configuration for prolonged G t activation. Time-dependent UV-vis spectroscopy, HPLC, and molecular mechanics analyses revealed an atypical thermal reisomerization of the 11,13-dicis to the 11-cis configuration on a slow timescale, which enables Rh6mr to function in a photocyclic manner similar to that of microbial Rhs. With this photocyclic behavior, Rh6mr repeatedly recruits and activates G t in response to light stimuli, making it an excellent candidate for optogenetic tools based on retinal analog-bound vertebrate Rhs. Overall, these comprehensive structure-function studies unveil a unique photocyclic mechanism of Rh activation by an 11-cis-to-11,13-dicis isomerization.is the visual pigment found in rod outer segments of vertebrate and invertebrate photoreceptors that mediates the transformation of light into vision (1-5). By contrast, microbial Rhs mediate both the energy conversion and cell signaling required for cell survival (2, 6, 7). All classes of Rhs feature a heptahelical transmembrane structure that incorporates a covalently bound retinal chromophore, but they differ in their ability to interconvert between their two spectral absorption states driven by light exposure (2). Although vertebrate Rh undergoes a one-way photobleaching reaction of the retinal chromophore after light absorption (8), microbial Rhs exhibit an intrinsic photocyclic behavior with no chromophore release (2,8). This makes vertebrate Rhs unsuitable for optogenetic applications that require reversible control over effector ligands. Nevertheless, all Rhs require a cis-trans/trans-cis isomerization of their chromophores to trigger a protein conformational change that mediates the downstream signaling cascade or energy conversion (2,8). Previous studies on bovine Rh regenerated with sixcarbon-ring retinal chromophores (Rh6mr) featuring a locked C 11 =C 12 cis-trans isomerization (SI Appendix, Fig. S1) have implied their ability to activate the G protein transducin (G t ). These results suggest an alternative mechanism that can propagate the downstream visual cascades (9)(10)(11)(12)(13)(14). In this study, we provide direct evidence that Rh6mr can activate G t by an atypical cis-to-dicis isomerization that is also photocyclic, undergoing an 11,13-dicis-to-11-cis reisomerization. Even though it is believed that cis-trans isomerization is required to achieve the conformational change in opsin needed for G t activation, our results generalize this prerequisite to any isomerization that can achieve the same con...

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Section: Discussionmentioning

confidence: 99%

Photocyclic behavior of rhodopsin induced by an atypical isomerization mechanism

Gulati

Jastrzębska

Banerjee

et al. 2017

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

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“…11,32,[54][55][56][57][58][59][60][61][62][63][64][65] This would involve a change in protonation states during the photocycle 33 and experimental work has confirmed that Glu 113 is the dark-state counterion and been suggestive, but not conclusive, that Glu 181 is active during the light-activated stages. 66,67 The simulation results are intriguing in suggesting that this counterion switch mechanism could be present as part of the relaxation mechanism of the protein to the retinal conformational change. We should emphasize that in our calculations there is no change in the charge state of Glu 181 during or after the forced isomerization.…”

Section: Counterion Switchmentioning

confidence: 99%

How a small change in retinal leads to G‐protein activation: Initial events suggested by molecular dynamics calculations

Stevens

Woolf

2006

Proteins

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Rhodopsin is the prototypical G-protein coupled receptor, coupling light activation with high efficiency to signaling molecules. The dark-state X-ray structures of the protein provide a starting point for consideration of the relaxation from initial light activation to conformational changes that may lead to signaling. In this study we create an energetically unstable retinal in the light activated state and then use molecular dynamics simulations to examine the types of compensation, relaxation, and conformational changes that occur following the cis-trans light activation. The results suggest that changes occur throughout the protein, with changes in the orientation of Helices 5 and 6, a closer interaction between Ala 169 on Helix 4 and retinal, and a shift in the Schiff base counterion that also reflects changes in sidechain interactions with the retinal. Taken together, the simulation is suggestive of the types of changes that lead from local conformational change to light-activated signaling in this prototypical system.

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“…Spectral decomposition was performed in the conformationally most sensitive spectral region between 1,800 and 1,600 cm Ϫ1 (marked in green). This range comprises the amide I vibrations of the protein backbone and the CAO stretch of protonated carboxylic acids Glu-122 and Asp-83, which are involved in hydrogen-bonded networks between H3 and H5 and between H1, H2, and H7, respectively (11,(23)(24)(25), and are sensitive markers for the conformational changes during receptor activation (12,(26)(27)(28).…”

Section: Changes In Interhelical Hydrogen-bonded Network In the Metamentioning

confidence: 99%

“…1A). The first switch entails disruption of a salt bridge between the retinal PSB on H7 and its complex counterion, consisting of Glu-113 on H3 and Glu-181 on extracellular loop 2 in the transmembrane part of the receptor (12), by deprotonation of the PSB and internal proton transfer to Glu-113 (13). The second switch is proton uptake by Glu-134 of the conserved E(D)RY motif at the cytoplasmic terminus of H3 (14), forming a salt bridge with Arg-135 that is part of an interhelical network between H3 and H6 in the cytoplasmic domain.…”

mentioning

confidence: 99%

Two protonation switches control rhodopsin activation in membranes

Mahalingam

Martínez‐Mayorga

Brown³

et al. 2008

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

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Activation of the G protein-coupled receptor (GPCR) rhodopsin is initiated by light-induced isomerization of the retinal ligand, which triggers 2 protonation switches in the conformational transition to the active receptor state Meta II. The first switch involves disruption of an interhelical salt bridge by internal proton transfer from the retinal protonated Schiff base (PSB) to its counterion, Glu-113, in the transmembrane domain. The second switch consists of uptake of a proton from the solvent by Glu-134 of the conserved E(D)RY motif at the cytoplasmic terminus of helix 3, leading to pH-dependent receptor activation. By using a combination of UV-visible and FTIR spectroscopy, we study the activation mechanism of rhodopsin in different membrane environments and show that these 2 protonation switches become partially uncoupled at physiological temperature. This partial uncoupling leads to Ϸ50% population of an entropy-stabilized Meta II state in which the interhelical PSB salt bridge is broken and activating helix movements have taken place but in which Glu-134 remains unprotonated. This partial activation is converted to full activation only by coupling to the pH-dependent protonation of Glu-134 from the solvent, which stabilizes the active receptor conformation by lowering its enthalpy. In a membrane environment, protonation of Glu-134 is therefore a thermodynamic rather than a structural prerequisite for activating helix movements. In light of the conservation of the E(D)RY motif in rhodopsin-like GPCRs, protonation of this carboxylate also may serve a similar function in signal transduction of other members of this receptor family.FTIR spectroscopy ͉ G protein-coupled receptor ͉ ionic lock ͉ membrane protein ͉ UV-visible spectroscopy G protein-coupled receptors (GPCRs) are 7-helical transmembrane proteins that exist in conformational equilibria between inactive and active conformations modulated by the binding of ligands (1). In the case of rhodopsin as a visual pigment, the ligand is the retinal chromophore, which is covalently linked to a lysine on transmembrane helix H7 by a protonated Schiff base (PSB) (2). The 11-cis retinal chromophore of the dark state is an inactivating inverse agonist that is converted by photoisomerization to the all-trans agonist, driving the conformational transitions leading to receptor activation. Within milliseconds several inactive intermediates are formed, such as Batho, BSI, Lumi, and Meta I, that can be examined by using time-resolved (3) or cryotrapping techniques (4). The early transitions involve mainly a relaxation of the isomerized retinal chromophore (5, 6), with only minor changes to the ␣-helix bundle of the receptor protein as revealed by electron crystallography of the Meta I state (7). Only in the transition from Meta I to the active receptor conformation Meta II is a rearrangement of the helix bundle observed, involving tilt movements of H6 (8-10) and presumably also of H5 (11).Activation of the receptor is proposed to involve 2 distinct protonation switches ...

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The Role of Glu181 in the Photoactivation of Rhodopsin

Cited by 102 publications

References 60 publications

Photocyclic behavior of rhodopsin induced by an atypical isomerization mechanism

Photocyclic behavior of rhodopsin induced by an atypical isomerization mechanism

How a small change in retinal leads to G‐protein activation: Initial events suggested by molecular dynamics calculations

Two protonation switches control rhodopsin activation in membranes

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