Zinc-induced Decrease of the Thermal Stability and Regeneration of Rhodopsin

Valle, Luís J. del; Ramon, Eva; Cañavate, Javier; Paulo, Dias; Garriga, Pere

doi:10.1074/jbc.m210760200

Cited by 34 publications

(48 citation statements)

References 41 publications

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“…Our studies confirm that the highaffinity metal coordination site in the TM domain is selective for Zn 2ϩ . Interestingly, at the solvent-accessible, low-affinity metal coordination site, recent studies suggest that high concentrations of either Zn 2ϩ or Cu 2ϩ can destabilize rhodopsin protein (13). This is similar to what has been observed with other proteins, where high-affinity and physiological metal coordination sites are selective, while low-affinity and non-physiological (or pathophysiological) metal coordination sites may be non-selective.…”

Section: Discussionsupporting

confidence: 71%

“…Interestingly, Zn 2ϩ deficiency is also known to cause retinal neurodegeneration and night blindness (10), symptoms reminiscent of retinitis pigmentosa. Furthermore, Zn 2ϩ has been shown to directly bind rhodopsin (11,12) and to reduce rhodopsin thermal stability and regeneration with 11-cis-retinal at higher Zn 2ϩ concentrations (50 -200 M) (13). While Zn 2ϩ is a well known structural and catalytic cofactor for a number of metalloenzymes and transcription factors, recent studies have identified Zn 2ϩ as an allosteric modulator of structure and function for a number of G protein-coupled receptors, including the dopamine (14), adrenergic (15,16), melanocortin (17,18), and chemokine (19) receptors.…”

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confidence: 99%

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Critical Role of Transmembrane Segment Zinc Binding in the Structure and Function of Rhodopsin

Stojanović

Stitham

Hwa

2004

Journal of Biological Chemistry

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Zinc deficiency and retinitis pigmentosa are both important factors resulting in retinal dysfunction and night blindness. In this study, we address the critical biochemical and structural relevance of zinc ions in rhodopsin and examine whether zinc deficiency can lead to rhodopsin dysfunction. We report the identification of a high-affinity zinc coordination site within the transmembrane domain of rhodopsin, coordinated by the side chains of two highly conserved residues, Glu 122 in transmembrane helix III and His 211 in transmembrane helix V. We also demonstrate that this zinc coordination is critical for rhodopsin folding, 11-cis-retinal binding, and the stability of the chromophore-receptor interaction, defects of which are observed in retinitis pigmentosa. Furthermore, a cluster of retinitis pigmentosa mutations is localized within and around this zinc binding site. Based on these studies, we believe that improvement in zinc binding to rhodopsin at this site within the transmembrane domain may be a pharmacological approach for the treatment of select retinitis pigmentosa mutations. Transmembrane coordination of zinc may also be an important common principle across G-protein-coupled receptors.Several neurodegenerative disorders, Alzheimer disease, Parkinson disease, familial amyotrophic lateral sclerosis, and the transmissible spongiform encephalopathies, share a common pathogenesis involving the misfolding and aggregation of specific proteins. Mounting evidence has demonstrated the direct binding of zinc (Zn 2ϩ ) to the ␤-amyloid (Alzheimer disease), ␣-synuclein (Parkinson disease), superoxide dismutase (amyotrophic lateral sclerosis), and prion (transmissible spongiform encephalopathies) proteins, linking either the gain or loss of Zn 2ϩ binding to the progression of these severe protein misfolding disorders. Zn 2ϩ promotes aggregation of the highly fibrillogenic prion peptide, PrP106 -126 (1), and of the ␤-amyloid protein (2-4) into amyloidogenic aggregates. Clioquinol, a metal chelating agent, is currently being investigated as a potential therapeutic solution to inhibit ␤-amyloid neurotoxicity (5). In contrast, in mutant superoxide dismutase protein, loss of affinity for Zn 2ϩ results in diminished protein activity (6), reduced protein stability (7), and formation of amyloid-like filaments (8). Thus, both zinc deficiencies and excesses can lead to neurodegenerative diseases.In the G protein-coupled photoreceptor rhodopsin, there have been over 100 distinct, heritable mutations identified, many of which induce an altered protein conformation, misfolding, aggregation, and cell death, followed by the clinical manifestation of the retinal neurodegenerative disorder retinitis pigmentosa (9). Interestingly, Zn 2ϩ deficiency is also known to cause retinal neurodegeneration and night blindness (10), symptoms reminiscent of retinitis pigmentosa. Furthermore, Zn 2ϩ has been shown to directly bind rhodopsin (11,12) and to reduce rhodopsin thermal stability and regeneration with 11-cis-retinal at higher Zn 2ϩ concen...

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Section: Discussionsupporting

confidence: 71%

mentioning

confidence: 99%

Critical Role of Transmembrane Segment Zinc Binding in the Structure and Function of Rhodopsin

Stojanović

Stitham

Hwa

2004

Journal of Biological Chemistry

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“…The mutant proteins in the dark are less stable than WT, with the stability decreasing with increasing size of the side chain. A number of factors are shown to affect the thermal stability of dark-state rhodopsin, including retinal disease-causing mutations (44) or zinc binding (45). Other factors mediating the stability of dark-state rhodopsin are proposed, like a conserved ion-pair interaction in the intradiscal loop E-2 of rhodopsin (46).…”

Section: Discussionmentioning

confidence: 99%

Structural and Functional Role of Helices I and II in Rhodopsin

Bosch

Ramon

Valle

et al. 2003

Journal of Biological Chemistry

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The naturally occurring mutations G51A and G51V in transmembrane helix I and G89D in the transmembrane helix II of rhodopsin are associated with the retinal degenerative disease autosomal dominant retinitis pigmentosa. To probe the orientation and packing of helices I and II a number of replacements at positions 51 and 89 were prepared by using site-directed mutagenesis, and the corresponding proteins expressed in COS-1 cells were characterized. Mutations at position 51 (G51V and G51L) bound retinal like wild-type rhodopsin but had thermally destabilized structures in the dark, altered photobleaching behavior, destabilized metarhodopsin II active conformations, and were severely defective in signal transduction. The effects observed can be correlated with the size of the mutated side chains that would interfere with specific interhelical interaction with Val-300 in helix VII. Mutations at position 89 had sensitivity to charge, as in G89K and G89D mutants, which showed reduced transducin activation. G89K showed a second absorbing species in the UV region at 350 nm, suggesting a charge effect of the introduced lysine. Increased formation of non-active forms of rhodopsin, like metarhodopsin III, may have some influence in the molecular defect underlying retinitis pigmentosa in the mutants studied. At the structural level, the effect of the mutations analyzed can be rationalized assuming a very specific set of tertiary interactions in the interhelical packing of the transmembrane segments of rhodopsin.

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“…GPCRs denaturation processes, however, involve large positive entropies of activation in the range of 100 to 300 entropy units, reflecting considerable structural randomization [32,33].…”

Section: Conformational Stability Of Gpcrsmentioning

confidence: 99%

“…An interesting case is that of the effect of zinc ions on Rho stability [33,55,56]. We previously reported a decrease in the thermal stability caused by zinc ions -measured as an increase in the rate of thermally-induced Schiff base hydrolysis (of purified Rho solubilized in the neutral DM detergent) [33]. Thermal bleaching is a widely used technique to test the stability of the Schiff base linkage and this should provide some indication on structural deviations from optimal packing of the retinal binding pocket of Rho.…”

Section: Transfer Modelmentioning

confidence: 99%

Stabilization of Membrane Proteins: the Case of G‐Protein‐Coupled Receptors

Reyes‐Alcaraz

Tzanov

Garriga

2008

Engineering in Life Sciences

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G-protein-coupled receptors are integral membrane proteins which constitute the largest family of signal transduction molecules participating in the majority of normal physiological processes. G-protein-coupled receptors are responsible for the control of enzyme activity, ion channels and vesicle transport, and they respond to a wide variety of stimuli, like signals involved in sensory systems such as vision, taste and olfaction, but also to a diverse set of chemical signals such as lipids, hormones, neurotransmitters, amino acids, nucleotides, peptides and proteins. This family of receptors is being widely studied because of its potential use as pharmacological targets in drug development, and recently also for its potential use in the development of novel biosensors. G-protein-coupled receptors are specifically designed to fold and function in a lipid bilayer environment, where these membrane proteins are remarkably stable and achieve their optimal performance. The currently used technology for the purification of G-protein-coupled receptors consists in their extraction from the cell membrane and solubilization into detergent micelles. A common drawback of this strategy is that G-proteincoupled receptors solubilized in typical detergents show rather poor conformational stability, which may result in relatively rapid inactivation. The poor stability of detergent-solubilized samples renders many membrane proteins biochemically intractable. This precludes the determination of a high-resolution structure and imposes severe limitations for the development of applications. Thus, the enhancement of the stability of G-protein-coupled receptors is a major issue in order to facilitate structural determination and to unravel their potential in biotechnological applications. This work provides a brief overview of some current advances in the experimental methods for stabilizing G-protein-coupled receptors that can also be extended to other types of membrane proteins. Structure of G-protein-Coupled Receptors (GPCRs)The distinctive structural feature of GPCRs is the bundle of seven transmembrane (7TM) a-helices that constitutes a relevant signature in structural biology. This conformational architecture, and its functional implications, is central to pharmacology and therapeutics. Nearly 2000 GPCRs have been reported since bovine opsin was cloned in 1983 and the b-adrenergic receptor in 1986. They are classified into over 100 subfamilies as a function of their sequence homology, ligand structure and receptor function. Significant differences exist among the members of the GPCRs superfamily, in spite of the high degree of amino acid homology found among the members of a particular subfamily. The visual photoreceptor rhodopsin (Rho) was the first GPCR whose crystal structure was determined at high-resolution [1][2][3][4][5][6][7]. The natural abundance and availability of Rho has made of this protein the prototypical GPCR for structural studies. As a consequence, Rho is often selected as a model protein for biochemical and b...

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Zinc-induced Decrease of the Thermal Stability and Regeneration of Rhodopsin

Cited by 34 publications

References 41 publications

Critical Role of Transmembrane Segment Zinc Binding in the Structure and Function of Rhodopsin

Critical Role of Transmembrane Segment Zinc Binding in the Structure and Function of Rhodopsin

Structural and Functional Role of Helices I and II in Rhodopsin

Stabilization of Membrane Proteins: the Case of G‐Protein‐Coupled Receptors

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