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J. Biol. Chem., Vol. 279, Issue 34, 35932-35941, August 20, 2004

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

Aleksandar Stojanovic, Jeremiah Stitham, and John Hwa

From the Departments of Pharmacology & Toxicology and Medicine (Section of Cardiology), Dartmouth Medical School, Hanover, New Hampshire 03755

Received for publication, April 6, 2004 , and in revised form, June 4, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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, Glu¹²² in transmembrane helix III and His²¹¹ 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 (Zn²⁺) 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 Zn²⁺ binding to the progression of these severe protein misfolding disorders. Zn²⁺ 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 Zn²⁺ 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, Zn²⁺ deficiency is also known to cause retinal neurodegeneration and night blindness (10), symptoms reminiscent of retinitis pigmentosa. Furthermore, Zn²⁺ has been shown to directly bind rhodopsin (11, 12) and to reduce rhodopsin thermal stability and regeneration with 11-cis-retinal at higher Zn²⁺ concentrations (50–200 µM) (13). While Zn²⁺ is a well known structural and catalytic cofactor for a number of metalloenzymes and transcription factors, recent studies have identified Zn²⁺ 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. Although a number of these studies have described the effect of engineered Zn²⁺ coordination sites in the transmembrane domain of rhodopsin and other G protein-coupled receptors (20, 21), and sites within intracellular and extracellular loops or at helical ends (14–19), our goal was to confirm and analyze the role of a native Zn²⁺ coordination site formed by residues within the transmembrane (TM)¹ domain, the first to be described among G protein-coupled receptors.

Upon analysis of the 2.8-Å resolution crystal structure of rhodopsin (22–24), we identified three putative zinc (Zn²⁺) ions bound to each rhodopsin monomer (Zn²⁺ ions 1, 2, and 3 on rhodopsin monomer A; Zn²⁺ ions 4, 5, and 6 on rhodopsin monomer B) with a seventh Zn²⁺ ion present at the interface between the two monomers of the rhodopsin dimer. We now report that the Zn²⁺ coordination site formed by residues within the rhodopsin transmembrane domain is physiologically relevant and critical to the structure and function of rhodopsin. Furthermore, we have confirmed that this site is specific for Zn²⁺ versus other similar divalent metals. This Zn²⁺ coordination site in rhodopsin lies within the 11-cis-retinal binding pocket (in the TM domain), 8 Å from the -ionone ring of 11-cis-retinal, coordinated by the transmembrane amino acids Glu¹²² (TM III; side chain carbonyl), Met¹⁶³ (TM IV; backbone carbonyl), and His²¹¹ (TM V; imidazole side chain). Through inductively coupled plasma mass spectrometry (ICP-MS), fluorescent detection of Zn²⁺ and site-directed mutagenesis, we confirm that Zn²⁺ is bound to rhodopsin and that the coordination site formed by residues Glu¹²² and His²¹¹ is a high-affinity zinc coordination site. In contrast to a low-affinity, solvent-accessible Zn²⁺ coordination site that destabilizes rhodopsin, the coordination of Zn²⁺ within the transmembrane domain is critical for dark state rhodopsin stability, 11-cis- retinal binding, and may play a vital role in transition to the active metarhodopsin II state.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Preparation of Rhodopsin Protein Samples—Amino acid substitutions were introduced into the synthetic rhodopsin gene through PCR mutagenesis, as described previously (25). COS-1 cells were transiently transfected with pMT4 vectors carrying the opsin genes. Cells were harvested and opsins regenerated with 11-cis-retinal and rhodopsin proteins purified by immunoaffinity chromatography. In experiments where rhodopsin was treated with EDTA (Sigma), CaCl₂ (Sigma), CoCl₂ (Sigma), CuCl₂ (Sigma), or ZnCl₂ (Fisher Scientific) or examined under various pH values, the respective phosphate-buffered saline buffer, with those changes, was prepared and utilized throughout the protein purification process. ROS (rod outer segments) from bovine retinas was a kind gift from Dr. Phillip Reeves (M.I.T.). The presence of rhodopsin protein, both from COS-1 cells and from ROS, was determined through UV-visible spectroscopy (500 nm absorbance) and Western blot analysis (25).

UV-visible Absorption Spectroscopy—UV-visible absorption spectra were recorded on a PerkinElmer Life Sciences -40 UV-visible spectrophotometer at 2 or 25 °C (25). Rhodopsin thermal stability was determined by monitoring the decay of the 500 nm absorbance at a constant temperature of 50 °C. Protein samples were allowed to equilibrate (2–5 min) to 50 °C prior to acquiring absorption spectra. Absorption spectra were obtained at 5-min intervals, until complete disappearance of the 500 nm absorbance. Initial values of the 500 nm absorbance were normalized between experiments to account for base-line shifts and protein concentration.

Fluorescent Zn²⁺ Detection—Purified rhodopsin protein or rhodopsin from ROS was incubated with a 1 µM concentration of the fluorescent Zn²⁺ indicator, FluoZin^TM-3 (Molecular Probes). Samples were excited in a CytoFluor II spectrofluorimeter (Applied Biosystems) at a wavelength of 485 nm (bandwidth of 20 nm), and the emission was measured at a wavelength of 508 nm (bandwidth of 20 nm). This was within the active range of FluoZin-3 (optimal excitation at 494 nm and emission at 515 nm). A standard curve of Zn²⁺ binding to FluoZin-3 was determined by the addition of 10-fold differing concentrations of Zn²⁺ to FluoZin-3 (six Zn²⁺ concentrations from 1.0 x 10^-10 M to 1.0 x 10^-5 M; n = 6). To exclude fluorescence due to binding of alternate cations, we measured fluorescence of FluoZin-3 upon the addition of Ca²⁺ (CaCl₂). To determine background Zn²⁺, the same concentration of FluoZin-3 was incubated with each solution in which rhodopsin was solubilized and purified. Rhodopsin was incubated with FluoZin-3 and assayed for fluorescence in either the dark or activated state. Three fluorescence scans were obtained and averaged for each cycle. For photoactivation, protein samples were illuminated for 30 s with a 150-watt fiber optic light. These samples were then returned to the dark for the remainder of the experiment.

ICP-MS—Purified rhodopsin protein was dissolved in a HNO₃ solution and digested in an MARS-5 microwave (300 watts for 10 min). ICP-MS analyses were performed on an Agilent 7500s ICP-MS equipped with an Agilent High Solids nebulizer. The solution was diluted 4- and 10-fold with deionized water and analyzed for zinc. Three isotopes of zinc (⁶⁶Zn, ⁶⁷Zn, and ⁶⁸Zn) were averaged for measurements and calculations. To check for contamination of the digestion procedure and sample manipulation, a blank solution was prepared and carried through each set of analyses. Gallium was used as internal standard for the determination of zinc.

Computer Modeling—The rhodopsin crystal structure (PDB ID: 1L9H [PDB] ) (24) was visualized with the Swiss PDB Viewer computer program (GlaxoSmithKline, Geneva, Switzerland) (26). Putative H-bonds were depicted in green. Zn²⁺ ions were depicted as gray spheres.

Data Analysis—Values for all experiments are expressed as mean ± S.E. All means and S.E. were calculated from at least three separate experiments using separate protein preparations. K rate constants, half-lives, and FluoZin-3 Zn²⁺ binding rates were acquired with nonlinear regression using GraphPad Prism® software. A 95% confidence interval was used for all curve-fitting procedures using GraphPad Prism. Where applicable, data were determined to be statistically significant using the unpaired t test (^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Through examination of the rhodopsin crystal structure (PDB ID: 1L9H [PDB] ) (24), we identified four putative Zn²⁺ coordination sites in each monomer of rhodopsin (Fig. 1A, Zn²⁺₁, Zn²⁺₂, Zn²⁺₃, and Zn²⁺₄). Interestingly, while many metal binding sites occur in solvent-exposed regions, there was a predicted Zn²⁺₃ coordination site within the TM 11-cis-retinal binding pocket of rhodopsin, 8 Å from the -ionone ring of 11-cis-retinal (Fig. 1B). Furthermore, we had previously identified a large clustering of retinitis pigmentosa mutations in this same region (9). As the rhodopsin crystallization procedure required supraphysiological concentrations of Zn²⁺ (100 mM), it was crucial to determine which Zn²⁺ coordination sites were physiologically and biochemically relevant and to determine the role played by the coordination of such Zn²⁺ ions. Determination of Zn²⁺ binding (ICP-MS and FluoZin-3) was followed by Zn²⁺ treatment (pre- and postprotein purification), biochemical removal of the solvent exposed Zn²⁺ binding sites (pH and EDTA), and mutagenic removal of the Zn²⁺ from the transmembrane site. These assays in combination with the rhodopsin crystal structure confirmed a structurally important, buried, high-affinity Zn²⁺ coordination site with the rhodopsin transmembrane domain and one or more low-affinity, solvent-exposed extracellular Zn²⁺ coordination sites.

View larger version (59K): [in this window] [in a new window]   FIG. 1. A, crystal structure of rhodopsin (PDB ID: 1L9H [PDB] ), indicating location of putative Zn2+ coordination sites: Zn2+1 (His195 and Glu197), Zn2+2 (Glu201 and Gln279), Zn2+3 (His211 and Glu122), and Zn2+4 (His100 of one monomer and Lys311, Arg314, and Asn315 of the second monomer). B, model of the Zn2+3 coordination site, as hypothesized in this study. Green dashed-lines represent putative hydrogen bonding.

View larger version (17K): [in this window] [in a new window]   FIG. 2. A, detection of Zn2+ bound to WT and His211 Cys rhodopsin, using ICP-MS and FluoZin-3. All experiments were performed at least three times, using separate protein preparations. Data were determined to be statistically significant using the unpaired t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). B, standard curve of Zn2+ (ZnCl2; solid line) and Ca2+ (CaCl2; dashed line) binding to the Zn2+-dependent fluorophore FluoZin-3. Inset, linear portion of the Zn2+ standard curve in B (from 10 e-7 to 10 e-6 M) in Y = MX + B form.

To validate the physiological relevance of our findings, rhodopsin from ROS was analyzed using FluoZin-3. FluoZin-3 chelated 0.18 ± 0.01 Zn²⁺ in the dark state and 0.20 ± 0.01 Zn²⁺ per rhodopsin in the photoactivated state. While these data were not found to be statistically significant, a small light-sensitive trend similar to that observed with rhodopsin purified from COS-1 cells was consistently observed with rhodopsin from ROS. Nevertheless, these studies were able to confirm that the low-affinity portion of Zn²⁺ bound to rhodopsin was less than half a Zn²⁺ per rhodopsin molecule in both COS-1-expressed and ROS rhodopsin. As a result of the much higher stability of rhodopsin in ROS versus the dodecyl maltoside system used for rhodopsin expressed in COS-1 cells, the Zn²⁺ ion in the TM domain may be less accessible and detectable by FluoZin-3 upon light activation.

Treatment of Rhodopsin with ZnCl₂ Reveals One Stabilizing and One Destabilizing Zn²⁺ Coordination Site
Previous studies have used high concentrations of Zn²⁺ after rhodopsin purification thus accessing only the solvent accessible site. The TM domain is buried in a highly hydrophobic region and only exposed either during protein folding or upon photoactivation, as observed with FluoZin-3 chelatability. To further support our hypothesis that the Zn²⁺₃ site and coordination of Zn²⁺ within the transmembrane domain is physiologically relevant, we both pretreated cells immediately following transfection (to promote access to the Zn²⁺₃ coordination site during protein folding and processing) in addition to the conventionally used posttreatment procedure (after rhodopsin purification). Control samples were left untreated. UV-visible absorption spectra, obtained from the three rhodopsin populations (untreated, pretreated (50 µM ZnCl₂ added to COS-1 cells), or posttreated (50 µM ZnCl₂ added to purified protein)) show identical formation of a 500 nm peak (not shown). Upon photoactivation, all three populations also show identical formation of a 380 nm peak (not shown). Immunoblotting of rhodopsin showed that, at concentrations as high as 50 µM ZnCl₂ in cell culture, no change in the expression or glycosylation of rhodopsin protein was observed (not shown).

Thermal stability of the rhodopsin dark state at 50 °C was measured as a rate of decay of the 500 nm absorbance (i.e. release of 11-cis-retinal) over time. The wild type rhodopsin dark state decayed with a k rate constant of 0.024 ± 0.001 min^-1 (Fig. 3A and Table I). Pretreatment of COS-1 cells with 10 µM ZnCl₂ consistently and significantly slowed the rate of rhodopsin decay to 0.021 ± 0.001 min^-1 (p < 0.05), while pretreatment with 50 µM ZnCl₂ accelerated the decay rate to 0.030 ± 0.001 min^-1 (p < 0.001; Fig. 3A and Table I). Posttreatment of rhodopsin protein with 10 µM ZnCl₂ increased the decay rate to 0.027 ± 0.002 min^-1, although t test analysis confirmed these data as not statistically significant. Posttreatment of rhodopsin protein with 50 µM ZnCl₂ markedly and significantly increased the decay rate to 0.052 ± 0.006 min^-1 (p < 0.01; Fig. 3A and Table I).

View larger version (36K): [in this window] [in a new window]   FIG. 3. A, thermal stability of rhodopsin treated with ZnCl2. , rhodopsin; , rhodopsin from COS-1 cells pretreated with 10 µM ZnCl2; •, purified rhodopsin posttreated with 50 µM ZnCl2. B, thermal stability of rhodopsin treated with EDTA. , rhodopsin; , rhodopsin treated with 1 mM EDTA. C, thermal stability of rhodopsin purified at varying pH levels. , pH 5.5; , pH 7.4; •, pH 8.0; , pH 8.5. D, thermal stability (t) as a function of buffer pH. GraphPad Prism software was used to fit the curve and to identify the half-way point. All experiments were performed at least n = 3 times, using separate protein preparations. E, to determine selectivity of the high-affinity Zn2+ binding site, pretreatment of wild type rhodopsin with 10 µM ZnCl2, CaCl2, CoCl2, and CuCl2 were performed and thermal decay rate constants (k) determined (*, p < 0.05; **, p < 0.01) on the purified protein.

View this table: [in this window] [in a new window]   TABLE I Thermal stability calculations for rhodopsin and rhodopsin mutant proteins Statistical significance for treatments with zinc, EDTA, and altered pH is compared with non-treated sample of same rhodopsin population (i.e. wild type versus wild type + 1 mM EDTA). Statistical significance (for mutants at pH 7.4) is compared with wild-type rhodopsin at pH 7.4 (i.e. wild type versus Glu122 Cys). ND, not detectable. ns, not statistically significant.

These data suggested the presence of a stabilizing Zn²⁺-selective coordination site whose occupancy could be detected with a 10 µM ZnCl₂ pretreatment and a destabilizing Zn²⁺ coordination site whose occupancy could be detected with a 50 µM ZnCl₂ posttreatment. These findings also indicated that only ZnCl₂ pretreatment, and not posttreatment of purified protein, allowed Zn²⁺ coordination at the stabilizing site. As we initially hypothesized, only the highly hydrophobic Zn²⁺₃ coordination site (Fig. 1A) would be inaccessible to ZnCl₂ posttreatment of purified rhodopsin. Altogether, our data suggested that the Zn²⁺₃ coordination site within the transmembrane domain was a higher affinity, protein-stabilizing site, while one of the solvent-exposed (Fig. 1A, Zn²⁺₁, Zn²⁺₂, and Zn²⁺₄) coordination sites was a lower affinity, protein-destabilizing site. To further support our hypothesis we used biochemical and mutagenic techniques to remove these coordination sites.

Removal of Zn²⁺ from a Solvent-exposed Zn²⁺ Coordination Site Stabilizes the Rhodopsin Dark State
To support the presence of a low-affinity, solvent-accessible, destabilizing site, we treated purified rhodopsin with EDTA, a strong Zn²⁺ chelator. We detected no fluorescence of FluoZin-3 in rhodopsin samples treated with 1 mM EDTA (pH 7.4), suggesting that EDTA chelated all Zn²⁺ that were solvent-accessible (not shown). Removal of Zn²⁺ from the solvent-accessible Zn²⁺ coordination site stabilized rhodopsin at 50 °C, as treatment with EDTA increased the thermal stability t of rhodopsin to greater than 60 min (Fig. 3B and Table I). While untreated rhodopsin began to immediately decay at 50 °C, EDTA-treated rhodopsin did not decay for the first 45 min. The protein then began to degrade at a rate constant of 0.025 ± 0.006 min^-1, similar to untreated rhodopsin (Fig. 3B and Table I). As EDTA chelates other divalent cations we also examined the use of pH.

The solvent-exposed Zn²⁺ coordination sites, Zn²⁺₁ and Zn²⁺₄, each has a histidine residue as a critical coordinating ligand (Zn²⁺₂ does not). Since protonated histidine has a much lower affinity for Zn²⁺, we altered solvent pH levels near the pK_a of histidine (pH 6.5) to determine whether coordination of Zn²⁺ occurred at one of these two sites. When we purified rhodopsin at pH 5.5 and 6.0 (equilibrium shifted toward protonated histidine), rhodopsin exhibited no detectable decay in the dark state for a period of at least 120 min (Fig. 3C and Table I). Purification of rhodopsin at pH 7.4, 8.0, and 9.0 (equilibrium shifted toward unprotonated histidine) destabilized rhodopsin in a pH-dependent manner, increasing the decay rates to 0.024 ± 0.001 min^-1 (p < 0.001), 0.040 ± 0.001 min^-1 (p < 0.001), and 0.049 ± 0.002 min^-1 (p < 0.001), respectively (Fig. 3C and Table I). In Fig. 3D, a plot of thermal decay t versus solvent pH displays a half-point at pH 6.7 ± 0.3, similar to the pK_a of the histidine side chain (pH 6.5). These data suggest that rhodopsin thermal stability is directly dependent on the state of histidine protonation. These data further support our hypothesis that coordination of Zn²⁺ at the solvent-exposed Zn²⁺₁ or Zn²⁺₄ coordination site destabilizes the rhodopsin dark state. Furthermore, it also indicated that coordination of Zn²⁺ at the Zn²⁺₂ coordination site was most likely not physiologically relevant. This ascertainment is supported by the fact that Zn²⁺₂ has no histidine or cysteine residues, two amino acids commonly found to compose Zn²⁺ coordination sites.

Mutation of the Zn²⁺₃ Coordination Site Transmembrane Domain Residues His²¹¹, Glu¹²², and Trp¹²⁶ Alters Spectral Properties, 11-cis-Retinal Binding, and Rhodopsin Thermal Stability
Mutagenesis studies were performed on the highly conserved residues that were predicted to form and stabilize the putative Zn²⁺ coordination site within the transmembrane domain. Characterization of such mutations, utilizing a variety of experimental techniques, would provide critical information on the contribution of each residue to Zn²⁺ coordination.

His²¹¹—A crystal structure-based model (Fig. 1B) suggested that His²¹¹ was the primary Zn²⁺₃ coordinating residue. More interestingly, FluoZin-3 data of the His²¹¹ Cys mutant indicated the important role of this residue to Zn²⁺₃ affinity. Site-directed mutagenesis to His²¹¹ Cys (a residue still able to coordinate Zn²⁺) and His²¹¹ Phe (a residue unable to coordinate Zn²⁺) was performed. His²¹¹ Cys and His²¹¹ Phe mutants bind 11-cis-retinal with an absorbance ratio lower than wild type, at both 2 and 25 °C (Fig. 4A and Table II). Fig. 4A also shows a shift in the _max from the wild type 500 to 493 nm, for both His²¹¹ Cys and His²¹¹ Phe. Such data suggest the destabilization and altered structure of the 11-cis-retinal binding pocket. Interestingly, the decrease in 11-cis-retinal binding was much more evident at the higher temperature (25 °C), indicating a potential role of this site in thermal stability of the native protein. Mutation to His²¹¹ Cys and His²¹¹ Phe also increased the thermal decay k rate constant from the wild type 0.024 ± 0.001 min^-1 to 0.157 ± 0.009 min^-1 (p < 0.01; Fig. 4B and Table I) and 0.201 ± 0.010 min^-1 (p < 0.01; data not shown), respectively.

View larger version (54K): [in this window] [in a new window]   FIG. 4. A, UV-visible absorption spectra of His211 mutant proteins. Black solid line, rhodopsin at 25 °C; dashed line, His211 Cys at 25 °C; gray solid line, His211 Phe at 25 °C. B, thermal stability of His211 Cys mutant protein. , rhodopsin; , His211 Cys; •, His211 Cys from COS-1 cells pretreated with 10 µM ZnCl2. C, UV-visible absorption spectra of Glu122 mutant proteins. Black solid line, rhodopsin at 25 °C; black dashed line, Glu122 Cys at 25 °C; gray solid line, Glu122 Leu at 25 °C. D, thermal stability of Glu122 Cys mutant protein. , rhodopsin; , Glu122 Cys; •, Glu122 Cys from COS-1 cells pretreated with 10 µM ZnCl2. E, UV-visible absorption spectra of Glu126 Asp mutant protein. Solid line, rhodopsin at 25 °C; dashed line, Trp126 Asp at 25 °C. F, thermal stability of Trp126 Asp mutant protein. , rhodopsin; , Trp126 Asp; •, Trp126 Asp from COS-1 cells pretreated with 10 µM ZnCl2. All experiments were performed at least n = 3 times, using separate protein preparations. Data were determined to be statistically significant using the unpaired t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Glu¹²²—Glu¹²² Cys (a residue still able to coordinate Zn²⁺) and Glu¹²² Leu (a residue unable to coordinate Zn²⁺) bind 11-cis-retinal with an absorbance ratio lower than wild type, at both 2 and 25 °C (Fig. 4C and Table II). In addition, there was a shift in the _max from the wild type 500 to 482 nm (Glu¹²² Cys) and 492 nm (Glu¹²² Leu; Table II). As with His²¹¹ mutants, the decrease in 11-cis-retinal binding was much more evident at the higher temperature (25 °C), indicating a potential role of Glu¹²² in thermal stability of the native protein. Glu¹²² Cys and Glu¹²² Leu mutants also increased the thermal decay rate to 0.041 ± 0.002 min^-1 (p < 0.01; Fig. 4D and Table I) and 0.038 ± 0.001 min^-1 (p < 0.001; data not shown), respectively. Pretreatment of cells expressing the Glu¹²² Cys mutant, with 10 µM ZnCl₂, improved the thermal stability of this rhodopsin mutant from a k rate constant of 0.041 ± 0.002 min^-1 to 0.029 ± 0.003 min^-1 (p < 0.05; Fig. 4D and Table I). While 10 µM ZnCl₂ pretreatment improved protein stability, a pretreatment of 50 µM ZnCl₂ showed no statistically significant effect (Table I). Such data indicate that, at 50 µM ZnCl₂ there is binding of Zn²⁺ at the lower affinity, destabilizing site as well. This hypothesis is supported by similar treatments in wild type rhodopsin, where a higher concentration of ZnCl₂ also bound to the solvent-accessible site. Such a hypothesis may also explain the absence of improved His²¹¹ Cys protein stability with additional ZnCl_2. Pretreatment of COS-1 cells expressing the Glu¹²² Leu mutant, with 10 or 50 µM ZnCl₂, showed no improvement in the decay rate of that rhodopsin mutant (data not shown). Posttreatment with ZnCl₂ did not improve the thermal stability of either Glu¹²² Cys (Table I) or Glu¹²² Leu (not shown).

Trp¹²⁶—Although tryptophan is unlikely to directly coordinate Zn²⁺, hydrophobic amino acids typically surround the metal binding site to facilitate metal coordination and to stabilize the site. The Trp¹²⁶ Asp mutant binds 11-cis-retinal with an absorbance ratio lower than wild type, at both 2 and 25 °C (Fig. 4E and Table II). In addition, there is a shift in the _max from the wild type 500 to 484 nm (Fig. 4E and Table II). Mutation of Trp¹²⁶ Asp also increased the rhodopsin decay rate to 0.070 ± 0.007 min^-1 (p < 0.05; Fig. 4F). Pretreatment of COS-1 cells expressing the Trp¹²⁶ Asp mutant, with 10 or 50 µM ZnCl₂, showed no improvement in the decay rate of that rhodopsin mutant (Fig. 4F). Posttreatment of Trp¹²⁶ Asp with either 10 or 50 µM ZnCl₂ also did not improve the thermal stability (data not shown).

Modeling of the Zn²⁺ Coordination Site in the Rhodopsin Transmembrane Domain
His²¹¹ and Glu¹²² are necessary for the full affinity of this Zn²⁺₃ coordination site. The thermal stability of His²¹¹ Cys and Glu¹²² Cys could be rescued by pretreating with 10 µM Zn²⁺ (although the results for the more severe His²¹¹ Cys had not reached statistical significance). Combined, these findings supported our FluoZin-3 and ICP-MS data showing a decrease in affinity for Zn²⁺ with His²¹¹ Cys. Molecular modeling of the Zn²⁺₃ coordination site (PDB ID: 1L9H [PDB] ) showed the structural basis for our results (Fig. 5). Computations performed by the Swiss PDB Viewer computer program proposed that mutation of Glu¹²² Cys would result in a Cys¹²² conformation that could not interact with Zn²⁺₃ (Fig. 5B). Such a conformation would account for the reduced Zn²⁺ affinity and reduced thermal stability that we observed with this mutant. However, an alternate conformation of Cys¹²² could coordinate Zn²⁺ more tightly (Fig. 5C). Our data suggest that, through the addition of exogenous Zn²⁺, we favored the formation of this alternate and more stable Cys¹²² conformation (Fig. 5C). Improvement in Zn²⁺₃ coordination correlated well with an increased thermal stability for this mutant. Pretreatment of Glu¹²² Leu did not show an improvement in thermal stability, as observed with the Glu¹²² Cys mutant. This was expected, since no conformation of leucine could participate in improved Zn²⁺ coordination.

View larger version (34K): [in this window] [in a new window]   FIG. 5. A, crystal structure-based model (PDB ID: 1L9H [PDB] ) of the Zn2+3 coordination site, as hypothesized in this study. Green dashed-lines represent putative hydrogen bonding. B, model of the Glu122 Cys Zn2+3 coordination site, as predicted by the Swiss PDB Viewer. C, model of an alternate Glu122 Cys Zn2+3 coordination site. The Swiss PDB Viewer was utilized to examine a second potential conformation of the mutant Cys122 residue. D, proposed model of a tetra-coordinated transition state, upon 11-cis-retinal isomerization. E, proposed model of the Zn2+3 site, immediately following His211 protonation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Glu¹²² and His²¹¹ Form a Zn²⁺₃ Coordination Site within the Transmembrane Domain—Formation of the Zn²⁺₃ coordination site is critical for rhodopsin dark state stability. Loss of Glu¹²², Trp¹²⁶, or His²¹¹ lowers the affinity of rhodopsin for Zn²⁺₃, resulting in a destabilized 11-cis-retinal binding pocket, altered spectral properties of the chromophore, and diminished retinal binding (Fig. 4 and Table II). His²¹¹ is obligatory to the formation of this Zn²⁺ coordination site, as any mutation of His²¹¹ results in a severely unstable protein. Previous studies have suggested the presence of such a zinc coordination site in the transmembrane domain. Fourier transform infrared studies on rhodopsin showed that the frequency of the C=O stretch of Glu¹²² was very low, indicating strong hydrogen bonding in the ground state (27). The most recent 2.6-Å rhodopsin crystal structure (PDB ID: 1L9H [PDB] ) identifies the nearest group with which Glu¹²² could hydrogen-bond, as the His²¹¹ imidazole nitrogen, 4.26 Å away (24), suggesting a water-mediated hydrogen bond or metal coordination rather than a direct hydrogen bond. Mutation of His²¹¹ Asn and His²¹¹ Phe caused large aberrations in the Fourier transform infrared spectral bands assigned to the Glu¹²² carboxyl side chain, as well as in bands that could not be attributed to the chromophore, His²¹¹, or Glu¹²², but rather to a"group X"(28). Additionally, the carbonyl group of Glu¹²² interacted directly with this group X. These studies support our model that Glu¹²² and His²¹¹ form the Zn²⁺₃ (group X) coordination site in rhodopsin (Fig. 1B). The importance of this coordination is further highlighted by the high degree of conservation. Glu¹²², Trp¹²⁶, and His²¹¹ are 80, 86, and 100% conserved, respectively, in vertebrate rhodopsin pigments. Interestingly, upon analysis of 227 PDB files displaying a Zn²⁺ coordination site, we found that a Zn²⁺ coordination site from the -toxin of Clostridium perfringens (PDB ID: 1CA1 [PDB] ) displayed a resemblance to the geometry assumed by the Zn²⁺₃ ion (His side chain-Glu side chain-backbone carbonyl). Such an observation suggests that the coordinated Zn²⁺₃ ion may play an important and novel catalytic role in rhodopsin.

Rhodopsin Loses Affinity for the Zn²⁺ Ion within the Transmembrane Domain in Meta II, Suggesting a Role in the Meta I-Meta II Transition—Past studies have concluded that more Zn²⁺ bind rhodopsin upon light activation, due to an increase in detected levels of Zn²⁺ (11, 12). An early study using atomic absorption spectrophotometry and chromatography showed that the stoichiometry of Zn²⁺ to rhodopsin from ROS was 0.28 in the dark state and 0.45 in the photoactivated state (12). Furthermore, Fourier transform infrared bands attributed to group X (discussed above) disappear in Meta II (28). We also report that the high-affinity transmembrane domain Zn²⁺ binding site is not detectable in the dark state, becoming exposed and of lower affinity upon light activation. As a result, we conclude that Zn²⁺₃ is an important transmembrane structural cofactor for the rhodopsin ground state that may be released and detectable only upon photoactivation.

It has been suggested that repositioning of the -ionone ring toward TM IV ultimately determines which activated state is assumed (27, 29, 30). Thus, structural changes in the region near Glu¹²² and His²¹¹ are required for proper repositioning of the -ionone ring (27). Previous studies have show a direct interaction between 11-cis-retinal and Gly¹²¹ in rhodopsin TM III (31–35). The increase in the size of Gly¹²¹ correlated directly with the increasing dark state activity. Additionally, a Gly¹²¹ Leu mutant was able to activate rhodopsin in the presence of -ionone alone, with no Schiff base formation. Based on our studies, upon photoactivation Gly¹²¹ is displaced along with its adjacent Glu¹²². We propose that such repositioning of Glu¹²² (3 Å away from the -ionone ring), would result in the association of the Glu¹²² carboxyl with Zn²⁺₃ (Fig. 5D). In support of this model, during Meta I, group X (Zn²⁺₃) undergoes a change in hydrogen bonding with an OH-bearing residue (28). The nearest –OH group for Zn²⁺₃ to associate with is the Glu¹²² carboxyl (4.28 Å). Zn²⁺ has no energetic barrier to multiple coordination geometries and can therefore assume four- or five-membered coordination with relative ease (36). In metalloenzymes, the coordination number of Zn²⁺ changes to stabilize high-energy intermediates and other transition states (37). An altered interaction between Glu¹²² and Zn²⁺₃ could modify the local pH, reduce the charge on Zn²⁺₃, and promote the Zn²⁺₃-catalyzed protonation of a nearby residue. Interestingly, protonation of His²¹¹ is coupled to the formation of Meta II (38). The midpoint of the Meta I-Meta II transition (pH 6.4) occurs near the pK_a of the histidine side chain (pH 6.5) (39). As a result, we propose that protonated His²¹¹ would lose the interaction with Zn²⁺₃ (Fig. 5E).

Our data suggest that, in the ground (dark) state, the Zn²⁺₃ coordination site in the rhodopsin transmembrane domain is a stabilizing mechanism, retaining rhodopsin in the inactive conformation and 11-cis-retinal as an inverse agonist. Upon photoactivation, this Zn²⁺ coordination is disrupted, promoting the dissociation of TM helix III from helices IV and V and allowing the unhindered formation of photoactivated intermediates. It has been hypothesized that protonated His²¹¹ interacted electrostatically with another amino acid to drive rearrangement of TM helices (38); our studies suggest that protonated His²¹¹ loses an interaction (with Zn²⁺₃) to drive this helical rearrangement. Interestingly, His²¹¹ is 100% conserved in vertebrate rhodopsin pigments, and Cys²¹¹ is 100% conserved in vertebrate cone pigments. The conserved nature of this site raises the possibility that the Zn²⁺₃-mediated Meta II formation may be an acquired feature that accounts for the 100-fold difference in light sensitivity between rod and cone pigments (40). The fact that Zn²⁺ deficiency results in abnormal dark adaptation and night blindness in humans and other animals (41) further supports the critical role of this site in rhodopsin structure and function.

Night Blindness and Protein Misfolding in Retinitis Pigmentosa—Naturally occurring mutations His²¹¹ Pro and His²¹¹ Arg give rise to severe opsin misfolding, leading to retinitis pigmentosa, a serious retinal degenerative disorder (reviewed in Refs. 9, 42, and 43). Interestingly one of the first manifestations of this disorder is night blindness. Moreover, a large cluster of retinitis pigmentosa mutations are located in the region near His²¹¹. We have recently shown that two of these mutations, Leu¹²⁵ Arg and Ala¹⁶⁴ Val, directly interfere with a Glu¹²²-Trp¹²⁶-His²¹¹ interaction (25). We now propose that the disrupted Zn²⁺₃ coordination site in the rhodopsin transmembrane domain directly leads to the protein misfolding and night blindness associated with these mutations. A number of studies have shown that folding of the transmembrane domain is coupled to the formation of a specific tertiary structure and the native Cys¹¹⁰-Cys¹⁸⁷ disulfide bond in the intradiscal domain (42, 44–46). Since loss of the Zn²⁺₃ coordination site and loss of the Cys¹¹⁰-Cys¹⁸⁷ disulfide bond promote rhodopsin unfolding, it is exciting to speculate that formation of the Zn²⁺₃ site could be coupled to the formation of this critical disulfide bond. Combined, our studies strongly support the importance of zinc in rhodopsin structure and function and also provide a possible molecular mechanism for night blindness and retinitis pigmentosa. Additionally, improvement in protein stability of the Glu¹²² Cys mutant with the addition of Zn²⁺ further highlights the importance of this site and suggests a possible therapeutic role for Zn²⁺ in retinitis pigmentosa.

Potential Mechanism for Neurodegeneration from Zinc Excesses and Deficiencies—Zn²⁺ promotes aggregation of the highly fibrillogenic prion peptide, PrP106–126 (1), and of the -amyloid protein (2–4) into amyloidogenic aggregates, leading to cellular apoptosis and neurodegeneration. In contrast, in the mutant superoxide dismutase protein, loss of affinity for Zn²⁺ results in diminished protein activity (6), reduced protein stability (7), and formation of amyloid-like filaments (8). The mechanisms for these observations are not well understood, particularly as both the loss and gain of Zn²⁺ binding can induce neurodegeneration. We observe that both a loss of physiological Zn²⁺ binding and a gain of non-physiological Zn²⁺ binding can induce rhodopsin destabilization and misfolding through distinct mechanisms. Loss of a native high-affinity Zn²⁺ coordination site, either through Zn²⁺ deficiencies or induced by naturally occurring mutations, renders the rhodopsin protein unstable, misfolded, and eventually aggregated. Conversely, supraphysiological Zn²⁺ concentrations binding to a non-native, low-affinity Zn²⁺ coordination site in rhodopsin can also result in biochemical defects.

Selectivity of trace metals in neurodegeneration has recently gained increasing interest. Our studies confirm that the high-affinity metal coordination site in the TM domain is selective for Zn²⁺. Interestingly, at the solvent-accessible, low-affinity metal coordination site, recent studies suggest that high concentrations of either Zn²⁺ or Cu²⁺ 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. High concentrations of Cu²⁺, similar to Zn²⁺, also promote aggregation of the highly fibrillogenic prion peptide, PrP106–126 (1), and of the -amyloid protein (2–4) into amyloidogenic aggregates. Our current study may help clarify the mechanisms by which a loss and/or gain of metal binding results in a wide variety of neurodegenerative diseases.

	FOOTNOTES

 
^* This work was supported in part by grants from the Department of Pharmacology & Toxicology at the Dartmouth Medical School (to J. H.), the Karl Kirchgessner Foundation (to J. H.), a PhRMA Foundation Predoctoral Fellowship (to A. S.), an American Heart Association Predoctoral Fellowship (to J. S.), and by an Albert J. Ryan Foundation Fellowship (to A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This article was selected as a Paper of the Week.

To whom correspondence should be addressed: Dept. of Pharmacology & Toxicology, 7650 Remsen, Dartmouth Medical School, Hanover, NH 03755. Tel.: 603-650-1813; Fax: 603-650-1129; E-mail: John.Hwa{at}Dartmouth.edu.

¹ The abbreviations used are: TM, transmembrane; ICP-MS, inductively coupled-plasma mass spectrometry; Meta I, metarhodopsin I; Meta II, metarhodopsin II; PDB, Protein Data Bank; ROS, rod outer segment; WT, wild type.

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