Mapping the Ligand Binding Pocket in the Cellular Retinaldehyde Binding Protein*,

Zhiping WuDagger §, Yanwu Yang, Natacha Shaw||, Sanjoy BhattacharyaDagger , Lin YanDagger , Karen WestDagger , Karen Roth**DaggerDagger, Noa Noy||, Jun Qin, and John W. CrabbDagger §§§

From the Dagger  Cole Eye Institute and  Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, the § Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115, the || Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853, and the ** former Adirondack Biomedical Research Institute, Lake Placid, New York 12946

Received for publication, December 16, 2002, and in revised form, January 16, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Retinoid interactions determine the function of the cellular retinaldehyde binding protein (CRALBP) in the rod visual cycle where it serves as an 11-cis-retinol acceptor for the enzymatic isomerization of all-trans- to 11-cis-retinol and as a substrate carrier for 11-cis-retinol dehydrogenase (RDH5). Based on preliminary NMR studies suggesting retinoid interactions with Met and Trp residues, human recombinant CRALBP (rCRALBP) with altered Met or Trp were produced and analyzed for ligand interactions. The primary structures of the purified proteins were verified for mutants M208A, M222A, M225A, W165F, and W244F, then retinoid binding properties and substrate carrier functions were evaluated. All the mutant proteins bound 11-cis- and 9-cis-retinal and therefore were not grossly misfolded. Altered UV-visible spectra and lower retinoid binding affinities were observed for the mutants, supporting modified ligand interactions. Altered kinetic parameters were observed for RDH5 oxidation of 11-cis-retinol bound to rCRALBP mutants M222A, M225A, and W244F, supporting impaired substrate carrier function. Heteronuclear single quantum correlation NMR analyses confirmed localized structural changes upon photoisomerization of rCRALBP-bound 11-cis-retinal and demonstrated ligand-dependent conformational changes for residues Met-208, Met-222, Trp-165, and Trp-244. Furthermore, residues Met-208, Met-222, Met-225, and Trp-244 are within a region exhibiting high homology to the ligand binding cavity of phosphatidylinositol transfer protein. Overall the data implicate Trp-165, Met-208, Met-222, Met-225, and Trp-244 as components of the CRALBP ligand binding cavity.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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In vivo studies show that the cellular retinaldehyde binding protein (CRALBP)1 functions in the retinal pigment epithelium (RPE) as an 11-cis-retinol acceptor in the isomerization step of the rod visual cycle (1). Following illumination, CRALBP knockout mice exhibit over a 10-fold delay in rhodopsin regeneration, 11-cis-retinal synthesis, and dark adaptation relative to WT animals (1). In vitro CRALBP can also serve as a substrate carrier (2, 3), stimulating the enzymatic oxidation of 11-cis-retinol by RPE 11-cis-retinol dehydrogenase (RDH5) and retarding its esterification by lecithin:retinol acyl transferase. In addition, CRALBP is required for in vitro hydrolysis of endogenous RPE 11-cis-reinyl ester (4) and is a component of an RPE visual cycle protein complex where it may serve other physiological roles (3). CRALBP is expressed in RPE, retinal Müller cells, ciliary epithelium, iris, cornea, pineal gland, and oligodendrocytes of the optic nerve and brain. The function of the protein in tissues other than RPE is not known, however, CRALBP has also been implicated as being important in an alternate visual cycle for cone visual pigment regeneration possibly involving Müller cells (1, 5). Mutations in the human CRALBP gene causing retinal pathology are addressed in the accompanying report (6).

As part of ongoing efforts to define functional domains in this key visual cycle protein, we are characterizing CRALBP-ligand interactions and the structure of the retinoid binding pocket. Ligand interactions in this water-soluble, 316-residue protein are noncovalent and localized to C-terminal residues 120-313 (7, 8). Previous analyses of human recombinant CRALBP (rCRALBP) and site-directed mutants support residues Gln-210 and Lys-221 as components of the retinoid binding pocket (8, 9). Preliminary NMR studies have suggested that rCRALBP undergoes limited structural changes upon photoisomerization of bound 11-cis-retinal and that Met and Trp residues may be associated with these ligand-dependent conformational changes (9, 10). As depicted in Fig. 1, four of the seven methionines in rCRALBP and both tryptophans are located in the C-terminal retinoid binding domain. To identify Met and Trp in the ligand binding cavity, we describe here the preparation, retinoid binding, and structural properties of rCRALBP mutants W165F, W244F, M158A, M208A, M222A, and M225A.


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Fig. 1.   The primary structure of rCRALBP. The amino acid sequence of human rCRALBP is shown with the Met and Trp altered by site-directed mutagenesis in shadow font (Met-158, Met-208, Met-222, Met-225, Trp-165, and Trp-244). Other Met are in boldface. Boxed residues Gln-210 and Lys-221 were previously localized to the CRALBP retinoid-binding pocket (8). CRALBP residues 1-119 and 314-316 may be removed by limited proteolysis without disrupting retinoid binding (7). The underlined region exhibits high homology with the ligand binding cavity of yeast phosphatidylinositol-transfer protein (27). The N-terminal His tag fusion sequence is shown in italics (9). Numbering begins with the N terminus (Ser) of the native protein.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
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Materials-- Human rCRALBP was produced in bacteria as described elsewhere (9, 11). 11-cis-Retinal was obtained from NEI, National Institutes of Health, and 9-cis-retinal was purchased from Sigma.

Site-directed Mutagenesis-- Six human rCRALBP mutants carrying a single substitution (M158A, M208A, M222A, M225A, W165F, and W244F) were created as described for the Altered-Sites mutagenesis kit (Promega). Briefly, WT human CRALBP cDNA in the pET19b vector (9) was denatured and co-annealed with a mutagenic primer and a primer that removes an ScaI restriction site from the vector. Second-strand synthesis was then completed by the addition of ligase, polymerase, and dNTPs. The mutagenesis reaction was cleaved with ScaI prior to transformation into the mismatch repair-deficient Escherichia coli strain ES1301. Plasmid DNA from the overnight ES1301 culture was digested with ScaI and transformed into DH5alpha cells. Plasmid DNA from DH5alpha resistant to ScaI digestion was evaluated for the presence of the desired mutation by sequence analysis using the ABI PRISM Dye Terminator Cycle Sequencing kit and the model 373A DNA sequencer (PerkinElmer Life Sciences, Applied Biosystems). The following oligonucleotides were used for mutagenesis with the underlined nucleotides indicating altered sites: ScaI selection primer, 5'-AATGACTTGGTTGAGTATTCACCAGTCACAGAA-3'; M158A, 5'-GCCGAGTGGTCGCGCTCTTCAACATTG-3'; W165F, 5'-GGTCATGCTCTTTAATATTGAGAACTTCCAAAGTCAAG-3'; M208A, 5'-AAGGGCTTTACCGCGCAGCAGGCTGCTAGCCTCCGGACTTC-3'; M222A, 5'-GATCTCAGGAAGGCGGTCGACATGC-3'; M225A, 5'-AGGAAGATGGTGGACGCGCTCCAGGATTCCTT-3'; W244F, 5'-ATCCACCAGCCATTCTACTTCACCACGA-3'. The sequence of each mutated cDNA was determined by automated DNA sequence analysis for both strands prior to transformation into E. coli strain BL21(DE3)LysS for the expression of the rCRALBP mutants.

Purification of Apo-rCRALBP-- To minimize work in the dark, apo-rCRALBP was purified under ambient room light prior to the addition of retinoid using a slightly modified procedure. Bacterial cell pellets (~3 g) containing rCRALBP were suspended in 9 ml of lysis buffer (50 mM sodium phosphate buffer, pH 7.8, 300 mM NaCl), incubated with DNase (2 units/per ml) at room temperature for 30-40 min, sonicated, and centrifuged at 22,000 × g for 1 h. The soluble cell lysate was incubated with 1 ml of agarose nickel affinity support (nickel-nitrilotriacetic acid, Qiagen) for 1 h, then the resin was washed with 2 liters of lysis buffer followed by 1 liter of lysis buffer containing 40 mM imidazole. rCRALBP was eluted with 20 ml of lysis buffer containing 250 mM imidazole; 1-ml fractions were collected, and the first 6-8 fractions were pooled, yielding 4-7 mg of purified rCRALBP. Alternatively, microgram amounts of apo-protein were purified using nickel-nitrilotriacetic acid silica spin columns as previously described (8). Following retinoid labeling, holo-rCRALBP was protected from light to prevent ligand photoisomerization.

Amino Acid Analysis, Electrophoresis, and Protein Quantification-- Phenylthiocarbamyl amino acid analysis was performed using an Applied Biosystems model 420H/130/920 automated analysis system (12). SDS-PAGE was performed according to Laemmli on acrylamide gels using a Mini-Protein II system (Bio-Rad). Protein was quantified using the Bio-Rad protein assay (13); for measuring rCRALBP, the Bio-Rad assay was calibrated with rCRALBP previous quantified by amino acid analysis.

Mass Spectrometry-- The masses of intact mutant rCRALBP (~5.0 µg of each) were determined by MALDI-TOF MS using a PE Biosystems Voyager DE Pro MALDI-TOF mass spectrometer with WT human rCRALBP and bovine serum albumin as external and internal calibration standards, respectively (14). Mutant rCRALBP were digested with trypsin or endoproteinase AspN and analyzed by MALDI-TOF MS with delayed extraction in reflector mode and with synthetic peptides as internal calibration standards (15, 16). For all MALDI-TOF MS, alpha -cyano-4-hydroxycinnamic acid was used as matrix (~5 mg/ml in acetonitrile/water/3% trifluoroacetic acid, 5:4:1), and each spectrum was accumulated for at least 250 laser shots. Tandem mass spectrometry was performed using the PE Sciex API 3000 triple quadrupole electrospray instrument fitted with a nanospray interface (Protana). Tryptic digests were eluted from ZipTips (Millipore) in 75% acetonitrile, 0.02% trifluoroacetic acid, and 2-5 µl sample volumes were infused at ~50 nl/min through gold-coated glass capillaries (4-µm inner diameter, New Objectives, Inc.) (15). Precursor ions were selected by their m/z value in Q1, and the resulting fragments were analyzed in Q3. The spectra was acquired in positive ion mode using a step size of 0.2 Da and 0.4-ms dwell time.

Retinoid Labeling and Analysis-- Purified WT or mutant apo-rCRALBP was labeled in the dark with either 11-cis-retinal or 9-cis-retinal by incremental addition of retinoid with mixing between additions to a total of about 1.2-fold molar excess retinoid over rCRALBP (11). After ~45 min of incubation, excess retinoid was removed by Sephadex G-25 spin chromatography (centrifuged at 2000 × g for 2 min). Bleaching was by exposure to ambient light for 20 min at 4 °C. Retinoid binding measurements by UV-visible spectrophotometry were performed with a Hewlett Packard 8453 diode array spectrophotometer. The ratios of the extinction coefficients for holo-rCRALBP with bound 11-cis-retinal (epsilon 280/epsilon 425 = 3.2) or 9-cis-retinal (epsilon 280/epsilon 400 = 2.2) were used to estimate rCRALBP retinoid binding stoichiometry from observed A280/A425 or A280/A400 absorbance spectral ratios, respectively (8, 9).

Fluorescence spectroscopy was used to measure rCRALBP affinity for retinoid ligand. The WT and mutant apo-proteins were excited at 280 nm, and tryptophan fluorescence emission was monitored at 340 nm (using a SPEX Industries Fluorolog 2 spectrofluorometer). Titrations with 11-cis- or 9-cis-retinal monitored the decrease in the intrinsic fluorescence of the apo-protein (0.5 µM rCRALBP), and equilibrium dissociation constants were calculated from the titration data as described previously (8, 17).

Analysis of Substrate Carrier Function-- Human recombinant 11-cis-retinol dehydrogenase (rRDH5) was expressed in Hi-5 insect cells using a baculovirus vector kindly provided by Dr. K. Palczewski (3, 18) and purified to apparent homogeneity by nickel affinity chromatography. RDH5 oxidation activity was measured at pH 7.5 using tritium-labeled 11-cis-retinol as the rCRALBP ligand according to Saari et al. (19). Tritium-labeled 11-cis-retinol was prepared by reducing 11-cis-retinal with triated sodium borohydrate (NaB(3H)4) (20). Kinetic parameters (Km and Vmax) were determined from Lineweaver-Burk plots using enzyme assays performed with purified rRDH5 and purified WT and mutant holo-rCRALBP as the sole source of retinoid substrate. Control assays with free retinoid as substrate were performed in the absence of any carrier protein.

Solution State Heteronuclear Single Quantum Correlation NMR-- 15N uniformly labeled WT rCRALBP was prepared by biosynthetic incorporation in E. coli strain BL21(DE3)LysS grown in defined minimal media containing 1 g/liter 15N ammonium chloride as the sole nitrogen source plus M9 salts, 2 mM MgCl2, 100 µM CaCl2, 1 µM FeCl3, 50 µM ZnSO4, 10 µg/ml biotin, 10 µg/ml folic acid, 0.1 µg/ml riboflavin, 5 µg/ml thiamine, and 20% glucose in D2O (10). [15N]Methionine-labeled rCRALBP was prepared by biosynthetic incorporation in the same E. coli strain grown in a modified M9 minimal media. Bacterial cells were first grown in LB media to mid log phase (A600 ~ 0.5) then centrifuged, resuspended, and grown in defined minimal media containing 2× M9 salts plus 1% casamino acid, 2 mM MgSO4, 100 µM CaCl2, 0.04% glucose, 1% glycerol, 1 g/liter vitamin B1, 10 mg/liter tryptophan, and 213 mg/liter [15N]methionine. For protein expression, when cultures reached mid log phase in minimal media, isopropyl-1-thio-beta -D-galactopyranoside was added to 0.5 mM, and the growth temperature was shifted from 37 °C to 25 °C. About 3 g of wet bacterial cell pellet was used for purification of 15N-labeled apo-rCRALBP by nickel affinity chromatography under ambient light conditions. Following labeling with 11-cis-retinal and removal of excess retinoid by Sephadex G-25 spin chromatography, the holo-protein was concentrated to about 0.3 mM by Centricon centrifugation in 50 mM phosphate buffer, pH 7.0, 100 mM NaCl, 1 mM dithiothreitol-EDTA. The holo-rCRALBP preparations were adjusted to 8% D2O (v/v) and transferred to 250-µl microcell NMR tubes (Shigemi Inc., Allison Park, PA). All NMR experiments were performed at 25 °C with a Varian INOVA 500-MHz spectrometer equipped with a triple resonance probe (21). Sensitivity enhanced two-dimensional 1H-15N heteronuclear single quantum correlation experiments were recorded using water-flip-back for water suppression. Data was processed on a Sun UltraSPARC workstation using NMR Pipe and Pipp software (22, 23). Unless otherwise indicated, holo-protein preparations were maintained in the dark or under dim red illumination to prevent retinoid photoisomerization.

    RESULTS
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Expression and Structural Verification of rCRALBP Site-directed Mutants-- To probe the structure of the CRALBP retinoid binding pocket, Met residues Met-158, Met-208, Met-222, and Met-225 were substituted with Ala, Trp residues Trp-165 and Trp-244 were substituted with Phe, and the mutant and WT recombinant proteins were produced in bacteria and purified. Mutants W165F, M208A, M222A, and M225A were present in the soluble cell lysate in amounts comparable to WT rCRALBP. Mutants W244F and M158A were less soluble, and amounts were reduced ~50 and ~70%, respectively, relative to the WT protein (Fig. 2A). rCRALBP mutant M158A was recovered in insufficient yield for further characterization. All the other mutants and WT rCRALBP were purified to apparent homogeneity (Fig. 2B) and characterized by amino acid analysis (Table I) and MALDI-TOF mass spectrometry (Supplemental Table SI). These analyses show that the amino acid compositions and intact molecular weights of the mutants are in excellent agreement with the theoretically expected values. About 70% of each mutant protein sequence was confirmed by MALDI-TOF MS peptide mass mapping (not shown), including the peptides containing the substitutions (Supplemental Table SI). The M222A and M225A rCRALBP mutations were verified by electrospray MS/MS sequence analysis (Supplemental Fig. S1).


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Fig. 2.   SDS-PAGE analysis of mutant and WT rCRALBPs. A, soluble crude bacterial lysates (~10 µg of total protein) from cells expressing the indicated mutant or WT rCRALBP, and B, the purified proteins (~1 µg of protein) were analyzed by SDS-PAGE on 10% or 12% acrylamide gels, respectively, and stained with Coomassie Blue. The lower solubility of rCRALBP mutants M158A and W244F is apparent in panel A. The arrow in panel A indicates the rCRALBP band.


                              
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Table I
Amino acid compositions of human rCRALBP mutants
Compositions were determined by phenylthiocarbamyl amino acid analysis of the purified wild type and mutant proteins; Cys and Trp were not determined (12).

Retinoid Binding Analyses by UV-visible Spectroscopy-- Purified WT and mutant apo-rCRALBPs were incubated with either 11-cis-retinal or 9-cis-retinal, excess retinoid was removed, and UV-visible spectra were recorded for each holo-protein. Human WT rCRALBP exhibits characteristic absorption maxima (8, 9) at 425 nm when complexed with 11-cis-retinal and at 400 nm when complexed with 9-cis-retinal (Fig. 3). Upon bleaching the ligand absorbance maxima shift to ~380 nm due to the formation of unbound all-trans-retinal. Complexed with 11-cis-retinal, the chromophore absorbance maxima closely resemble that of the WT protein (Fig. 3) for rCRALBP mutants M208A (lambda max = 425.5 ± 0.3 nm, n = 4 separate preparations), W165F (lambda max = 424.3 ± 0.2 nm, n = 4), and W244F (lambda max = 424.3 ± 0.2 nm, n = 4). Binding stoichiometries of 0.9-1.0 mol of 11-cis-retinal were determined for these mutants (Fig. 3). Complexed with 9-cis-retinal, the ligand absorbance maxima for these mutants are similar (within ~7 nm) to that of WT rCRALBP (M208A lambda max = 400.5 ± 0.6 nm, n = 4; W165F lambda max = 393.3 ± 0.7 nm, n = 4; and W244F lambda max = 404.5 ± 0.8 nm, n = 4), but binding stoichiometries are estimated to be ~40% lower than for the WT protein (Fig. 3). Relative to WT rCRALBP, chromophore maxima are significantly shifted (~16-28 nm) toward the UV for mutants M222A and M225A complexed with either 11-cis-retinal (M222A lambda max = 396.3 ± 0.9 nm, n = 4; M225A lambda max = 408.4 ± 0.4 nm, n = 5) or 9-cis-retinal (M222A lambda max = 378.3 ± 0.4 nm, n = 4; M225A lambda max = 373.3 ± 1.1 nm, n = 4). Appropriate extinction coefficients for holo-rCRALBP at these wavelengths are unavailable, and ligand binding stoichiometries for M222A and M225A remain to be determined. Upon bleaching the expected shift in ligand absorbance to ~380 nm could be verified for all the mutants with both the 9-cis and 11-cis ligands except for M222A and M225A with 9-cis-retinal. Fluorescence titration results confirmed that M222A and M225A bind 9-cis-retinal.


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Fig. 3.   Retinoid binding analysis of mutant and WT rCRALBP. UV-visible absorption spectra are shown for the indicated mutant and WT purified rCRALBPs before and after exposure to bleaching illumination. Approximate binding stoichiometries are: WT, 1.0 mol for both 11-cis-retinal and 9-cis-retinal; M208A, W165F, and W244F, 0.9-1.0 mol for 11-cis-retinal and 0.6 mol for 9-cis-retinal. Major shifts in chromophore maxima obscure binding stoichiometries for M222A and M225A.

Retinoid Binding Analyses by Fluorescence Spectroscopy-- Apparent equilibrium dissociation constants (Kd) of complexes of mutant rCRALBPs with 11-cis- or 9-cis-retinal were determined by fluorescence titration of the apo-proteins, monitoring the decrease in the intrinsic fluorescence of the protein upon ligand binding (Table II). Except for mutant W165F, where ligand affinities were weak and undetectable as measured, the apparent Kd values for mutant M208A, M222A, M225A, and W244F and WT rCRALBP were in the nanomolar range for 11-cis- and 9-cis-retinal, consistent with previous analyses and within experimental error for the methodology (8). However, relative to WT rCRALBP, the determined Kd values for the mutants complexed with 11-cis-retinal were 2.0- to 2.6-fold higher and, when complexed with 9-cis-retinal, between 1.3- and 2.7-fold higher. The measurements demonstrate that all the mutants exhibit weaker affinity for both these ligands than the WT protein.


                              
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Table II
Equilibrium dissociation constants of rCRALBP with retinoids
Mean Kd values ± S.E. are shown for n titrations.

Kinetic Properties of rRDH5 Interaction with Mutant rCRALBPs-- Kinetic parameters of recombinant 11-cis-retinol dehydrogenase (rRDH5)-catalyzed oxidation of 11-cis-retinol were compared using free retinoid or rCRALBP-bound retinoid as substrate (Table III). When rRDH5 was assayed with WT rCRALBP, about 3-fold lower Km values and ~20% higher Vmax values were obtained relative to assays with free retinoid as substrate. Mean Km values determined for the rRDH5-catalyzed oxidation of 11-cis-retinol bound to rCRALBP mutants W165F, M208A, M222A, M225A, and W244F were similar to that for WT rCRALBP, but 8-24% greater, suggesting lower rRDH5 affinity for mutant-bound substrate (Table III). Average Vmax values determined for the enzyme reaction with the mutants were 10-29% slower than for the WT protein, and mutants M222A, M225A, and W244F yielded Vmax values slower than the reaction with free retinoid. Statistically, the determined Km for M225A and the Vmax values for M222A, M225A, and W244F are significantly different than the values for WT rCRALBP (Student's t test assuming equal variance, p values = 0.001-0.04, single-sided).


                              
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Table III
Kinetic parameters of mutant rCRALBP substrate carrier function for rRDH5
Enzymatic oxidation of rCRALBP-bound 11-cis-retinol was measured with purified rRDH5 as described under "Experimental Procedures." Mean Km and Vmax values ± S.D. are shown for n = 4 determinations.

Structural Analyses by Heteronuclear NMR-- To further explore CRALBP-ligand interactions, HSQC NMR analyses were performed with mutants M208A, M222A, and M225A labeled with [15N]Met and with mutant W165F and WT rCRALBP uniformly labeled with 15N. The biosynthetic incorporation of [15N]Met was verified by MALDI-TOF mass spectrometry prior to NMR analysis as illustrated in Supplemental Fig. S2. Monoisotopic peak intensities for Met-containing peptides were compared with and without isotopic labeling; increased intensities of the 15N-containing isotopic peak after biosynthetic incorporation was used to demonstrate significant labeling with [15N]Met. Significant 15N incorporation during uniform labeling of W165F and WT rCRALBP was also achieved, evidenced by a substantial increase (~1.9 kDa) in the molecular mass of the intact proteins (not shown).

Assignment of rCRALBP residues Met-208, Met-222, and Met225 in HSQC NMR spectra was achieved by comparing mutant and WT spectra using the [15N]Met labeled proteins. Fig. 4A shows the HSQC spectrum of [15N]Met labeled WT rCRALBP, which reveals seven major signals predicted to correspond to the seven Met in rCRALBP. Each of the mutant spectra (Fig. 4, B-D) lack one signal that corresponds to the altered Met residue. Superpositioning of the spectra allowed definitive assignment of the Met-208, Met-222 and Met-225 amide NH signals and tentative, random assignment of the other four Met in HSQC spectra of WT rCRALBP (Fig. 4A).


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Fig. 4.   Assignment of Met in HSQC NMR analysis of rCRALBP. HSQC NMR spectra are shown for [15N]Met-labeled WT rCRALBP and mutants M208A, M222A, and M225A with bound 11-cis-retinal. Each of the spectra for the mutants lacks one of the signals in the spectra for WT rCRALBP corresponding to the altered Met residue thus allowing assignment of Met-208, Met-222, and Met-225 in panel A. Other major signals are randomly labeled Ma, Mb, Mc, and Md and tentatively assigned to the other four methionines in WT rCRALBP. The asterisks denote unknown minor signals, two of which are paired with strong Met signals suggesting that they originate from a minor component of the protein containing [15N]Met.

Assignment of rCRALBP residues Trp-165 and Trp-244 was based on characteristic Trp side-chain NH signals in the downfield chemical shift region. Only one Trp signal exists in the HSQC spectrum of uniformly 15N-labeled mutant W165F (Supplemental Fig. S3), allowing assignment of this signal to Trp-244. WT rCRALBP contains two Trp and uniformly 15N-labeled WT rCRALBP HSQC NMR spectra contain two downfield signals characteristic of Trp, one at about 10.7/133.5 ppm for Trp-244 and the other at about 10.85/136 ppm for Trp-165 (Fig. 5).


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Fig. 5.   HSQC NMR analysis of 15N-rCRALBP with and without bound 11-cis-retinal. This experiment correlates directly bonded 1H-15N pairs in WT rCRALBP. The 1H-15N correlation spectrum for rCRALBP with bound 11-cis-retinal was recorded in the dark (red). The sample was then exposed to bleaching illumination and re-analyzed to obtain the correlation map without bound ligand (blue). The majority of the signals were unaffected by ligand isomerization, however, about 6-12% were perturbed, indicating that rCRALBP undergoes a localized conformational change upon removal of the retinoid ligand. Arrows highlight Met-208, Met-222, Met-225, Trp-165, and Trp-244 before bleaching, and all these residues except Met-225 clearly exhibit chemical shifts of varying degrees upon bleaching. Arrows also indicate tentative assignments for the other four Met in rCRALBP, three of which do not undergo chemical shifts upon bleaching (i.e. Ma, Mb, and Mc). Residue assignments were determined in Fig. 4 and Supplemental Fig. S3.

HSQC NMR spectra before and after bleaching of 15N uniformly labeled WT rCRALBP with bound 11-cis-retinal are shown in Fig. 5. Most residues remain unchanged upon bleaching, however, some 20-40 residues exhibit significant chemical shift changes. Ligand-dependent conformational changes appear to be associated with Met-208 and Met-222, which both underwent significant chemical shift changes, and with Trp-165 and Trp-244, which exhibit small chemical shift changes upon bleaching. The Met-225 amide NH signal is situated in the central crowded area of the spectra and difficult to interpret. Other evidence (Fig. 3, Tables II and III) suggests that Met-225 may also interact with ligand. Three tentative Met resonances do not undergo chemical shifts upon bleaching and may correspond to Met-(-1), Met-9, and Met-68 (Fig. 1), because they are not required for ligand binding (7). Met resonance Md does change upon bleaching and may correspond to Met-158.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Retinoid interactions determine the functions of CRALBP in the visual cycle. The visual cycle is the enzymatic processes by which the light-absorbing chromophore 11-cis-retinal is regenerated from the all-trans isomer following bleaching of rod and cone visual pigments. Although many details of the rod visual cycle are understood, the retinoid isomerization chemistry remains controversial (2, 24, 25). Possible mechanisms for cone visual pigment regeneration are only now emerging (5). Photoisomerization of 11-cis- to all-trans-retinal occurs in the photoreceptor cells, however, the 11-cis isomer is synthesized in the RPE for rhodopsin regeneration and perhaps in Müller cells for cone visual pigment regeneration (5). CRALBP is expressed in both the RPE and Müller cells and may serve as an 11-cis-retinol acceptor and substrate carrier in cone pigment regeneration as it does in the rod visual cycle (1, 5). As an approach to better understanding visual cycle mechanisms, we are identifying CRALBP functional domains and characterizing CRALBP ligand interactions. Preliminary NMR studies suggested that Met and Trp residues undergo ligand-dependent conformational changes (9, 10). Here we identify Met and Trp residues that appear to participate in CRALBP ligand interactions.

Recombinant CRALBP mutants M208A, M222A, M225A, W165F, and W244F were produced in bacteria and purified to apparent homogeneity, and their primary structural integrity was confirmed by amino acid analysis and mass spectrometry. The solubility of the mutant rCRALBPs was comparable to that of the WT protein except for W244F and uncharacterized mutant M158A, which were significantly less abundant in the soluble cell fraction. All the tested mutant proteins bound 11-cis- and 9-cis-retinal and therefore were not grossly misfolded. Based on UV-visible spectra, M208A, W165F, and W244F bound 11-cis-retinal-like WT rCRALBP but bound 9-cis-retinal with small differences in chromophore absorbance maxima and lower apparent stoichiometries. Previously, lower binding stoichiometries for 9-cis-retinal but comparable UV-visible spectra with 11-cis-retinal were observed for rCRALBP mutants Q210R and K221A (8). Relative to WT rCRALBP, substantial shifts in ligand absorbance maxima were observed for mutants M222A and M225A complexed with either 11-cis- or 9-cis-retinal. Fluorescence titrations yielded apparent equilibrium dissociation constants for retinoid interactions with the mutants and WT rCRALBP, demonstrating that all the tested mutants exhibited lower affinities for 9-cis- and 11-cis-retinal relative to the WT protein. These measured alterations in absorption spectra and retinoid affinity suggest that Trp-165, Met-208, Met-222, Met-225, and Trp-244 may be closely associated or directly interact with the ligand. Substitution of Met-225 with Lys leads to human blindness (26), the molecular basis of which is probed in the accompanying report (6).

To evaluate the possible effect of Met and Trp on CRALBP substrate carrier function, we examined the kinetic parameters of the rRDH5-catalyzed oxidation of 11-cis-retinol complexed with the Met and Trp mutants. The greater Km value determined for the M225A mutant relative to the WT protein suggests that this substitution lowers the affinity of rRDH5 for rCRALBP-bound 11-cis-retinol. The effect of the mutations was more obvious with regard to rRDH5 reaction velocity, which was significantly decreased relative to WT rCRALBP for mutants M222A, M225A, and W244F. Furthermore, Vmax for the rRDH5 oxidation reaction with any of the mutants was equivalent to or lower than that with free 11-cis-retinol, whereas the Vmax for the reaction with WT rCRALBP was significantly greater than that with free retinoid. These results are consistent with the accompanying study of disease-associated rCRALBP mutants M225K and R233W (6). Overall, the kinetic analyses suggest that all the Met and Trp mutations may impair the substrate carrier function of CRALBP and strongly support the possibility of ligand interactions with CRALBP residues Met-222, Met-225, and Trp-244.

Two-dimensional HSQC NMR provides a very sensitive, nonperturbing technique for probing protein-ligand interactions by correlating directly amide-bonded 1H and 15N (21). Current HSQC NMR analyses of 15N uniformly labeled WT holo-rCRALBP before and after bleaching (Fig. 5) show a wide range of chemical shifts in the 1H and 15N dimension with very good resolution of the individual cross-peaks outside of the central crowded region. These signals are particularly useful for comparative analysis of the protein before and after light exposure. Most residues remained unchanged upon bleaching, whereas some 20-40 residues experienced significant chemical shift changes. These results confirm preliminary observations (10) suggesting that rCRALBP exhibits localized conformational changes upon photoisomerization of the retinoid ligand.

Previous one-dimensional NMR analyses of 19F-Trp- and [13C]Met-labeled WT rCRALBP suggested that Trp and Met residues might be involved in the interaction with retinoid (9, 10). The unique distribution of these residues in the protein provide potentially useful markers for mapping the ligand binding pocket by NMR. The HSQC NMR spectrum of [15N]Met-labeled WT rCRALBP was well resolved with seven major signals apparently corresponding to the seven Met in the protein (Fig. 4). Resonances for Met-208, Met-222, and Met-225 were definitively assigned by comparison with HSQC NMR spectra from the [15N]Met-labeled mutant proteins (Fig. 4). Three Met in the N-terminal region of rCRALBP (see Fig. 1) are not required for retinoid binding (7), and three apparent Met signals show no chemical shift upon bleaching (Fig. 5). A fourth Met, namely Met-158, may be part of the ligand binding pocket, but mutant M158A was not analyzed by NMR due to poor solubility and low yield. CRALBP contains two Trp, and resonances for Trp-165 and Trp-244 were assigned by comparison with HSQC NMR spectra of 15N-uniformily labeled WT rCRALBP and mutant W165F (Fig. 5 and Supplemental Fig. S3).

Upon bleaching of 15N-uniformily labeled WT rCRALBP, chemical shifts were clearly observed by HSQC NMR for Met-208, Met-222, Trp-165, and Trp-244 (Fig. 5), supporting possible ligand interactions for these residues. We predict Met-225 also undergoes a chemical shift upon bleaching, but detection was obscured by the crowded central region of the spectra. Comparison of HSQC spectra from [15N]Met-labeled WT rCRALBP before and after bleaching showed the disappearance of four amide signals after light exposure, including those for Met-208, Met-222, and Met-225 (not shown). The reason for the decreased signal intensity for these residues is not clear but may be due to aggregation of the apo-protein, which was more apparent with the [15N]Met-selectively labeled preparations (not shown). Overall, the HSQC NMR analyses strongly support possible ligand interactions with rCRALBP Met-208, Met-222, Trp-165, and Trp-244 and suggest similar interactions with Met-225.

In summary, we have found that Met-208, Met-222, Met-225, Trp-165, and Trp-244 influence CRALBP retinoid interactions and demonstrated that ligand-dependent changes in protein conformation are associated with most if not all of these residues. In further support of these ligand interactions, CRALBP residues Met-208, Met-222, Met-225, and Trp-244 are situated within a region exhibiting high homology to the ligand binding cavity in the crystal structure of phosphatidylinositol-transfer protein (8, 27). Including Gln-210 and Lys-221 (8), this study expands to seven the number of residues proposed as components of the CRALBP retinoid binding pocket. We anticipate over 20 residues will eventually be localized to this important functional domain, however, more definitive determination of the ligand binding pocket structure awaits crystallographic analysis.

    ACKNOWLEDGEMENT

We thank Dr. John C Saari for useful discussions and for reviewing the manuscript prior to publication.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants EY6603, EY14239, HL58758, and CA68150, by a Research Center grant from The Foundation Fighting Blindness, and by funds from the Cleveland Clinic Foundation. A preliminary report of this work was presented at The Annual Meeting of the Association for Research in Vision and Ophthalmology, April 29 through May 4, 2001, Ft. Lauderdale, FL (28).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. S1-S3 and Supplemental Table SI.

Dagger Dagger Present address: LaSalle School, 391 Western Ave. Albany, NY 12203.

§§ To whom correspondence should be addressed: Cole Eye Institute, i31, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-0425; Fax: 216-445-3670; E-mail: crabbj@ccf.org.

Published, JBC Papers in Press, January 20, 2003, DOI 10.1074/jbc.M212775200

    ABBREVIATIONS

The abbreviations used are: CRALBP, cellular retinaldehyde binding protein; HSQC, heteronuclear single quantum correlation; MALDI-TOF MS, matrix assisted laser desorption ionization time-of-flight mass spectrometry; RDH5, 11-cis-retinol dehydrogenase 5; RPE, retinal pigment epithelium; rCRALBP, recombinant CRALBP; WT, wild type.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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