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INTRODUCTION |
The cellular retinaldehyde-binding protein
(CRALBP)1 is thought to play
a fundamental role in vitamin A metabolism in the retina and retinal
pigment epithelium (RPE). Notably, mutations in the human CRALBP gene
can result in autosomal recessive retinitis pigmentosa (1). In
vitro CRALBP serves as a substrate carrier protein for enzymes of
the mammalian visual cycle, modulating whether
11-cis-retinol (11-cis-Rol) is stored as an ester
in the RPE or oxidized by 11-cis-Rol dehydrogenase to
11-cis-retinal (11-cis-Ral) for visual pigment
regeneration (2). In the RPE and Müller cells of the retina,
CRALBP carries endogenous 11-cis-retinoids, the isomers of
vitamin A utilized for phototransduction. However, CRALBP is not always
associated with a retinoid ligand and more than one physiological role
for the protein appears likely (3). The protein is also present in
ciliary body, cornea, pineal gland, optic nerve, brain,
transiently in iris, but not in
the rod and cone photoreceptors. CRALBP is expressed in developing
retina and RPE before the tissues contain 11-cis-retinoids
or the enzyme responsible for generating 11-cis-retinoids
(3). Apparently the protein serves functions unrelated to visual
pigment regeneration in brain and tissues not involved in the visual
cycle and may bind ligands other than retinoids.
CRALBP was first detected in retina about 20 years ago and shown to
carry 11-cis-Rol and 11-cis-Ral as endogenous
ligands (4, 5). Structure function studies have defined ligand
stereoselectivity and photosensitivity (6), developed a topological
and epitope map (7), established in vitro evidence for a
substrate carrier function in RPE (8, 9) and produced human recombinant
CRALBP (10, 11). The primary structures of bovine (12), human (13, 14),
and mouse CRALBP (15) have been determined and are ~87% identical in
protein sequence. Investigation of cis-elements and transcription factors required for tissue specific expression of CRALBP
are in progress (16).
As part of ongoing efforts to better understand the normal functions of
CRALBP, we are characterizing CRALBP-ligand interactions and the
retinoid binding pocket. Recent studies have localized the
retinoid-binding domain to the C-terminal region (17) and characterized
ligand interactions by NMR (18) and circular dichroism (11). The report
linking CRALBP mutations with autosomal recessive retinitis pigmentosa
also demonstrated that rCRALBP mutant R150Q lacks retinoid binding
function (1). Here we demonstrate that 11-cis-Ral does not
appear to be covalently attached to CRALBP. We further show by chemical
modification, site-directed mutagenesis, and retinoid binding analyses
that Gln-210 and Lys-221 are likely components of the CRALBP retinoid
binding pocket.
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EXPERIMENTAL PROCEDURES |
Materials--
Native bovine CRALBP was purified from frozen
bovine dark-adapted retinas (J. Lawson, Lincoln, NE) according to Saari
and Bredberg (19). Human recombinant CRALBP (rCRALBP) was produced in
Escherichia coli as described elsewhere (10, 11).
11-cis-Ral was obtained from the National Eye Institute, NIH
through R. Crouch and 9-cis-Ral was purchased from
Sigma.
Analysis of Covalent versus Noncovalent CRALBP Ligand
Interactions--
To explore the possibility that
11-cis-Ral was covalently attached to CRALBP by a Schiff
base linkage, CRALBP was labeled with
[3H]11-cis-Ral as described previously (9) and
mixed in the dark (7.5 µM final concentration of CRALBP)
at room temperature with either 50 mM NaBH4 in
50 mM Tris, pH 7.4, or 300 mM borane
dimethylamine in 5 mM MOPS, 30 mM sodium
acetate, 4 mM sodium phosphate, pH 4.0. Various denaturants
were then added in the dark to give the following final conditions: pH
4, pH 10, 1% SDS, 1% Triton X-100, 8 M urea, and 50%
ethanol. After 20 min of further incubation at room temperature, an
equal volume of ice-cold ethanol was added followed by 3 volumes of
hexane. The upper phase was removed, and the extraction with hexane
repeated. Radioactivity was determined in the combined upper phases by
liquid scintillation counting. Extraction of the retinoid from the
protein in the absence of reducing and denaturing agents served as the
control. For some experiments, the hexane extract was dried in a stream
of flowing argon, dissolved in 68% aqueous acetonitrile, and analyzed
by reverse phase HPLC (5). In a second approach exploring the possibility of a Schiff base linkage, a solution of CRALBP complexed with 11-cis-Ral was prepared in 10 mM MOPS
buffer at pH 7 and a UV-visible absorption spectrum obtained. SDS and
HCl were added to the solution under red illumination to give final
concentrations of 1% and pH 4, respectively. A second spectrum was
then obtained and a difference spectrum generated.
Identification of Solvent-accessible and -inaccessible Lysine
Residues by Chemical Modification--
Reductive methylation of native
bovine CRALBP with bound 11-cis-Ral was performed
essentially according to Longstaff and Rando (20). The CRALBP retinoid
complex was incubated overnight in the dark with 2 mM
formaldehyde and 20 mM pyridine/borane, 10 mM
PIPES at pH 6.5. After 24 h, fresh formaldehyde and
pyridine/borane were added. Spectral analysis after 48 h of
methylation demonstrated no change in the 425- and 280-nm absorbance,
indicating the protein-retinoid complex was still intact. The protein
was then denatured in the light by dialysis against 6 M
guanidine HCl, 0.1 M borate, pH 9, and acetylated with 4 mM [3H]acetic anhydride for 30 min at room
temperature followed by exhaustive dialysis to remove background
[3H] (21). To identify acetylation sites, the modified
CRALBP (20 µg) was digested with subtilisin in 25 mM
Tris-Cl, pH 8.5, 2 M urea at 37 °C for 2.5 h with
4% protease (w/w), peptides purified by RP-HPLC and radioactive
chromatography fractions characterized by Edman degradation and
electrospray mass spectrometry (ESMS). To measure the amount of
acetylation, the modified protein was quantified by amino acid analysis
and radioactive incorporation was determined by liquid scintillation
counting. Total acetylation was found to be about 2.9 mol of
[3H]acetate/mol of CRALBP based upon the specific
activity of [3H]acetic anhydride (~35 dpm/pmol).
Radioactive peptide recovery following RP-HPLC was estimated by
phenylthiohydantoin-derivative yield from Edman analysis of
chromatography fractions.
Site-directed Mutagenesis--
Four human rCRALBP mutants
carrying a single substitution (K152A, I162V, K221A, and K294A) were
prepared by co-annealing a mutagenic primer and a selection primer to
remove the ScaI restriction site from the wild type rCRALBP
pET19b vector essentially as described by Deng and Nicholoff (22) and
in the Altered-Sites Mutagenesis kit (Promega). The ScaI
selection primer was 5'-AATGACTTGGTTGAGTATTCACCAGTCACAGAA-3'. The K152A
mutagenic primer was 5'-CTCTCAGTCGGGACGCGTATGGCCGAGTGGTC-3'. The I162V
mutagenic primer was 5'-GTCATGCTCTTCAACGTTGAGAACTGGCAAAGT-3'. The K221A
mutagenic primer was 5'-ACTTCAGATCTCAGGGCGATGGTGGACATGCTC-3'. The K294A
mutagenic primer was 5'-GGGGGCACGCTGCCCGCGTATGATGGCAAGGCC-3'. rCRALBP
mutant Q210R was generated by sequential steps of PCR according to
Ausubel et al. (23) using the following primers: forward,
5'-CGTCGCGCTCCAGCGAAAGCGGTCCTCGCC-3'; reverse,
5'-GCGCTCATCGTCATCCTCGGCACCGTCACC-3'; 5'-mutagenic,
5'-ACCATGCAGCGGGCTGCTAGTCTCCGG-3'; 3'-mutagenic, 5'-ACTAGCAGCCCGCTGCATGGTAAAGCC-3'. Two partial cDNA fragments with
overlapping sequences were generated in the first PCR step using the
wild type rCRALBP pET3a vector as template with primer combinations
forward and 3'-mutagenic or reverse and 5'-mutagenic. The two fragments
were purified, blunted with T4 DNA polymerase (Promega), and combined
to generate the full-length mutant cDNA using primer combination
forward and reverse in the second PCR step. PCR was performed for 25 cycles on a model 480 Perkin-Elmer Cetus thermal cycler using the
following conditions: 94 °C for 1 min, 55 °C for 2 min, and
72 °C for 3 min. The final PCR product containing the Q210R
substitution was subcloned into the pCRII vector (Invitrogen) and
amplified in E. coli strain DH5
. For expression, the
insert was excised, subcloned into the pET19b vector (Novagen), and
transformed into E. coli strain BL21(DE3)LysS. Primers were
synthesized on a model 392 DNA/RNA synthesizer and the structures
of all mutants verified by automated DNA sequence analysis using a
model 373 DNA sequencer (Perkin-Elmer, Applied Biosystems
Division instrumentation) (1).
Purification of rCRALBP Using Ni-NTA Spin Columns--
Details
for producing and purifying milligram amounts of holo-rCRALBP have been
described elsewhere (10, 11). The following procedure was used for
purifying microgram amounts of either holo- or apo-rCRALBP. Ni-NTA
silica spin columns (Qiagen) were washed and equilibrated with spin
column buffer (50 mM sodium phosphate, pH 7.8, 300 mM sodium chloride) and used for purification of rCRALBP essentially according to the manufacturer. Bacterial cell pellets (0.3 g) containing rCRALBP were suspended in 1 ml of spin column buffer
containing 15 mM imidazole, sonicated, and centrifuged, and
the clarified soluble lysate fraction was transferred to another tube.
The clarified lysate (600 µl) was applied directly to a Ni-NTA spin
column and centrifuged at 2000 rpm for 4 min at room temperature. The
column was sequentially washed by centrifugation with 600-µl aliquots
of (i) the spin column buffer containing 15 mM imidazole,
(ii) the buffer containing 50 mM imidazole (2×), (iii) the
buffer containing 90 mM imidazole (2×), and (iv) finally eluted twice with 85 µl of the buffer containing 250 mM
imidazole. Apoprotein was purified under ambient room light conditions;
holoprotein was purified under dark room conditions with dim red
illumination to prevent photoisomerization of the retinoid. Buffer and
protein preparations were kept on ice during the purification procedure except during centrifugation steps. Approximately 150 µg of rCRALBP was typically recovered by spin column purification.
Retinoid Labeling and Analysis--
For purification of
holoprotein, either 11-cis-Ral or 9-cis-Ral
(~72 nmol or ~1.2-fold molar excess over rCRALBP) was incubated with the clarified bacterial lysate for 15 min at 4 °C in the dark
prior to spin column purification. For retinoid labeling of purified
apo-rCRALBP, either 11-cis-Ral or 9-cis-Ral
(1.2-fold molar excess over CRALBP) was incubated for 15 min at 4 °C
with the protein in the Ni-NTA spin column buffer containing 250 mM imidazole. Excess retinoid was removed from apoprotein
preparations prior to UV-visible absorbance measurements by molecular
sieve chromatography using Sephadex G-15. Retinoids were added from a
concentrated solution (1-2 mg/ml) in ethanol (11). Bleaching of
rCRALBP samples and retinoid binding measurements by scanning UV-visible spectrophotometry (with either a Hitachi U-2000
spectrophotometer or a Hewlett Packard 8452A diode array
spectrophotometer) were as described previously (11). The ratio of the
extinction coefficients for 11-cis-Ral-labeled rCRALBP
(
280/
425 = 3.2) and for
9-cis-Ral-labeled rCRALBP
(
280/
400 = 2.2) approximate the spectral
ratio values for rCRALBP fully saturated with these retinoids (11).
Observed absorbance spectral ratios
(A280/A425 and
A280/A400) were used to
estimate rCRALBP retinoid binding stoichiometries.
Fluorescence spectroscopy was also used to evaluate apo wild type and
apo mutant rCRALBP interactions with retinoid by monitoring the
quenching of tryptophan fluorescence upon ligand binding. The amount of
quenching is a function of the location and orientation of the ligand
relative to the tryptophan residues. The protein was excited at 280 nm,
and tryptophan fluorescence emission monitored at 340 nm (using a SPEX
industries fluorolog 2 spectrofluorometer). Titrations with
11-cis- and 9-cis-Ral were carried out by
monitoring the decrease in the intrinsic fluorescence of the apoprotein
(0.5 µM rCRALBP) and analyzed by fitting the data to an
equation derived from simple binding theory as described elsewhere
(24).
Amino Acid Analysis, Edman Degradation, Electrophoresis, and
Protein Quantification--
Phenylthiocarbamyl amino acid analysis was
performed using an Applied Biosystems model 420H/130/920 automated
analysis system (25), and Edman degradation was performed using an
Applied Biosystems gas phase protein sequencer model 470/120/900 (12).
To identify [3H]acetyl-lysine, sequencer fractions from
each Edman cycle were analyzed for tritium by liquid scintillation
counting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was performed on 12% acrylamide gels according to Laemmli
using a Mini-Protein II slab gel system (Bio-Rad). Apo-rCRALBP was
quantified using the Bio-Rad protein assay (26) calibrated with rCRALBP previously quantified by amino acid analysis.
Mass Spectrometry--
ESMS and liquid chromatography ESMS
(LC-ESMS) were performed with a Perkin-Elmer Sciex API 300 triple
quadrupole mass spectrometer (Concord, Ontario, Canada) fitted with an
articulated ion spray plenum and an atmospheric pressure ionization
source (27). Initial tuning and calibration was with a standard mixture
of polypropylene glycol from Perkin-Elmer Sciex. Resolution was
adjusted to about 50% valley between adjacent isotope peaks in a
singly charged cluster, allowing singly charged ions to be identified
by apparent spacing between peaks and doubly charged ions to be
distinguished from those with higher charge states. Nitrogen was used
as the nebulization gas (at 40 p.s.i.) and curtain gas and was
supplied from a Dewar (XL-45, Taylor Wharton) of liquid nitrogen
(Merriam-Graves, Claremont, NH). LC-ESMS were acquired in positive
ion mode at an orifice potential of 50 V. For LC-ESMS of intact
proteins a scan range of m/z 800-1750 was used
with 0.1 atomic mass unit steps and a scan time of 7.5 s; for
analysis of peptides a scan range of m/z
250-2200 was used with 0.25 atomic mass unit steps and a scan time of
6 s. Synthetic peptides of known mass were used to verify correct
calibration. RP-HPLC for LC-ESMS was performed at a flow rate of 5 µl/min on a Perkin-Elmer C18 capillary column (0.5 × 150 mm)
using an Applied Biosystems model 140D HPLC system and aqueous
acetonitrile/trifluoroacetic acid solvents. To verify the structures of
the mutant rCRALBPs (11), purified mutant rCRALBP (40 µg) were
reduced with dithiothreitol and alkylated with iodoacetamide in the
presence of 8 M urea, 400 mM ammonium bicarbonate, pH 8. The carboxyamidomethylated proteins were digested with trypsin (Promega) in 1 M urea, 50 mM
ammonium bicarbonate (2.5% trypsin by weight, overnight at 37 °C)
and the tryptic digest (~4 µg) analyzed by LC-ESMS. A portion
(50%) of the mutant I162V tryptic digest was further fragmented with
endoproteinase Asp-N (Boehringer Mannheim) (2% protease by weight,
6 h at 37 °C) under the same conditions and analyzed by
LC-ESMS. Following subtilisin digestion of modified bovine CRALBP,
portions of select chromatography fractions containing
[3H]acetyl-lysine were dried, resuspended in 0.2-5%
formic acid, 50% methanol and analyzed by infusion at 5 µl/min to
confirm the presence of modified amino acids.
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RESULTS |
CRALBP Ligand Interactions Are Noncovalent--
Attempts to trap a
putative Schiff base were based on previous studies (8, 28)
demonstrating that the aldehyde of 11-cis-Ral was not
accessible to water soluble reagents when bound to CRALBP. Reducing
agents (NaBH4, borane dimethylamine) were added in the dark
to a solution of [3H]11-cis-Ral bound to
CRALBP and then protein denaturants were added in the dark. Following a
20-min incubation period, retinoids were extracted into hexane and
radioactivity determined. The results of these experiment are shown in
Fig. 1. Nearly quantitative amounts of
retinoid were extracted from CRALBP regardless of the denaturant or
reducing agent (Fig. 1). HPLC analysis of the extracted retinoid from
several of the treatments at neutral pH showed that
[3H]11-cis-Rol was the only retinoid present
(not shown). Treatments at low pH resulted in considerable destruction
of the retinoid, but again 11-cis-Rol was the major product
detected. If the CRALBP was bleached with white light in the presence
of the reducing agents, the product of the reaction was
all-trans-Rol, as reported previously (5). In another
approach to identify a Schiff base, the absorption spectrum of CRALBP
complexed with 11-cis-Ral was obtained for the unbleached
native protein at pH 7 (Fig. 2). Under red illumination the pH of the solution was rapidly reduced to 4, SDS
was added to a final concentration of 1%, and a second spectrum
obtained. The spectra (Fig. 2) show a conversion of the 425-nm
absorbing species, characteristic of 11-cis-Ral bound to the
native protein, to a 380-nm species, characteristic of free 11-cis-Ral. These results do not support the presence of a
Schiff base because protonated Schiff bases of retinal absorb at
approximately 440 nm and are stable at low pH (29).

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Fig. 1.
Extraction of retinoids following treatment
of CRALBP with reducing agents and denaturants. CRALBP complexed
with [3H]11-cis-Ral was incubated with 50 mM NaBH4 (panel A) or 300 mM borane dimethylamine (panel B) under red
illumination. The indicated denaturants or additives were then added,
and the incubation continued for 20 min at room temperature. Retinoids
were then extracted into hexane and radioactivity determined. Samples
marked CRALBP contain the binding protein without denaturant or
reducing agent.
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Fig. 2.
Spectral analysis of CRALBP before and after
denaturation. A, the solid trace shows the
absorption spectrum of native CRALBP complexed with
11-cis-Ral at pH 7. The dashed trace shows the
spectrum of the protein at pH 4 in the presence of 1% SDS.
B, the difference spectrum obtained by subtracting the
solid trace from the dashed trace is shown.
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Identification of Buried and Solvent-exposed Lysine Residues in
CRALBP--
Native bovine CRALBP was reductively methylated then
denatured in 6 M guanidine HCl, radioactively acetylated,
fragmented with subtilisin, and the peptides fractionated by RP-HPLC
(Fig. 3A). Approximately 87%
of the radioactivity applied to the HPLC was accounted for in the
chromatography fractions (Fig. 3B). Acetylated residues were
identified by sequence and mass spectrometric analysis of the
radioactive chromatography fractions (Fig. 3B). Radioactive lysine residue 221 was identified in chromatography fractions 10 and 22 within peptide DLRKM (residues 218-222); it was acetylated but not
methylated based on ESMS analysis and therefore was completely inaccessible to solvent (observed mass = 703.5, calculated
mass = 703.3). Radioactive lysine residue 152 was identified in
chromatography fractions 10, 27, 28, and 30 within peptide residues
152-154, 148-152, 148-155, and 148-156. Radioactive lysine residue
294 was identified in chromatography fractions 35, 42, and 46 within peptide residues 287-294, 287-296, and 287-298. Peptides containing lysine residues 152 and 294 were found by ESMS to exhibit masses either
about 42 or 56 Da greater than the calculated masses for the unmodified
peptides, consistent with the presence of either N
-acetyl-lysine or
N
-acetyl-N
-methyl-lysine
(not shown). In addition, during Edman analysis, the
phenylthiohydantoin amino acid encountered at the lysine positions with
the larger mass modification eluted later than
phenylthiohydantoin-acetyl-lysine (not shown). These results suggest
that residues 152 and 294 may be partially inaccessible to solvent.
Thirteen of the 16 lysine residues were not recovered in peptides with
substantial radioactivity and are considered to be methylated and fully
exposed to solvent (Fig. 4). Lysine
residues fully accessible to solvent appear to exist in bovine CRALBP
at positions 27, 46, 48, 54, 90, 103, 185, 204, 235, 254, 258, 260, and
298.

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Fig. 3.
RP-HPLC fractionation of CRALBP subtilisin
peptides. The UV profile (A220)
(A) and the radioactivity profile (total cpm, 3H
acetylation) (B) from the primary fractionation of bovine
CRALBP subtilisin peptides (~20 µg, 555 pmol protein). RP-HPLC was
performed with an Applied Biosystem model 130 HPLC and 5 Vydac C18
column (2.1 × 250 mm) at 150 µl/min using the indicated
gradient. Solvent A was 0.1% trifluoroacetic acid and solvent B was
84% acetonitrile containing about 0.07% trifluoroacetic acid. Labeled
fractions were rechromatographed (not shown) and analyzed by Edman
sequencing and/or electrospray mass spectrometry.
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Fig. 4.
Lysine topological map of CRALBP. Total
incorporation of 3H from acetylation at each lysine residue
was estimated from Edman sequence analysis of chromatography fractions
in Fig. 3 and plotted versus CRALBP residue number. Tritium
incorporation was blocked by methylation at 13 solvent accessible
lysine residues in the native structure. Lys-152, Lys-221, and Lys-294
were acetylated following denaturation in 6 M guanidine
HCl, suggesting these residues are buried or partially buried within
the native protein structure.
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Mutagenesis Strategy and Structural Verification of rCRALBP
Site-directed Mutants--
A previous report (17) described a three
point rCRALBP mutant with reduced retinoid binding capability that
resulted from random PCR misincorporation and contained mutations at
residues 9 (Met to Thr), 162 (Ile to Val) and 210 (Gln to Arg).
Positions 162 and 210 are within the retinoid-binding domain (17), and therefore rCRALBP mutants I162V and Q210R were prepared to determine which of these substitutions influenced retinoid binding. To probe the
possibility that lysine may reside within the retinoid binding pocket
and influence ligand interactions (as in visual pigments and CRBP),
buried or partially buried lysine residues (Fig. 4) were substituted
with alanine and mutant rCRALBPs K152A, K221A, and K94A produced for
retinoid binding studies. Typical SDS-PAGE profiles of purified rCRALBP
mutants are shown in Fig. 5. All intact
mutants were structurally characterized by amino acid analysis (Table
I) and electrospray mass spectrometry
(Table II) and found to exhibit
compositions and molecular weights in excellent agreement with the
known values. Each mutation was confirmed by identification of peptides
containing the relevant substitutions (Table II).

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Fig. 5.
SDS-PAGE of purified wild type and mutant
rCRALBPs. SDS-PAGE analysis of wild type rCRALBP (WT)
and rCRALBP mutants I162V, Q210R, K152A, K221A, and K294A following
retinoid labeling and protein purification. Absorption spectra of these
protein preparations are shown in Figs. 6-8.
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Table I
Amino acid compositions of rCRALBP mutants
Compositions were determined by phenylthiocarbamyl amino acid analysis
of the purified mutant proteins and average error determined as
described elsewhere (25). Cys and Trp were not determined. WT
represents the wild type fusion rCRALBP (10, 11).
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Table II
Masses of human rCRALBP mutants and mutant peptides
Masses were determined by LC-ESMS as described under "Experimental
Procedures." Calculated masses are chemical average masses except for
those less than 2000, which are monoisotopic. Error refers to the
difference between the observed and calculated masses. Residues refer
to the fusion human rCRALBP structure (which contains an N-terminal
His-tag extension). Mutant designations reflect substitution positions
in the native protein. Mutant peptides are from tryptic digestion
unless marked with an asterisk (*). Peptide I162V* was from digestion
with trypsin and endoproteinase Asp-N. Substituted amino acid residues
are shown in bold italics within peptide sequences.
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Retinoid Binding Analyses by UV-visible Spectroscopy--
Retinoid
binding analyses performed by labeling rCRALBP with either
11-cis-Ral or 9-cis-Ral in bacterial lysates
followed by protein purification are shown in Figs.
6-8. Wild type rCRALBP absorption spectra
(Fig. 6) exhibit the characteristic chromophoric maxima at 425 nm for
bound 11-cis-Ral and at 400 nm for bound 9-cis-Ral. Upon exposure to light, the chromophore
absorbance maxima shift to ~380 nm because of the production of
unbound all-trans-Ral. rCRALBP mutant I162V was found to
bind both 11-cis-Ral and 9-cis-Ral in a manner
similar to wild type rCRALBP; mutant Q210R exhibits wild type-like
binding with 11-cis-Ral but reduced binding with 9-cis-Ral (Fig. 7). rCRALBP
mutants K152A, K221A, and K294A also exhibit wild type-like binding
with 11-cis-Ral, and mutants K152A and K294A exhibit wild
type binding with 9-cis-Ral (Fig.
8). Of the three lysine mutants, only
K221A exhibits reduced binding with 9-cis-Ral (Fig. 8).
Retinoid binding analyses with 11-cis-Ral and
9-cis-Ral were also performed with apo-rCRALBP by labeling the protein with retinoid after purification. Results of
UV-visible spectral analyses of the apoprotein preparations agree
overall with the results in Figs. 6-8. Approximate binding
stoichiometries obtained with the apo-rCRALBP preparations for
n = 2 measurements were: wild type, 1.0 mol
11-cis, 0.6 mol 9-cis; mutant I162V, 1.1 mol
11-cis, 1.0 mol 9-cis; mutant Q210R, 0.5 mol
11-cis, 0.3 mol 9-cis; mutant K152A, 0.8 mol
11-cis, 0.7 mol 9-cis; mutant K221A, 0.8 mol
11-cis, 0.3 mol 9-cis; and mutant K294A, 1.0 mol 11-cis, 0.8 mol 9-cis.

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Fig. 6.
Wild type rCRALBP absorption spectra.
UV-visible absorption spectra are shown for purified wild type fusion
rCRALBP before and after exposure to bleaching illumination.
Approximate binding stoichiometries are ~1.0 mol 11-cis
(maximum 425 nm) and ~1.0 mol 9-cis (maximum 400 nm). Upon
bleaching, the chromophore absorbance maxima shift to ~380 nm because
of photoisomerization of the retinoid and production of unbound
all-trans-Ral.
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Fig. 7.
Retinoid binding analysis of rCRALBP mutants
I162V and Q210R. UV-visible absorption spectra before and after
exposure to bleaching illumination are shown following retinoid
labeling in cell lysates and protein purification. Approximate binding
stoichiometries from the above spectra are: mutant I162V, 1.2 mol
11-cis; 0.7 mol 9-cis; mutant Q210R, 1.1 mol
11-cis; 0.3 mol 9-cis. Similar results were
obtained from at least three of each protein preparation.
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Fig. 8.
Retinoid binding analysis of rCRALBP mutants
K152A, K221A, and K294A. UV-visible absorption spectra before and
after exposure to bleaching illumination are shown following retinoid
labeling in cell lysates and protein purification. Approximate binding
stoichiometries from the above spectra are: mutant K152A, 1.1 mol
11-cis; 0.9 mol 9-cis; mutant K221A, 1.1 mol
11-cis; 0.3 mol 9-cis; and mutant K294A, 1.1 mol
11-cis; 0.7 mol 9-cis. Similar results were
obtained from at least three of each protein preparation.
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Retinoid Binding Analyses by Fluorescence
Spectroscopy--
Equilibrium dissociation constants
(Kd) of complexes of wild type and mutant rCRALBP
with 11-cis-Ral and 9-cis-Ral were measured with
multiple apoprotein preparations by fluorescence titrations monitored
by following the decrease in the intrinsic fluorescence of the proteins
upon ligand binding. Apparent Kd values extracted
from the titration data were in the nanomolar range for both retinoids
(Table III). Consistent with an earlier report (6), the fluorescence titrations (Table III) corroborate a lower
affinity of wild type rCRALBP for 9-cis-Ral
(Kd ~53 nM) than for
11-cis-Ral (Kd ~21 nM). The
titrations also show that the affinity of rCRALBP mutants Q210R and
K221A for 9-cis-Ral (Kd ~70
nM) is reduced relative to the wild type protein.
Extraction of binding sites from the titration data yielded average
number of binding sites = 0.4 ± 0.05 for both
11-cis-Ral (n = 38) and 9-cis-Ral
(n = 32).
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Table III
Equilibrium Dissociation Constants of rCRALBP with Retinoids
Mean Kd values ± S.E. are shown for
n titrations. Average values are from the combined
fluorescent titration data for all wild type and mutant rCRALBP
preparations and reflect the overall greater affinity of rCRALBP for
11-cis-Ral than for 9-cis-Ral.
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DISCUSSION |
Retinoid interactions determine the function of CRALBP in the RPE
where the protein is thought to serve a substrate carrier role in the
synthesis of 11-cis-Ral for visual pigment regeneration (2).
In vitro, CRALBP affects the partitioning of
11-cis-Rol between two competing enzymatic reactions;
esterification by lecithin:retinol acyltransferase is retarded, and
oxidation by RPE 11-cis-Rol dehydrogenase is stimulated (2).
Molecular features of ligand interactions in bovine CRALBP and several
mutants and wild type human rCRALBP have been compared using chemical
modification, UV-visible and fluorescence spectroscopy. These
comparisons reveal no covalent interactions between CRALBP and retinoid
and further define the structure of the retinoid-binding domain,
including topological properties and the identification of Gln-210 and
Lys-221 as residues likely within the ligand binding cavity.
Chromophore Absorbance and Lack of Evidence for a Schiff
Base--
The 11-cis-Ral in rhodopsin, the visual pigment
of rod photoreceptor cells, displays an absorption maximum of 493 nm,
red shifted relative to the maximum of the free retinoid in ethanol (383 nm). This bathochromic shift results in part from formation of a
protonated Schiff base with Lys-296 of opsin and positioning of the
counterion provided by the carboxylate of Glu-113 (30). The
CRALBP·11-cis-Ral complex exhibits an absorption maximum
of 425 nm (28), suggesting that a Schiff base mechanism might be involved in the bathochromic shift. The aldehyde of
11-cis-Ral bound to CRALBP is not accessible to water
soluble chemical-reducing agents (8). Thus, denaturation of holo-CRALBP
in the presence of water soluble reducing agents should expose a Schiff
base (if present) and result in covalent attachment of the retinoid to the protein via a secondary amine linkage. However, attempts to trap a
Schiff base were unsuccessful (Fig. 1). The retinoid was nearly
quantitatively extracted in hexane regardless of reducing agent or
denaturing agent, suggesting the lack of covalent interactions between
ligand and protein.
Schiff bases of 11-cis-Ral are stable at low pH where they
exhibit a spectral maximum of 444 nm (29). However, a rapid lowering of
the pH of a CRALBP solution (with red illumination) to pH 4 and
addition of SDS produced a spectrum of free 11-cis-Ral with an absorption maximum of 380 nm (Fig. 2). The possibility remains that
a Schiff base is present whose rate of hydrolysis is faster than its
rate of reduction. However, it is unlikely that a Schiff base would not
be trapped at low pH. Thus, 11-cis-Ral does not appear to be
complexed to CRALBP via a Schiff base, and the bathochromic shift in
chromophore absorbance from 380 to 425 nm must be accomplished by other
interactions.
What accounts for the CRALBP red shift in chromophore absorbance?
Mutagenesis studies have shown that the covalent bond between 11-cis-Ral and Lys-296 in rhodopsin is not essential for
ligand binding or formation of a long wavelength absorption maximum
when 11-cis-Ral is presented in the form of an exogenous
Schiff base with an n-alkylamine (31). Electrostatic
interactions with the counterion appear to be the stabilizing force in
the resulting noncovalent rhodopsin complex. In addition, recent
studies have shown hydroxyl-bearing amino acids increase the red shift
of 11-cis-Ral bound to human red and green cone pigments
(32). We speculate that electrostatic interactions and Ser, Thr, and
Tyr amino acids also influence chromophore absorbance in CRALBP
(11). [19F]-Trp NMR observations suggest a more open,
flexible protein structure when CRALBP is in the unliganded form
compared with the holo-form (11). Conformational mobility with
associated positioning effects of amino acid side chains
(e.g. hydroxyl and ionizable groups) also likely influence
CRALBP chromophore absorbance.
The CRALBP Retinoid-binding Domain and Topology--
Previously a
CRALBP retinoid binding fragment was identified and shown to retain the
425 nm absorbance maxima characteristic of CRALBP bound
11-cis-Ral (17). Limited chymotryptic proteolysis removes
119 residues from the N terminus and 3 residues from the C terminus of
holo-CRALBP, degrading about 39% of the protein and limiting the
retinoid-binding domain to 194 residues. As expected for a hydrophobic
ligand, the retinoid-binding domain contains more nonpolar than polar
residues. However, 10 of the 15 lysine in human CRALBP reside in the
retinoid-binding domain, and as determined by chemical modification
(Fig. 3), all but 1 (i.e. Lys-221) are solvent accessible.
The lysines at positions 152 and 294 may be located in flexible regions
of the molecule as peptides containing these residues were recovered
with double modifications (Fig. 4), suggesting they may be partially
exposed. An earlier topological study demonstrated that residues 30-99 and 176-229 in native bovine CRALBP were not accessible to several specific antipeptide antibodies (7). Current observations are of higher
resolution and supplement the previous study.
Retinoid Binding Properties of rCRALBP Mutants--
Two rCRALBP
mutants (I162V and Q210R) were prepared to identify substitutions
altering the retinoid binding properties of a PCR misincorporation
mutant (17) and three others (K152A, K221A, and K294A) to investigate
possible retinoid interactions with buried or partially buried lysine
residues (Figs. 3 and 4). The structural integrity of all the mutant
proteins was confirmed (Tables 1 and 2; Fig. 5) and their retinoid
binding properties evaluated by UV-visible and fluorescence
spectrometry. When labeled with retinoid before purification, all of
the rCRALBP mutants bound stoichiometric amounts of
11-cis-Ral like the wild type protein, indicating that the
proteins were not grossly misfolded (Figs. 6-8). However, of the 5 mutants tested, Q210R and K221A exhibit altered UV-visible absorption
spectra when presented with 9-cis-Ral and bound markedly
less than stoichiometric amounts of 9-cis-Ral (~0.3 mol of
retinoid/mol of protein). Essentially the same UV-visible spectral
results were obtained whether the retinoid labeling was performed in
bacterial lysates or with the apoproteins. The physiological significance of CRALBP's ability to bind 9-cis-Ral is not
yet understood; however, it was shown previously that bovine CRALBP exhibits greater relative affinity for 11-cis- than
9-cis-Ral (6). The lower binding stoichiometries observed
with Q210R and K221A and 9-cis-Ral are likely because of
losses of retinoid during protein chromatography and reflect reduced
affinity for the 9-cis-isomer (Table III). These results
implicate Gln-210 and Lys-221 as potential components of the CRALBP
retinoid binding pocket. In other recent studies,
ligand-dependent rCRALBP conformational changes have been
demonstrated by NMR and shown to be associated in part with Met
residues by 13C NMR (11, 18). The close proximities of
Gln-210 with Met-208 and Lys-221 with Met-222 further support the
possible association of Gln-210 and Lys-221 with the retinoid binding
cavity.
Overall, the apparent equilibrium dissociation constants determined for
wild type apo-rCRALBP (Table III) reveal lower affinities for
9-cis-Ral than for 11-cis-Ral and for the Q210R
and K221A mutants, even lower affinities for interactions with
9-cis-Ral. The lower affinity for the
9-cis-isomer suggests that the Q210R and K221A mutations
have changed the structural integrity of the ligand binding pocket. The
apparent Kd values determined for wild type rCRALBP
(~21 nM for 11-cis-Ral and ~53
nM for 9-cis-Ral) are similar in magnitude to
Kd values reported for other protein-retinoid
interactions. For example, for binding with all-trans-Rol, Kd ~10-40 nM have been reported for
CRBP (33). For delipidated interphotoreceptor RBP,
Kd ~50 nM have been reported for
binding with all-trans-Rol and Kd
~29-36 nM for binding with 11-cis-Ral (24).
Notably, the low solubility and lability of retinoids in aqueous
solution contribute to the difficulty and variability in retinoid
affinity measurements (33). Variable protein stability also cannot be
ruled out as a contributing factor in the present measurements as
apo-CRALBP is structurally less stable than the holoprotein (9, 11).
Furthermore, it is difficult to accurately assess the affinity of
protein-retinoid complexes when the Kd is
significantly lower than the protein concentration required for the
measurement. In these cases, Kd values extracted
from fluorescence titrations can reflect upper limits rather than
precise estimates (34, 35). Yet recent analyses suggest that
fluorescence titrations can accurately measure Kd
values in the range of 15-20 nM. For example, for the
retinoid X receptor complex with 9-cis-retinoic acid, Kd ~15 nM was found by both
fluorescence titrations (35) and a charcoal binding assay (36).
However, fluorescence titration also yield Kd ~ 15 nM for the retinoic acid receptor complex with
all-trans-retinoic acid (35) although a much higher binding
affinity has been established (Kd in the
sub-nanomolar range) (36). Thus, the apparent Kd
values in Table III may reflect upper limits and the actual
Kd values may be lower, particularly for the
complexes with 11-cis-Ral. Nevertheless, the present data
demonstrate that rCRALBP has a lower affinity for 9-cis-Ral
than for 11-cis-Ral and that the Q210R and K221A mutants
display lower affinities for 9-cis-Ral as compared with wild
type rCRALBP.
Ligand Interactions in Phosphatidylinositol-transfer
Protein--
Recently the crystal structure of yeast Sec14
(phosphatidylinositol-transfer protein, PITP) was reported to 2.5 Å resolution (37). This 35-kDa protein shares sequence similarity with
CRALBP (38) and the three-dimensional structures of the two proteins are likely to be similar. In vitro, PITP catalyzes exchange
between membrane bilayers of phosphatidylinositol and
phosphatidylcholine and in vivo is required for vesicle
budding from the Golgi complex (37). The ligand binding cavity of PITP
is lined with hydrophobic residues that make van der Waals contacts
with the acyl chains of two molecules of the detergent
n-octyl-
-D-glucopyranoside in the crystal
structure. Consistent with the current results, CRALBP residues Gln-210
and Lys-221 are located within a segment of sequence that exhibits high
homology to part of the ligand binding cavity of PITP (Fig.
9).

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Fig. 9.
Comparison of PITP ligand-binding pocket
sequence with CRALBP. Yeast PITP residues, shown on black
background, line the ligand binding cavity in the crystal structure
(37). Homologous sequence from human CRALBP (38) include residues
Gln-210 and Lys-221, demonstrated to be important in CRALBP
interactions with 9-cis-Ral, and likely to be components of
the retinoid binding pocket.
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Ligand Interactions in Other Retinoid-binding Proteins--
No
Schiff base linkage or other covalent bonds attach retinoid to the
cellular retinol-binding proteins (CRBP and CRBP II), which bind 1 mol
of all-trans-Rol or all-trans-Ral per mol of protein and also exhibit red shifted chromophore absorbance. CRBP and
CRBP II serve intracellular transport and substrate carrier functions
and belong to a family of small intracellular lipid and
retinoid-binding proteins containing about 130 amino acids (33). X-ray
crystallographic structures have been determined for both CRBP and CRBP
II and detailed comparisons with other known structures are available
(33, 39, 40). CRBP and CRBP II exhibit the
-clam motif common to the
lipid-binding protein family and are more flattened in shape than the
classic
-barrel structure of serum retinol-binding protein (RBP).
Although CRALBP shares no significant sequence homology with RBP or the
other cellular retinoid-binding proteins, the three-dimensional motifs characteristic of RBP and CRBP tolerate significant sequence diversity, allowing in the case of CRBP and insect fatty acid-binding protein, superimposable crystal structures with only 12% sequence identity (40). Notably, 23 residues are within about 5 Å of retinoid in the
ligand binding cavities of CRBP and CRBP II, including Gln and Lys
residues critical for retinoid binding (33, 39, 40). The
-amino
group of Lys-40 in CRBP and CRBP II is within van der Waals distance of
bound all-trans-Rol and may participate in electrostatic
interactions with the
electrons of the isoprene arm of the
retinoid, which is in a solvent inaccessible environment. Amino-aromatic hydrogen bonding interactions in CRBP between Gln-108 and Phe-4 have been proposed as the basis for the protein's higher affinity for all-trans-Rol than for
all-trans-Ral. The specific roles of Lys-221 and Gln-210 in
CRALBP are not yet known, but the above relationships provide plausible
types of interactions.
CRALBP mutation R150Q has been associated with autosomal recessive
retinitis pigmentosa and shown to abolish 11-cis-Ral binding as well as to significantly decrease rCRALBP solubility (1). Notably, a
single Arg residue can modulate ligand specificity and affinity in the
cellular retinoic acid-binding protein, intestinal fatty acid-binding
protein (41), and CRBP II. Mutating Arg-111 to Gln in CRABP destroys
the protein's ability to bind retinoic acid (42). In FABP,
substituting corresponding Arg-106 with Gln reduces the protein's
affinity for fatty acids and converts the molecule to a
retinoid-binding protein. In CRBP II, Gln-108 occupies the
corresponding position, which when substituted with Arg, converts the
molecule to a fatty acid-binding protein with decreased affinity for
retinoids (43). Whether CRALBP Arg-150 is part of the ligand binding
pocket is not clear; however, this residue appears to play an important
stabilizing role in the CRALBP structure.
In summary, the lack of a covalent bond between CRALBP and retinoid is
consistent with the proposed physiological function(s) of CRALBP, in
which rapid association and dissociation of the ligand would appear
important. The apparent substrate carrier function of CRALBP in the RPE
resembles that of CRBP more than serum RBP or the visual pigments;
however, a precise determination of the structure of the CRALBP ligand
binding cavity awaits crystallographic analysis. In this study, we have
found that Gln-210 and Lys-221 influence CRALBP retinoid interactions.
These residues are likely to be components of the retinoid binding
pocket and may participate in key noncovalent interactions. The
function of CRALBP in tissues such as cornea, ciliary body, and
oligodendrocytes of the brain and optic nerve and whether ligands other
than cis-retinoids associate with the protein remain to be
determined.
We gratefully acknowledge Valerie Oliver and
Marina LaDuke for manuscript preparation.