From the 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
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ABSTRACT |
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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.
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.
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 DH5 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,
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
(
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- 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).
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 ( 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.
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).
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).
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).
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-( 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells. Plasmid DNA from DH5
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.
-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.
280/
425 = 3.2) or
9-cis-retinal (
280/
400 = 2.2)
were used to estimate rCRALBP retinoid binding stoichiometry from
observed A280/A425 or
A280/A400 absorbance
spectral ratios, respectively (8, 9).
-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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
Amino acid compositions of human rCRALBP mutants
max = 425.5 ± 0.3 nm, n = 4 separate preparations),
W165F (
max = 424.3 ± 0.2 nm, n = 4), and W244F (
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
max = 400.5 ± 0.6 nm,
n = 4; W165F
max = 393.3 ± 0.7 nm, n = 4; and W244F
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
max = 396.3 ± 0.9 nm, n = 4;
M225A
max = 408.4 ± 0.4 nm, n = 5)
or 9-cis-retinal (M222A
max = 378.3 ± 0.4 nm, n = 4; M225A
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.
View larger version (20K):
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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.
Equilibrium dissociation constants of rCRALBP with retinoids
Kinetic parameters of mutant rCRALBP substrate carrier function for
rRDH5
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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.
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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.
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Dr. John C Saari for useful discussions and for reviewing the manuscript prior to publication.
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FOOTNOTES |
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* 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.
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
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ABBREVIATIONS |
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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.
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REFERENCES |
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