(Received for publication, November 18, 1994)
From the
Cellular retinoic acid-binding protein type II (CRABP-II) is one
of two small molecular weight, cytosolic proteins which specifically
bind retinoic acid (RA). Crystallographic and site-directed mutagenesis
studies of several related proteins have indicated that either one or
two conserved amino acid residues, homologous to positions Arg and Arg
of CRABP-II, are important for the binding
of the hydrophobic ligand. In this report we have prepared
site-directed mutations of these two positions of CRABP-II, Arg
and Arg
, as well as Lys
to determine
the role of these residues in the binding of RA. Recombinant wild type
and mutant CRABP-II proteins were expressed and purified, and the
affinity for retinoids was determined by fluorometric titration and
binding of
H-labeled compounds. K82A displayed an identical K
for all-trans-RA as wild type
CRABP-II and the K
for
all-trans-RA of R111A was only slightly higher. On the other
hand, the two Arg
mutants, R132A and R132Q, of CRABP-II
demonstrated undetectable binding of all-trans-RA. Taken
together these data demonstrate that Arg
is a critical
amino acid residue for the binding of RA by CRABP-II.
Retinoic acid (RA), ()a vitamin A metabolite, is a
necessary component of many biological processes including growth,
differentiation, and morphogenesis (for review, see (1) ). The
actions of RA have been suggested to be mediated by two classes of
proteins. The first is the family of nuclear retinoic acid receptors
(RAR-
, RAR-
, and RAR-
) (2, 3, 4) and nuclear retinoid X receptors
(RXR-
, RXR-
, and
RXR-
)(5, 6, 7) . The RARs and RXRs,
which belong to the multigene family of steroid/thyroid hormone
receptors, are RA-inducible transcriptional regulatory proteins which
transduce the RA signal by altering the rate of transcription of
specific genes (for review, see (8) ). The second class
consists of the small, cytoplasmic proteins (CRABP-I and CRABP-II) (for
review, see (9) ). The function of these proteins is unclear.
Although it is not known whether the CRABPs are necessary for RA
action, it has been suggested that they play an important role in
sequestering RA within the cell and directing the metabolism of bound
RA(10, 11, 12) .
CRABP-II was first purified from whole rat pups(13) , and later both the mouse (14) and human (15, 16) cDNA clones were isolated. Comparison of human and mouse CRABP-II demonstrates 93.5% amino acid sequence identity. However, human and mouse CRABP-I display 73% amino acid sequence identity with human and mouse CRABP-II, respectively. CRABP-I and CRABP-II specifically bind RA while having no detectable affinity for either retinol or retinal(13, 17) . Both CRABP-I and CRABP-II display similar ligand binding properties for RA and several of its metabolites; however, CRABP-II appears to have a 3-fold lower affinity for stoichiometrically binding retinoids when compared to CRABP-I(13, 17) . Finally, during embryogenesis both CRABP-I and CRABP-II are widely expressed; however, in the adult the expression of CRABP-II becomes restricted to the skin(14, 15, 16, 18, 19, 20) .
CRABP-II belongs to a large superfamily of low molecular weight,
cytoplasmic, small hydrophobic molecule-binding proteins often referred
to as lipid-binding proteins (LBPs). The crystal structures of several
LBPs including CRBP-I(21) , CRBP-II(22) , intestinal
fatty acid-binding protein(23, 24, 25) , P2
myelin protein(21, 26) , bovine heart fatty
acid-binding protein(27) , adipocyte lipid-binding
protein(28) , and muscle fatty acid-binding protein (29) have been reported. All of the LBPs whose structures have
been determined contain 10 antiparallel -strands arranged in two
orthogonal
-sheets and two short
-helices. Either one or both
of the residues at the homologous positions to Arg
and
Arg
of CRABP-II are involved in ligand binding.
No
crystallographic information is presently available for either CRABP-I
or CRABP-II. Site-directed mutagenesis studies of CRABP-I have
demonstrated that replacement of either Arg or
Arg
(homologous to the Arg
and Arg
of CRABP-II) with Gln results in a significantly lower affinity
for RA, suggesting that these two residues are involved in interacting
with the ligand(30) . In this report we have examined the role
of the two conserved Arg residues, Arg
and
Arg
, of CRABP-II for binding of RA. Our studies
demonstrate that mutation of Arg
of CRABP-II to either an
Ala or a Gln results in markedly decreased binding of
all-trans-RA and no acquisition of all-trans-retinol
binding when compared to that of the wild type protein. On the other
hand, mutation of Arg
of CRABP-II to an Ala resulted in a
near wild type affinity for all-trans-RA. Taken together these
data suggest that Arg
is critical for the binding of RA
by CRABP-II.
The mutants were prepared by PCR site-directed mutagenesis(31) . All oligonucleotides were purchased from the Oligonucleotide Synthesis Laboratory at Temple University School of Medicine or Ransom Hill Biosciences, Inc. Sense primers are indicated as s, antisense primers as as, and the mutant codon of the mutagenic primers in bold and underline. EcoRI-linearized pKSmcrabp-II was used as a template for preparation of the wild type and mutant constructs. For K82A, two PCR fragments were synthesized using the primer pairs CRABPII-5s (5`-CCGCCGGATCCTAACTTTTCTGGCAAC-3`) plus K82A-as (5`-TTTCACCAAACTGGCACAGGGTCTCCC-3`) and CRABP-II-3as (5`-GGGAGGGTGCAGGTACCCGGGCTTAAGATAAA-3`) plus K82A-s (5`-GGGAGACCCTGTGCCAGTTTGGTGAAA-3`), respectively. The two PCR fragments were purified, annealed, and amplified in a second PCR reaction using the primers CRABPII-5s and CRABPII-3as. The amplified DNA was digested with BamHI and EcoRI and ligated into BamHI/EcoRI digested pRSETB (Invitrogen). The remaining mutants were prepared in exactly the same manner except that the mutagenic primers for R111A were R111A-as (5` GGTCAGTTCAGCGCTCCAGGA 3`) and R111A-s (5`-TCCTGGAGCGCTGAACTGACC 3`), for R132A were R132A-as (5`- TCGGACGTAGACGGCGGTGCACACAAC 3`) and R132A-s (5`-GTTGTGTGCACCGCCGTCTACGTCCGA 3`), and for R132Q were R132Q-as (5` TCGGACGTAGACCTGGGTGCACACAAC-3`) and R132Q-s (5`- GTTGTGTGCACCCAGGTCTACGTCCGA- 3`). Wild type CRABP-II was subcloned into BamHI/EcoRI-digested pRSETB after PCR amplification using the CRABPII-5s and CRABPII-3as primers followed by digestion of the DNA product with BamHI and EcoRI.
Since enterokinase was found to be inactive when a Pro was near the
cleavage site, Pro of CRABP-II was changed to Leu. Wild
type P1L was prepared by PCR amplification of EcoRI-digested
pKSmcrabp-II DNA utilizing the primer pairs, P1L-s
(5`-GCGCGAGATCTTAACTTTTCTGGCAACTGG-3`) and CRABPII-3as. The resulting
PCR fragment was digested with BglII and EcoRI and
subcloned into BamHI/EcoRI-digested pRSETB. The
mutants were prepared by replacing the PstI/ApaI
fragment of each with that of the Leu
wild type DNA.
Each clone was verified by DNA sequence analysis of all the nucleotides encoding the entire fusion protein by Sanger methodology (32) using Sequenase Version II. No codon mutations were found in the entire CRABP-II fusion protein coding sequences of each construct except for the desired mutations.
The cell pellet was
thawed on ice and resuspended in sonication buffer (50 mM sodium phosphate, pH 8.0, 100 mM KCl) containing 10
mM -mercaptoethanol and 1 mM phenylmethylsulfonyl fluoride at a concentration of 1 g of cell
paste/10 ml of buffer. All subsequent steps were performed at 4 °C.
The cells were sonicated for 30 min followed by centrifugation at
20,000
g for 60 min. The clear supernatant was loaded
onto Ni-NTA (Qiagen) column (2
14 cm) at a flow rate of 1
ml/min. After applying the sample, the column was washed extensively
first with sonication buffer, pH 8.0 (approximately 1.6 liters),
followed by sonication buffer, pH 6.0 (approximately 500 ml). The
recombinant CRABP-II fusion protein was eluted from the column with
sonication buffer, pH 4.0. Fractions were monitored by measuring the
absorbance at 280 nm. The CRABP-II fusion protein fractions were
dialyzed against 20 mM Tris-HCl, pH 7.5, and 5 mM EDTA to remove any Ni
associated with the
protein followed by dialysis against 50 mM bis-Tris-HCl, pH
7.5, 0.2 M NaCl, 2 mM
-mercaptoethanol, 0.05%
sodium azide and stored at a concentration of approximately 1 mg/ml at
4 °C. Purity of protein was monitored by SDS-polyacrylamide gel
electrophoresis (PAGE)(34) . The protein concentration was
determined with the Bio-Rad protein assay using crystalline bovine
serum albumin as the standard.
For cleavage of the CRABP-II fusion proteins, aliquots of each fusion protein were dialyzed against 10 mM Tris, pH 8.0, and then incubated with 1:50 w/w enterokinase (Biozyme) for 10 h at 37 °C. Efficiency of cleavage was monitored by SDS-PAGE. After completion of the digestion, the leader sequence and any uncleaved fusion protein was removed by batch treatment with the Ni-NTA resin followed by dialysis overnight against 10 mM Tris, pH 7.4, 2 mM dithiothreitol using dialysis tubing with a molecular weight cut off of 3500. The protein concentration of the cleaved protein was then determined as described above.
The recombinant apoCRBP-I (35) which was used as a positive control in the retinol binding studies was a generous gift of Dr. William S. Blaner, Columbia University, College of Physicians and Surgeons, New York.
Wild type CRABP-II cDNA and four mutant cDNAs (K82A, R111A,
R132A, and R132Q) were prepared in the prokaryotic expression plasmid,
pRSETB. This expression vector encodes a Ni binding
site which facilitates purification of the fusion protein and an
enterokinase cleavage site. The codon for Pro
of CRABP-II
was cloned in frame directly adjacent to the codons for the
enterokinase cleavage site. This resulted in the production of a fusion
protein containing a 3000-dalton leader peptide at the amino-terminal
end of CRABP-II.
Fig. 1A shows a representative gel demonstrating the expression and purification of the CRABP-II fusion proteins. All fusion proteins were efficiently produced in E. coli after the addition of IPTG (compare lanes 1 and 2). In addition, the CRABP-II fusion proteins were recovered in the supernatant (greater than 90% for wild type and each mutant except R111A in which only approximately 30% was recovered in the supernatant) (Fig. 1A, lane 3) and each of the fusion proteins were readily purified by affinity chromatography over Ni-NTA (Fig. 1, A, lane 4, and B). The purity of all protein samples was estimated to be greater than 95% by SDS-PAGE. The yield of each recombinant CRABP-II fusion protein was approximately 5 mg/liter except for the R111A which was always lower (approximately 0.5 mg/liter) principally due to loss of protein in the cell pellets.
Figure 1:
Expression, purification, and
cleavage of CRABP-II. Protein samples were analyzed with a 15%
SDS-polyacrylamide gel stained with Coomassie Blue. Molecular weight
standards were ovalbumin, 43,000; carbonic anhydrase, 29,000;
-lactoglobulin, 18,400; lysozyme, 14,300; bovine trypsin
inhibitor, 6,200. Panel A, induction and purification of wild
type CRABP-II fusion protein. Lane 1, whole cell lysate from
bacteria transformed with pRSETB/CRABP-II before addition of IPTG; lane 2, whole cell lysate from bacteria transformed with
pRSETB/CRABP-II 90 min after addition of IPTG; lane 3, 20,000
g supernatant of bacterial lysates; lane 4,
purified CRABP-II fusion protein after Ni-NTA chromatography. Panel
B, Purified wild type and mutant CRABP-II fusion proteins. Lane 1, wild type; lane 2, K82A; lane 3,
R111A; lane 4, R132A, and lane 5, R132Q. Panel
C, cleavage of wild type CRABP-II fusion protein with
enterokinase. Lane 1, CRABP-II fusion protein; lanes
2-6, CRABP-II fusion protein after 2, 4, 6, 8, and 10 h,
respectively, of incubation with
enterokinase.
Figure 2:
Fluorometric titration of wild type and
mutant CRABP-II fusion proteins with all-trans-RA. Panel
A, titration of 1 µM of each CRABP-II fusion protein
with the indicated concentrations of all-trans-RA was
monitored by measuring fluorescence quenching at 340 nm with excitation
at 280 nm. Panel B, linearization of the data in Panel A by the method of Cogan et al.(36) . Equations of
lines are: wild type, y = 0.985x -
0.104; K82A, y = 0.996x - 0.106; and
R111A, y = 1.025x - 0.163. The R value for each line was greater than
0.96.
To confirm
the results from the fluorometric titration assay, we have measured the
binding of all-trans-[H]RA to wild type
and each of the mutant CRABP-II fusion proteins. As shown in Fig. 3, the fusion proteins for wild type CRABP-II and the two
mutants, K82A and R111A, demonstrated saturable and specific binding of
all-trans-RA, while both the R132A and R132Q fusion proteins
showed no specific binding. Scatchard analysis of these data yielded a K
value for all-trans-RA of 109 ±
10 nM, 119 ± 11 nM and 148 ± 14 nM for wild type, K82A, and R111A fusion proteins, respectively, and
no detectable binding for either of the two Arg
mutants.
Figure 3:
Titration of wild type and mutant CRABP-II
fusion proteins with all-trans-[H]RA. Panel A, specific binding of
all-trans-[
H]RA to 100 nM of
wild type and each mutant CRABP-II fusion proteins. Panel B,
Scatchard analysis of the saturation kinetic data for wild type and
mutant CRABP-II fusion proteins. Equations of the lines are: wild type, y = -0.009x + 0.851; K82A, y = -0.008x + 0.709; and R111A, y = -0.007x + 0.554. The R
value for each line was greater than
0.94.
In summary, two independent methods of assessing the binding of
all-trans-RA to wild type and mutant CRABP-II fusion proteins
yielded essentially the same results. It should be noted that the K value of wild type CRABP-II fusion protein was
slightly higher than that of 65 nM reported for rat pup
CRABP-II (13) or 14 nM for mouse recombinant
CRABP-II(17) . This is most likely due to the presence of the
extra 3000-dalton leader peptide associated with our fusion protein.
Fig. 1C shows the cleavage of the fusion protein by enterokinase. Approximately 50% of the fusion protein was cleaved after 2 h of incubation with enterokinase, and greater than 95% was cleaved after 10 h of incubation. The 3000-dalton leader peptide cannot be seen on this gel because it runs with the dye front.
Due
to limited amounts of cleaved proteins and since the two methods to
measure binding of all-trans-RA to CRABP-II described above
yielded similar K values, we have measured the
affinity for all-trans-RA of the cleaved wild type, R111A,
R132A, and R132Q proteins by measuring binding of
all-trans-[
H]RA. Fig. 4demonstrates that both the cleaved wild type and cleaved
R111A CRABP-II proteins displayed specific and saturable binding of
all-trans-[
H]RA, while again both of the
Arg
mutant proteins were unable to bind
all-trans-RA. The K
for
all-trans-RA of wild type CRABP-II was 33 ± 4 nM similar to that previously reported for
CRABP-II(13, 17) . The K
for
all-trans-RA of R111A CRABP-II was 45 ± 7 nM which is quite comparable to that of wild type CRABP-II. However,
we were still unable to determine a K
for the two
cleaved Arg
mutants due to the lack of detectable binding
of all-trans-[
H]RA.
Figure 4:
Titration of cleaved wild type, R111A,
R132A, and R132Q CRABP-II proteins with
all-trans-[H]RA. Specific binding of
all-trans-[
H]RA to 10 nM of
cleaved wild type, R111A, R132A, and R132Q mutant CRABP-II proteins. Inset shows the Scatchard analysis of the saturation kinetic
data for wild type CRABP-II and R111A CRABP-II. Equation of the line
for wild type is y = -0.031x +
0.228 and for R111A is y = -0.022x + 0.200. The R
value for each line was
greater than 0.95.
Figure 5:
Titration of wild type, R132A, and R132Q
CRABP-II fusion proteins with all-trans-retinol. Panel
A, 1 µM of each fusion protein was titrated with the
indicated concentrations of all-trans-retinol and protein
fluorescence quenching was measured at 340 nm with excitation at 280
nm. Panel B, specific binding of
all-trans-[H]retinol to 50 nM of each fusion protein. CRBP-I is shown as a positive control for
the retinol in the binding assay.
In this report we demonstrate that mutation of Arg of CRABP-II to either an Ala or Gln results in a markedly
decreased binding of all-trans-RA, while mutation of two other
positively charged amino acids of CRABP-II, Arg
and
Lys
, to Ala had little effect on all-trans-RA
binding. Although Arg
of CRABP-II appears to be critical
for binding of RA, mutation of this residue to either an Ala or a Gln
did not increase the binding of the all-trans isomer of
retinol when compared to wild type CRABP-II. The wild type and all the
mutant fusion apoproteins demonstrated similar fluorescence spectra and
values however we can not fully eliminate the
possibility that the lack of RA binding displayed by the Arg
mutants is due to a global conformational change in these
proteins. Taken together these data suggest that from among the two
conserved residues shown to be important for ligand binding within the
LBP family, Arg
alone plays a critical role in the
binding of all-trans-RA to CRABP-II.
A major factor in the
binding of RA to CRABP-II involves the electrostatic interactions
between the carboxylate group of all-trans-RA and positively
charge amino acid residues in the protein(9) . Since mutation
of Arg (one of the two positively charged amino acid
demonstrated to interact with the carboxylate group of some related
family member proteins) to both a neutral and a polar amino acid
abolished measurable binding of all-trans-RA to CRABP-II, it
is likely that the carboxylate group of all-trans-RA interacts
with this Arg of CRABP-II. However, we cannot eliminate other possible
indirect interactions. Arg
may indirectly affect
all-trans-RA binding through charge stabilization mediated by
either bound water molecules or a nearby polar group of an amino acid
such as Tyr
(9) . Also, mutation of Arg
could result in a general perturbation of the ligand binding
pocket displacing side chains of other amino acid residue(s) which
interact directly with all-trans-RA.
It is interesting that
Arg of CRABP-II appears to be critical for the binding of
all-trans-RA while Arg
appears to be
unimportant. Data from both three-dimensional structural analyses and
site-directed mutagenesis studies of LBPs suggest that either one or
both of the amino acid residues at the homologous position to 111 and
132 of CRABP-II are important for high affinity binding of ligand. For
example, three dimensional analysis of several proteins which bind
fatty acids, including myelin P2 protein, adipocyte lipid-binding
protein and heart fatty acid-binding protein indicates that both of the
two conserved Arg residues (Arg
and Arg
which are homologous to Arg
and Arg
of CRABP-II) are associated with the binding of
ligand(21, 26, 27, 28) . In
addition, mutagenesis of either Arg
or Arg
of CRABP-I (30) results in a dramatic decrease in ligand
binding. On the other hand, both structural analysis and mutagenesis
studies of intestinal fatty acid-binding protein strongly indicates
that Arg
(homologous position to Arg
of
CRABP-II) is critical for fatty acid binding (23, 24, 25, 39) while structural
analysis of muscle fatty acid-binding protein indicates that
Arg
(homologous position to Arg
of
CRABP-II) forms hydrogen bonds with the carboxylate group of fatty
acids. Structural analysis of both CRBP-I and CRBP-II suggest that
Gln
(homologous position to Arg
of
CRABP-II) is essential for ligand binding(21, 22) .
However, mutagenesis of Gln
of CRBP-I to Arg results in
just a 3-fold decrease in retinol binding (40) while there is
no mutagenesis data available concerning Gln
(homologous
position to Arg
of CRABP-II). Finally, mutagenesis of
either Gln
or Gln
of CRBP-II (41) results in a dramatic reduction in ligand binding. Thus,
it is possible that different members of the LBPs have evolved into
distinct subgroups depending on which of these two amino acid positions
govern binding of ligand as suggested by Jakoby et
al.(39) . If this is the case then CRABP-II and muscle
fatty acid-binding protein may form one subgroup in which only the
Arg
/Arg
position is critical for ligand
binding.
Several investigators have explored the potential of
changing ligand specificity of LBPs since Gln is in the conserved
position of proteins which bind retinol and/or retinal while Arg is in
the same position of proteins which bind ligands with a carboxylate
group. In our studies we were unable to change the retinoid specificity
of CRABP-II by mutation of the critical Arg (Arg) to a
Gln. Zhang et al.(30) with CRABP-I and Cheng et
al.(41) with CRBP-II were also unable to observe retinoid
specificity changes in their mutagenesis studies. However, mutation of
Gln
of CRBP-I to an Arg resulted in an increased binding
of retinal and RA (40) , mutation of Gln
of
CRBP-II to an Arg resulted in binding of fatty acids but not
RA(41) , and mutation of Arg
of intestinal fatty
acid-binding protein to Gln allowed for binding of either retinol or
retinal(39) . These data suggest that the ligand specificity of
each of these proteins is governed not only by these two conserved
amino acid position but is also related to the other amino acid
residues of each protein within the vicinity of the ligand binding site
and the flexibility of the ligand.
Finally, comparison of our data concerning CRABP-II with the mutagenesis data of CRABP-I reported by Zhang et al.(30) suggests that there are differences in the amino acid residues involved in the binding of all-trans-RA by these two proteins. Confirmation of this will have to await the availability of three-dimensional structural information. These data suggest that CRABP-II has evolved a somewhat distinct binding site when compared to CRABP-I which may account for the 3-fold lower affinity of CRABP-II for ligand compared to CRABP-I observed by Fiorella et al.(17) . It is possible that CRABP-II has a function or requirement within the specific tissues in which it is expressed that is different from that of CRABP-I analogous to what has been suggested for intestinal fatty acid-binding protein versus muscle fatty acid-binding protein(29) .