©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Arginine 132 of Cellular Retinoic Acid-binding Protein (Type II) Is Important for Binding of Retinoic Acid (*)

(Received for publication, November 18, 1994)

Lan X. Chen (1) Zhen-ping Zhang (2) Angela Scafonas (2) R. Christopher Cavalli (2)(§) Jerome L. Gabriel (2) (3) Kenneth J. Soprano (1) (3) Dianne Robert Soprano (2) (3)(¶)

From the  (1)Department of Microbiology and Immunology, (2)Department of Biochemistry, and (3)Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 ^3H-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.


INTRODUCTION

Retinoic acid (RA), (^1)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-alpha, RAR-beta, and RAR-) (2, 3, 4) and nuclear retinoid X receptors (RXR-alpha, RXR-beta, 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 beta-strands arranged in two orthogonal beta-sheets and two short alpha-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.


MATERIALS AND METHODS

Plasmid Constructs and Site-directed Mutants

The mouse CRABP-II cDNA clone was generously provided by Dr. Vincent Giguere, The Hospital for Sick Children, Toronto, Canada, as a full-length cDNA encoding mouse CRABP-II cDNA in pBluescript KS vector (pKSmcrabp-II) (14) . Four mutants of wild type CRABP-II were prepared in which Lys was replaced with an Ala (K82A), Arg was replaced with an Ala (R111A), Arg was replaced with an Ala (R132A), and Arg was replaced with a Gln (R132Q).

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^1 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^1 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.

Protein Expression and Purification

The wild type and mutant plasmid expression vectors were transformed into Escherchia coli K12 strain BL21(DE3)pLysS (Novagen)(33) . A single colony picked from a freshly streaked plate was used to inoculate 100 ml of 2 times YT broth containing 150 µg/ml ampicillin (Sigma) and 35 µg/ml chloramphenicol (Sigma) and grown overnight at 37 °C. The next morning, 20 ml of this culture were used to inoculate 10 liters of 2 times YT broth that contained the same antibiotics as above. The fermentation was carried out at 37 °C in a MF-14 Microferm fermentor (New Brunswick Scientific) until the A of the culture reached approximately 0.6. At this time the cells were induced to express the recombinant protein by adding isopropyl-1-thio-beta-D-galactopyranoside (IPTG) to a final concentration of 0.6 mM. Ninety min later the cells were harvested by centrifugation at 5000 times g for 30 min, and the cell pellet was stored at -80 °C.

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 beta-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 times g for 60 min. The clear supernatant was loaded onto Ni-NTA (Qiagen) column (2 times 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 beta-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.

Fluorescence Binding Assays

An SLM DMX-1000 fluorometer equipped with an integrator was used with emission and excitation slit widths of 5 nm. Various concentrations of the all-trans isomer of RA or retinol prepared in ethanol were added to approximately a 1 µM solution of protein in 20 mM Tris, pH 7.4, such that the final ethanol concentration did not exceed 1% (v/v). The following molar absorption coefficients were used to calculate the retinoid concentrations of the stock solutions: RA, 45,000 M cm at 350 nm; retinol, 46,000 M cm at 325 nm(36) . Protein quenching was monitored at excitation and emission wavelengths of 280 nm and 340 nm, respectively. At least 10 concentrations of each retinoid were used. The averages of three readings, with each value determined by the integrator over 8 s, were used for all calculations. Calculations of K(d) were performed by the method of Cogan et al.(36) . Briefly, the fraction of free binding sites on CRABP-II (alpha) was determined from alpha = (F - F(max))/(F(0) - F(max)), where F(max) = fluorescence intensity upon saturation of all the apoprotein molecules, F = fluorescence at each R(0) (retinoid concentration), and F(0) = initial fluorescence intensity. A straight line with a slope of 1/n and an ordinate intercept of K(d)/n is obtained when P(0)alpha is plotted versusR(0)(alpha/1 - alpha), where P(0) = total CRABP-II concentration and R(0) = retinoid concentration. Each K(d) value represents the mean ± S.D. of a minimum of three independent determinations utilizing at least two different batches of each protein.

Binding of all-trans-[^3H]RA and all-trans-[^3H]Retinol

The binding of all-trans-[^3H]RA and all-trans-[^3H]retinol was performed essentially as described by Siegenthaler et al.(37) . All reactions were performed in the dark. For the binding assay, 50 or 100 nM fusion CRABP-II proteins (see figure legends) and 10 nM cleaved CRABP-II proteins in the binding buffer (10 mM Tris, pH 7.4, 2 mM dithiothreitol) was incubated with various concentrations of all-trans-[^3H]RA (DuPont-NEN; 47.5 Ci/mmol) or all-trans-[^3H]retinol (DuPont-NEN; 37.3 Ci/mmol) ranging from 5 to 500 nM at 25 °C for 90 min. After incubation, an aliquot was removed for determination of total radioactivity and the remaining solution was mixed with an equal volume of charcoal dextran suspension (1.6% charcoal, 0.16% dextran T70, 0.25 M sucrose, 1 mM EDTA, and 10 mM Tris, pH 7.5). After 15 min of incubation at 4 °C, the tubes were centrifuged at 10,000 times g for 15 min. Supernatants were counted for radioactivity in a Beckman LS 6000BC liquid scintillation counter. Specificity of binding was demonstrated by competition with 200-fold excess unlabeled all-trans-RA or all-trans-retinol. Background radioactivity was determined by preparing duplicate samples except for the addition of protein. Specific binding was defined as the total binding minus the background binding. The K(d) values were determined by Scatchard plot analysis (38) using Cricket graphics. Each K(d) value represents the mean ± S.D. of at least three independent determinations utilizing at least two different batches of each protein.


RESULTS

Expression and Purification of Wild Type and Mutant CRABP-II Fusion Proteins

To identify amino acid residues of CRABP-II important for RA binding, we have focused our attention on two highly conserved Arg residues, Arg and Arg. Both residues either singly or together have been demonstrated to be important for ligand binding by the LBPs(21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32) . We have also chosen to examine another positively charged amino acid, Lys, which is conserved among family members but has not been implicated in ligand binding by these proteins. Crystallographic analysis of a family member, P2 myelin protein, indicates that Lys (homologous position to Lys in CRABP-II) is located in the betaF strand at top of the barrel on the opposite side of the fatty acid binding pocket (Arg and Arg of P2 myelin protein)(21) . In order to evaluate the role of each of these positively charged amino acids in the binding of RA by CRABP-II, we chose to mutate each to the neutral amino acid, Ala. Since R132A did not display any measurable binding of RA (as shown below), we also mutated this amino acid residue to Gln to determine if we could alter the retinoid specificity of CRABP-II (see below).

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^1 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; beta-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 times 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.



Binding of all-trans-RA to Wild Type and Mutant CRABP-II Fusion Proteins

The affinity of wild type and mutant CRABP-II fusion proteins for all-trans-RA was determined by two independent methods. Fig. 2A shows the quenching of fluorescence of wild type and mutant CRABP-II fusion proteins at 340 nm upon addition of increasing amounts of all-trans-RA. Measurement of the fluorescence spectra of the wild type and mutant fusion apoproteins upon excitation at 280 nm revealed very similar spectra with (max) at 340 nm for each protein (data not shown). The fluorescence of wild type and K82A CRABP-II fusion proteins decreased similarly upon addition of increasing amounts of all-trans-RA until the ligand binding site of each was saturated. At saturation, the binding of all-trans-RA quenched the fluorescence of the two proteins approximately 70%. The fluorescence of R111A fusion protein was also quenched upon addition of all-trans-RA to approximately 60% of the initial fluorescence intensity. In contrast, the addition of all-trans-RA to the two Arg mutant fusion proteins, R132A and R132Q, resulted in only a minimal amount of fluorescence quenching. Fig. 2B depicts the linearization of the data from Fig. 2A which allows for the calculation of the K(d) for all-trans-RA and the number of independent binding sites. Approximately 1 (0.97-1.01) independent binding sites for all-trans-RA were observed for each of the wild type, K82A, and R111A fusion proteins, similar to previously published data(13, 17) . The K(d) for all-trans-RA of wild type and K82A CRABP-II fusion proteins was identical, 106 ± 3 nM, while the R111A mutant demonstrated a slightly reduced affinity for all-trans-RA with a K(d) value of 159 ± 4 nM. On the other hand, for both of the two Arg mutants, the minimal fluorescence quenching observed resulted in a large degree of scatter in the linear least squares plots (R^2 = 0.08) making it impossible to calculate from the fluorescence data K(d) value for all-trans-RA.


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^2 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-[^3H]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(d) 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-[^3H]RA. Panel A, specific binding of all-trans-[^3H]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^2 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(d) 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.

Binding of all-trans-RA to Cleaved Wild Type, R111A, R132A, and R132Q CRABP-II

We next prepared cleaved wild type, R111A, R132A, and R132Q CRABP-II proteins to ensure that the leader sequence was not responsible for the inability of the two Arg mutant proteins to bind all-trans-RA and to assure that the fusion protein portion of R111A did not mask any reduction in the affinity of this protein for RA. In our initial experiments we were unable to cleave the leader peptide because the first amino acid of CRABP-II directly adjacent to the enterokinase cleavage site was Pro. In order to cleave the leader sequence, we mutated Pro^1 to Leu^1 in the wild type and each of the mutant cDNA constructs. It should be noted that the K(d) values for all-trans-RA of wild type CRABP-II and the mutant fusion proteins indicated in Fig. 2and Fig. 3were obtained using the Pro^1 proteins. However, exactly the same results were obtained when Pro^1 was replaced with Leu as the first amino acid of the CRABP-II portion of these fusion proteins (data not shown).

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(d) 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-[^3H]RA. Fig. 4demonstrates that both the cleaved wild type and cleaved R111A CRABP-II proteins displayed specific and saturable binding of all-trans-[^3H]RA, while again both of the Arg mutant proteins were unable to bind all-trans-RA. The K(d) 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(d) 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(d) for the two cleaved Arg mutants due to the lack of detectable binding of all-trans-[^3H]RA.


Figure 4: Titration of cleaved wild type, R111A, R132A, and R132Q CRABP-II proteins with all-trans-[^3H]RA. Specific binding of all-trans-[^3H]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^2 value for each line was greater than 0.95.



Binding of all-trans-Retinol to Wild Type and Arg Mutants of CRABP-II

Since Arg appears to be very important for binding of all-trans-RA to CRABP-II, we next wished to determine if the ligand specificity of CRABP-II could be altered by mutation of Arg. In particular, we were interested in determining if the R132Q mutant would allow specific binding of retinol since Gln is the amino acid found in the homologous position of CRBP-I and CRBP-II. Fig. 5, Panel A, shows the fluorometric titration of each fusion protein by monitoring the fluorescence quenching of the protein upon addition of increasing concentrations of all-trans-retinol. Since all-trans-RA can quench the fluorescence of CRABP-II when it is bound in the binding site, it is presumed that all-trans-retinol will act in a similar fashion if it binds in the CRABP-II ligand binding site. Panel B shows the binding of all-trans-[^3H]retinol to the wild type and Arg fusion proteins. By both methods, no specific binding of all-trans-retinol was observed for the wild type, R132A, and R132Q CRABP-II fusion proteins. The lack of binding of all-trans-retinol to the wild type CRABP-II is consistent with the report of Fiorella et al. (11) using similar fluorescence quenching techniques. As a positive control for retinol in the binding assay, the binding of all-trans-retinol to CRBP-I was measured. CRBP-I binding of retinol was specific and saturable with a K(d) of 43 nM for the fluorometric assay and 48 nm for the all-trans-[^3H]retinol binding assay which is similar to the previously published value(36) .


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-[^3H]retinol to 50 nM of each fusion protein. CRBP-I is shown as a positive control for the retinol in the binding assay.




DISCUSSION

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 (max) 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) .


FOOTNOTES

*
This work was supported by the National Institutes of Health Grant DK44517 (to D. R. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a National Institutes of Health Training Grant 5 T32 DK07162 and National Institutes of Health Grant DK44841.

Recipient of National Institutes of Research Career Development Award HD01076. To whom correspondence should be addressed: Dept. of Biochemistry, Temple University School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140. Tel.: 215-707-3266; Fax: 215-707-7536.

(^1)
The abbreviations used are: RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; CRBP, cellular retinol-binding protein; CRABP, cellular retinoic acid-binding protein; LBP, lipid-binding protein; PCR, polymerase chain reaction; IPTG, isopropyl-1-thio-beta-D-galactosidase; PAGE, polyacrylamide gel electrophoresis; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.


ACKNOWLEDGEMENTS

We thank Dr. William S. Blaner for the recombinant apoCRBP-I, Dr. Vincent Giguere for the mouse CRABP-II cDNA clone, F. Hoffmann-LaRoche and Co. (Nutley, NJ) for the retinoids, and Dr. Parkson Chong for the use of the fluorometer and helpful discussions.


REFERENCES

  1. Gudas, L., Sporn, M. B., and Roberts, A. B. (1994) in The Retinoids (Sporn, M. B., Roberts, A. B., and Goodman, D. S., eds) 2nd Ed., pp. 443-520, Raven Press, NY
  2. Giguere, V., Ong, P., Segui, P., and Evans, R. M. (1987) Nature 330, 624-629 [CrossRef][Medline] [Order article via Infotrieve]
  3. Petkovich, M., Brand, N. J., Krust, A., and Chambon, P. (1987) Nature 330, 444-450 [CrossRef][Medline] [Order article via Infotrieve]
  4. Zelent, A., Krust, A., Petkovich, M., Kastner, P., and Chambon, P. (1989) Nature 339, 714-717 [CrossRef][Medline] [Order article via Infotrieve]
  5. Mangelsdorf, D. J., Ong, E. S., Dyck, J. A., and Evans, R. M. (1990) Nature 345, 224-229 [CrossRef][Medline] [Order article via Infotrieve]
  6. Yu, V. C., Delsert, C., Andersen, B., Holloway, J. M., Devary, O. V., Naar, A. M., Kim, S. Y., Boutin, J. M., Glass, C. K., and Rosenfeld, M. G. (1991) Cell 67, 1251-1266 [Medline] [Order article via Infotrieve]
  7. Leid, M., Kastner, P., Lyons, R., Nakshatri, H., Saunders, M., Zacharewski, T., Chen, J.-Y., Staub, A., Garnier, J., Sylvie, M., and Chambon, P. (1992) Cell 68, 377-395 [Medline] [Order article via Infotrieve]
  8. Mangelsdorf, D. J., Umesono, K., and Evans, R. M. (1994) in The Retinoids (Sporn, M. B., Roberts, A. B., and Goodman, D. S., eds) 2nd Ed., pp. 319-350, Raven Press, NY
  9. Ong, D. E., Newcomer, M. E., and Chytil, F. (1994) in The Retinoids (Sporn, M. B., Roberts, A. B., and Goodman, D. S., eds) 2nd Ed., pp. 283-317, Raven Press, New York
  10. Boylan, J. F., and Gudas, L. J. (1991) J. Cell Biol. 112, 965-979 [Abstract]
  11. Fiorella, P. D., and Napoli, J. L. (1991) J. Biol. Chem. 266, 16572-16579 [Abstract/Free Full Text]
  12. Kakkad, B., and Ong, D. E. (1988) J. Biol. Chem. 263, 12916-12919 [Abstract/Free Full Text]
  13. Bailey, J. S., and Siu, C. H. (1988) J. Biol. Chem. 263, 9326-9332 [Abstract/Free Full Text]
  14. Giguere, V., Lyn, S., Yip, G., Siu, C. H., and Amin, S. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6233-6237 [Abstract]
  15. Astrom, A., Tavakkol, A., Petersson, U., Cromie, M., Elder, J. T., and Voorhees, J. J. (1991) J. Biol. Chem. 266, 17662-17666 [Abstract/Free Full Text]
  16. Eller, M. S., Oleksiak, M. F., McQuaid, T. J., McAfee, S. G., and Gilchrest, B. A. (1992) Exp. Cell. Res. 199, 328-336
  17. Fiorella, P. D., Giguere, V., and Napoli, J. L. (1993) J. Biol. Chem. 268, 21545-21552 [Abstract/Free Full Text]
  18. Dolle, P., Ruberte, E., Leroy, P., Morriss-Kay, G., and Chambon, P. (1990) Development 110, 1133-1151 [Abstract]
  19. Ruberte, E., Dolle, P., Chambon, P., and Morriss-Kay, G. (1991) Development 111, 45-60 [Abstract]
  20. Ruberte, E., Friederich, V., Morriss-Kay, G., and Chambon, P. (1992) Development 115, 973-987 [Abstract/Free Full Text]
  21. Cowan, S. W., Newcomer, M. E., and Jones, T. A. (1993) J. Mol. Biol. 230, 1225-1246 [CrossRef][Medline] [Order article via Infotrieve]
  22. Winter, N. S., Bratt, J. M., and Banaszak, J. J. (1993) J. Mol. Biol. 230, 1247-1259 [CrossRef][Medline] [Order article via Infotrieve]
  23. Sacchettini, J. C., Gordon, J. I., and Banaszak, L. J. (1988) J. Biol. Chem. 263, 5815-5819 [Abstract/Free Full Text]
  24. Scapin, G., Gordon, J. I., and Sacchettini, J. C. (1992) J. Biol. Chem. 267, 4253-4269 [Abstract/Free Full Text]
  25. Eads, J., Sacchettini, J. C., Kromminga, A., and Gordon, J. I. (1993) J. Biol. Chem. 268, 26375-26385 [Abstract/Free Full Text]
  26. Jones, T. A., Bergfors, T., Sedzik, J., and Unge, T. (1988) EMBO J. 7, 1597-1604 [Abstract]
  27. Muller-Fahrnow, A., Egner, U., Jones, T. A., Rudel, H., Spener, F., and Saenger, W. (1991) Eur. J. Biochem. 199, 271-276 [Abstract]
  28. Xu, Z., Bernlohr, D. A., and Banaszak, L. J. (1992) Biochemistry 31, 3484-3492 [Medline] [Order article via Infotrieve]
  29. Zanotti, G., Scapin, G., Spadon, P., Veerkamp, J. H., and Sacchettini, J. C. (1992) J. Biol. Chem. 267, 18541-18550 [Abstract/Free Full Text]
  30. Zhang, J., Liu, Z.-P., Jones, T. A., Gierasch, L. M., and Sambrook, J. F. (1992) Proteins Struct. Funct. Genet. 13, 87-99 [Medline] [Order article via Infotrieve]
  31. Higuchi, R., Krummel, B., and Saiki, R. K. (1988) Nucleic Acids Res. 16, 7351-7367 [Abstract]
  32. Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J. H., and Roe, B. A. (1980) Mol. Biol. 143, 161-178
  33. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Methods Enzymol. 185, 60-89 [Medline] [Order article via Infotrieve]
  34. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  35. Levin, M. S., Locke, B., Yang, N. C., Li, E., and Gordon, J. I. (1988) J. Biol. Chem. 263, 17715-17723 [Abstract/Free Full Text]
  36. Cogan, U., Kopelman, M., Mokady, S., and Shinitzky, M. (1976) Eur. J. Biochem. 65, 71-78 [Abstract]
  37. Siegenthaler, G., Saurat, J-H, Hotz, R., Camenzind, M., and Merot, Y. (1986) J. Invest. Dermatol. 86, 42-45 [Abstract]
  38. Scatchard, G. (1946) Ann. N. Y. Acad. Sci. 51, 660-672
  39. Jakoby, M. G., Miller, K. R., Toner, J. J., Bauman, A., Cheng, L., Li, E., and Cistola, D. P. (1993) Biochemistry 32, 872-878 [Medline] [Order article via Infotrieve]
  40. Stump, D. G., Lloyd, R. S., and Chytil, F. (1991) J. Biol. Chem. 266, 4622-4630 [Abstract/Free Full Text]
  41. Cheng, L., Qian, S.-J., Rothschild, C., d'Avignon, A., Lefkowith, J. B., Gordon, J. I., and Li, E. (1991) J. Biol. Chem. 266, 24404-24412 [Abstract/Free Full Text]

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