Identification of sulfhydryl-modified cysteine residues in the ligand binding pocket of retinoic acid receptor beta.

The diverse biological functions of retinoic acid (RA) are mediated through retinoic acid receptors (RARs) and retinoid X receptors. RARs contain a high affinity binding site for RA which is sensitive to treatment with sulfhydryl modification reagents. In an attempt to identify which Cys residues are important for this loss of binding, we created three site-specific RARβ mutants: C228A, C258A, and C267A. The affinity for RA of all three mutant receptors was in the range of that of the wild type protein, suggesting that none of these Cys residues are critical for RA binding. Rather, these modified Cys residue(s) function to sterically hinder RA binding; however, the modified Cys residues critical for the inhibition of binding differ depending on the reagent employed. Only modification of Cys228 is necessary to inhibit RA binding when RARβ is modified by reagents which transfer large bulky groups while both Cys228 and Cys267 must be modified when a small functional group is transferred. These data suggest that both Cys228 and Cys267 but not Cys258 lie in the ligand binding pocket of RARβ. However, Cys228 lies closer to the opening of the RARβ ligand binding pocket whereas Cys267 lies more deeply buried.

The diverse biological functions of retinoic acid (RA) are mediated through retinoic acid receptors (RARs) and retinoid X receptors. RARs contain a high affinity binding site for RA which is sensitive to treatment with sulfhydryl modification reagents. In an attempt to identify which Cys residues are important for this loss of binding, we created three site-specific RAR␤ mutants: C228A, C258A, and C267A. The affinity for RA of all three mutant receptors was in the range of that of the wild type protein, suggesting that none of these Cys residues are critical for RA binding. Rather, these modified Cys residue(s) function to sterically hinder RA binding; however, the modified Cys residues critical for the inhibition of binding differ depending on the reagent employed. Only modification of Cys 228 is necessary to inhibit RA binding when RAR␤ is modified by reagents which transfer large bulky groups while both Cys 228 and Cys 267 must be modified when a small functional group is transferred. These data suggest that both Cys 228 and Cys 267 but not Cys 258 lie in the ligand binding pocket of RAR␤. However, Cys 228 lies closer to the opening of the RAR␤ ligand binding pocket whereas Cys 267 lies more deeply buried.
Retinoic acid is the potent mediator of the biological effects of vitamin A that include growth, differentiation, and morphogenesis (for review, see Ref 1). These actions of retinoic acid are mediated by two evolutionarily distinct groups of nuclear receptors that belong to the multigene family of steroid/thyroid hormone receptors called retinoic acid receptors (RARs) 1 and retinoid X receptors (RXRs) (for review, see Ref. 2). The RARs and RXRs are each made up of three receptor types, designated ␣, ␤, and ␥ (2,(5)(6)(7)(8)(9)(10)(11). In dimeric form, these proteins function as ligand-dependent transcriptional regulatory factors by binding to DNA sequences located in the promoter of target genes called retinoic acid-responsive elements (RAREs) or retinoid X-responsive elements (RXREs). In vitro binding studies have demonstrated that both all-trans-RA (RA) and 9-cis-RA are ligands for the RARs, whereas only 9-cis-RA has been shown to be a ligand for the RXRs (12,13).
Like other members of the steroid/thyroid hormone superfamily, RARs and RXRs consist of six functionally distinct domains designated A-F (2). Unique functions have been described for several of these domains. The A and B domains are important for the ligand-independent transactivation function (AF-1). The C domain, which contains two zinc fingers, is important for both DNA binding and receptor dimerization. The E domain is functionally complex. In addition to containing all the information necessary for high affinity ligand binding, it also contains a ligand-dependent transactivation function (AF-2) and accessory dimerization sequences.
Recently there have been major advances in understanding the nature of the ligand binding domain of RARs and RXRs. High resolution crystal structures of the ligand binding domains of apo-RXR␣ and holo-RAR␥ have demonstrated that these receptors share a similar overall fold (14,15). Furthermore, several amino acids within the ligand binding domains of RARs have been reported to be functionally important for high affinity binding of RA. Previous studies from this laboratory have demonstrated that the positively charged amino acid residues, Arg 269 and Lys 220 , are critical for the high affinity binding of RA and retinoid specificity of RAR␤ (16,17). Ostrowski et al. (18) have reported that Ala 225 /Ile 232 of RAR␤ and the homologous amino acid residues Ser 232 /Thr 239 of RAR␣ are important for discrimination of subtype-specific synthetic ligands, and Lupisella et al. (19) have demonstrated using fluorescence quenching techniques that Trp 227 is within the ligand binding site of RAR␥. Finally, Dallery et al. (20) and Sani et al. (21) have demonstrated a loss of RA binding after treatment of the EF domain of RAR␣ and chicken skin RARs, respectively, with several sulfhydryl-modifying reagents suggesting that Cys residue(s) may be located in the ligand binding pocket of these receptors. The goal of this work was to identify which Cys residue(s) are important for this loss of RA binding and to address the role that this Cys residue(s) plays in the binding of RA.
In the current report we have shown that, similar to RAR␣, RA binding to both RAR␤ and RAR␥ is inhibited by the sulfhydryl-specific modifying reagents DTNB and MMTS and that the sulfhydryl group of the reactive Cys residue(s) of RAR␤ lies in or near the retinoid binding pocket. These Cys residue(s) alone do not play a critical role in RA binding. Rather, these modified Cys residue(s) of RAR␤ function to sterically hinder the binding of RA. The modified Cys residues critical for the inhibition of RA binding differ depending on the reagent em-* This work was supported by 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 be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ployed. Only modification of Cys 228 is necessary when RAR␤ is treated with reagents that transfer large bulky groups to reactive Cys residues, whereas both Cys 228 and Cys 267 must be modified when a small functional group is transferred such as thiomethyl or thiocyanide to fully inhibit RA binding. Taken together these data suggest that Cys 228 most likely lies closer to the opening of the ligand binding pocket of RAR␤, whereas Cys 267 lies more deeply buried within the ligand binding pocket.

MATERIALS AND METHODS
Plasmid Constructs and Site-directed Mutagenesis-Mutants were created according to the PCR site-directed mutagenesis technique described by Higuchi et al. (22). pSG5-RAR␤, a gift from Professor Pierre Chambon, Strasbourg, France, was linearized with XbaI and used as a template for the preparation of each mutant. Both sense (s) and antisense (as) oligonucleotide primers were purchased from Ransom Hill BioScience (La Jolla, CA). The GCT codon was used to encode the mutant Ala residue indicated in bold and underlined in the mutagenic primers.
The entire coding sequences of mouse RAR␣ and RAR␥ (gift from Professor Pierre Chambon) were cloned in frame into the BamHI/ HindIII (RAR␣) or NotI (RAR␥) restriction sites of pET29a(ϩ).
Expression of RAR␤ and Preparation of Receptor Extracts-Each RAR expression construct was transformed into Escherchia coli K12 strain BL21(DE3) cells (Novagen) (24). Ten l from a frozen glycerol stock was used to inoculate 5 ml of LB medium containing 30 g/ml kanamycin and shaken at 37°C until the A 600 was 0.6 -1.0. This culture was stored overnight at 4°C, and the following morning 1 ml of cells was used to inoculate 50 ml of LB medium containing 30 g/ml kanamycin. This culture was incubated with shaking at 37°C until the A 600 reached 0.6 to 1.0. At this time the cells were induced to express the recombinant S-Tag RAR fusion proteins by the addition of isopropyl-1thio-␤-D-galactopyranoside to a final concentration of 1 mM. Ninety minutes later the cells were harvested by centrifugation at 5000 ϫ g for 15 min.
The receptor extracts were prepared from a 50-ml culture by resuspending the cell pellet in lysis buffer (50 mM Tris, pH 8.0, 0.1% Triton X-100, and 200 g/ml lysozyme) at a final concentration of 1 ml of lysis buffer/10 ml of induced culture, freezing at Ϫ70°C followed by rapid thawing at 37°C, sonication until the solution lost its viscosity, and centrifugation at 17,000 ϫ g for 30 min. The supernatant was aliquoted and stored frozen at Ϫ80°C. Total protein concentration of the receptor extracts was determined with the Bio-Rad protein assay kit using crystalline bovine serum albumin as the standard. The production of the recombinant S-Tag wild type and mutant RAR fusion proteins in the receptor extracts was monitored using the S-Tag Western blot Kit (Novagen).
Retinoic Acid Binding Assays-The receptor extracts were diluted in binding buffer (40 mM HEPES, pH 7.9, 120 mM KCl, 10% glycerol, 0.1% (w/v) gelatin, 1 mM EDTA, 4 mM dithiothreitol (DTT), and 5 g/ml each of the protease inhibitors aprotinin and leupeptin) to a final concentration of 10 -30 g of total protein (0.1-0.3 pmol of wild type). Similar total protein concentrations were used for the mutant proteins. Total RA binding was determined in the diluted protein extracts by adding Dilutions of RA were made in ethanol. In a duplicate set of reactions nonspecific binding was determined in the presence of 200-fold molar excess unlabeled RA (generous gift from Hoffmann-LaRoche). The contribution of ethanol to the total volume was the same for each RA concentration and was equal to 2%. Bound RA was separated from free by extraction with 3% (w/v) equal particle size charcoal-dextran prepared as described by Dukoh et al. (25). All steps in the procedure were performed under yellow light. Specific RA binding was determined by subtracting the nonspecific binding from the total binding. The nonspecific binding was always less than 12% of the total binding. No specific RA binding was detected in receptor extracts prepared from cells containing pET29a-RAR␤ which were not induced to express RAR with isopropyl-1-thio-␤-D-galactopyranoside or in receptor extracts prepared from cells containing the empty pET29a plasmid and treated with isopropyl-1-thio-␤-D-galactopyranoside. Apparent equilibrium dissociation constants (K d ) were determined for the wild type and each mutant protein by Scatchard analysis (26).
Chemical Modification of Receptors-The receptor extracts were diluted in the same binding buffer as described above for K d determinations except that DTT was omitted. Stock solutions of modifying or reducing agents were made up as 100 ϫ stocks in the following solvents: 5,5Ј-dithiobis(2-nitrobenzoic acid) (DTNB) (Sigma) in ethanol; methyl methanethiosulfonate (MMTS) (Sigma) in ethanol; ␤-mercaptoethanol (BME) (Fisher) in binding buffer; DTT (Sigma) in binding buffer; sodium arsenite (Fisher) in binding buffer; N-ethylmaleimide (NEM) (Sigma) in ethanol, p-hydroxymercuribenzoate (p-HMB) (Sigma) in binding buffer, tri-n-butyl-phosphine (TBP) (Aldrich) in ethanol; and sodium cyanide (NaCN) in 0.225 M potassium phosphate, pH 7.2. In all cases, the chemical modification reactions were performed on ice for 5 min. For the chemical reversal or termination of a reaction, chemically modified extract was incubated with reducing agent for an additional 5 min on ice. Total RA binding was determined in duplicate reactions for each treatment group by incubating the sample in the presence of a saturating concentration of 15 nM [ 3 H]RA as determined from the saturation binding experiments (Fig. 3) as described above. The specific [ 3 H]RA binding was determined for each treatment group and expressed as the percent of RA binding in the absence of the chemicalmodifying reagent for each protein (% of control binding). The amount of specific bound RA at 100% relative binding was in the range of 0.1-0.3 pmol. Nonspecific binding was always less than 12% of the total bound RA.

RESULTS
The Effect of Sulfhydryl Modification of RAR␣, RAR␤, and RAR␥ on RA Binding- Fig. 1, A-F, depicts the results of the treatment of the full-length recombinant RAR␣, RAR␤, and RAR␥ fusion proteins with the Cys-modifying reagents DTNB and MMTS. Similar to what has been previously reported (20,21), full-length RAR␣ exhibits a dramatic reduction in RA binding upon treatment with both of the sulfhydryl-modifying reagents (Fig. 1, A and B). Furthermore, this loss of RA binding is highly specific and is not due to the disruption of structural disulfide bonds in the protein since it is reversible to a level comparable with that of the untreated RAR␣ upon the addition of the reducing agents BME or DTT. Likewise, RA binding to RAR␤ and RAR␥ was similarly affected by both DTNB (Fig. 1, C and E) and MMTS (Fig. 1, D and F). In all cases this inhibition of RA binding was also highly specific since it was reversed by reducing agents. Note that RA binding by RAR␥ following MMTS treatment was reversible to approximately 50% of that of untreated RAR␥ using DTT. However, treatment of the MMTS modified RAR␥ with the more hydrophobic reducing reagent TBP (27) restored RA binding to a level comparable with that of the untreated RAR␥.
We next wished to determine if any of these receptors contain reactive vicinal dithiols which are responsible for this loss of RA binding. For these experiments we used sodium arsenite which has been demonstrated to cross-link two thiol groups that are spatially close (vicinal dithiols) (28,29). Treatment of the recombinant RAR␣, RAR␤, and RAR␥ fusion proteins with 10 mM sodium arsenite did not result in any significant inhibition of RA binding (data not shown).
In order to determine if the modified Cys residues following DTNB and MMTS treatment are located within the RA binding pocket of RAR␤, we tested the ability of RA to protect critical Cys residues from chemical modification. In this experiment we used the alkylating agent NEM since the modification reaction can be terminated but not reversed by the addition of a reducing agent. Fig. 2 demonstrates that similar to DTNB and MMTS, RA binding to apo-RAR␤ is highly sensitive to modification by NEM. Furthermore, Fig. 2, inset, shows that 82% of the RA binding activity was maintained when holo-RAR␤ was treated with 1 mM NEM. This demonstrates protection of the critical Cys residue(s) to NEM modification when RAR␤ is liganded with RA (holo-RAR) prior to NEM treatment. This result strongly suggests that the target Cys residue(s) in RAR␤, whose modification by sulfhydryl-modifying reagents including NEM, DTNB, and MMTS results in inhibition of RA binding, lie in or near the retinoid binding site.
Creation of Site-specific Mutants-Chemical modification of critical Cys residues located within the retinoid binding site of RAR␤ could result in loss of RA binding by either the obliteration of a direct interaction of one or more Cys residues with RA or steric hindrance resulting in the inability of RA to access the binding pocket. In order to identify the critical Cys residue(s) and to determine which of these mechanisms are important in the inhibition of RA binding to RAR␤ following sulfhydryl modification, we have created three site-specific mutants of RAR␤ in which Cys 228 , Cys 258 , and Cys 267 were individually replaced with an Ala residue (C228A, C258A, and C267A). We have chosen to focus on these residues because Dallery et al. (20) have demonstrated that RA binding to the RAR␣ EF domains is sensitive to chemical modification of sulfhydryls. The EF domains of RAR␣ contain six Cys residues that are absolutely conserved among the three RARs. Of these six residues, only three (Cys 228 , Cys 258 , and Cys 267 of RAR␤) lie within the NH 2 -terminal 100 amino acid portion of the E domain previously demonstrated to be sufficient for high affinity binding of RA (30 -32).
Western blot analysis of receptor extracts containing the wild type and mutant RAR␤ fusion proteins demonstrated a major band that migrated at the same position (approximate molecular mass of 55 kDa) along with several smaller molecular weight degradation products also of similar size. In addition, the wild type and mutant RAR␤ fusion proteins displayed a similar level of expression (data not shown).
Effect of Site-specific Mutation on RA Binding- Fig. 3 shows representative saturation curves and corresponding Scatchard plots for wild type and each mutant RAR␤ fusion protein.
Average apparent K d values were determined for each receptor using at least three separately prepared receptor extracts (Table I). The K d value of 0.6 nM RA for wild type RAR␤ fusion protein is in good agreement with previous reports (16,17). Both C258A and C267A displayed a similar affinity for RA comparable with that of wild type RAR␤. Interestingly C228A, which has a K d value of 4.4 nM, displayed a small decrease (approximately 7-fold) in affinity for RA when compared with that of wild type RAR␤.
Effect of Treatment of C228A, C258A, and C267A Mutants with DTNB and p-HMB-Since the three mutant RAR␤s have an affinity for RA in the range of that of the wild type RAR␤, it is unlikely that sulfhydryl modification of RAR␤ inhibits RA binding by obliterating a critical direct interaction between the ligand and any one of these Cys residues in the ligand binding domain of the receptor. Therefore, the alternative mechanism of inhibition involving steric blocking of RA entry into the binding pocket upon sulfhydryl modification of RAR␤ was explored. We tested this hypothesis by subjecting each of the Cys mutants to sulfhydryl modification with the assumption that if one or more chemically modified Cys residues mediate the inhibition of RA binding, then replacement of the reactive Cys with an unreactive Ala residue should result in a receptor with RA binding activity comparable with that of the unmodified receptor. Fig. 4A shows the results of DTNB treatment of each of the mutants, plotted along with wild type RAR␤ for comparison. The profiles for inactivation of RA binding by C258A and C267A after DTNB treatment are similar to that of wild type RAR␤ suggesting that Cys 258 and Cys 267 are either not accessible to modification by DTNB or that modification of either of these residues by DTNB is not sufficient to interfere with RA binding. On the other hand, RA binding to C228A is not affected by DTNB modification. This strongly suggests that modification of Cys 228 alone with DTNB is responsible for the inhibition of RA binding following DTNB treatment.
In order to confirm this conclusion we have also used the sulfhydryl-specific modifying reagent p-HMB to test each mutant in a similar fashion. Modification of a sulfhydryl by p-HMB involves the transfer of a substituted benzyl ring which is similar in size and structure to the thiobis(2-nitrobenzoic acid) D, and F) for 5 min on ice. In a duplicate set of reactions the DTNB modification was reversed with 20 mM BME, and the MMTS modification was reversed with either 10 mM DTT or 10 mM TBP. In all cases, the reducing agent was added at the end of the 5-min incubation with the modifying reagent, and the samples were incubated on ice for an additional 5 min. Following treatment RA binding was determined using 15 nM [ 3 H]RA as described under the "Materials and Methods." The specific RA binding was determined for each treatment group and was expressed as the percent of RA binding in the absence of the chemical modifying reagent for each protein (% of control binding). Values are mean Ϯ S.E. for at least three independent experiments performed in duplicate. moiety which is transferred during DTNB modification. As can be seen in Fig. 4B, the profiles of inactivation of RA binding by the wild type and mutant RAR␤s treated with p-HMB are essentially identical to that of their respective DTNB-modified receptor. These results further demonstrate that modification of Cys 228 alone with either DTNB or p-HMB is sufficient to inhibit RA binding.
Effect of Treatment of C228A, C258A, and C267A Mutants with MMTS-In order to determine if the size of the modifying group is important, we performed similar experiments using MMTS. Unlike DTNB and p-HMB, MMTS treatment of a protein results in the transfer of the small thiomethyl-blocking group to reactive sulfhydryl groups (27). Fig. 5A shows the RA binding activity of the wild type and mutant fusion proteins after modification with MMTS. C258A demonstrated a loss of RA binding upon MMTS treatment which was identical to that of wild type RAR␤ further demonstrating that Cys 258 is not the Cys residue whose modification inhibits the binding of RA. C228A modified with MMTS displayed RA binding which was approximately 85% of that of the unmodified C228A. This is consistent with the findings obtained with DTNB and p-HMB which demonstrate that Cys 228 is an important reactive Cys. Interestingly the MMTS-treated C267A RAR␤ displayed approximately 60% of the RA binding activity of the unmodified C267A (Fig. 5A). Furthermore, this inhibition of RA binding by the unmodified C267A was not reversible with DTT but was reversible with the hydrophobic reducing agent TBP (Fig. 5B).
Since both C228A and C267A retained a considerable amount of RA binding following treatment with MMTS, we  Table I. with a small functional group compared with a large bulky group on RA binding, we have examined the effect of cyanolysis of DTNB-modified C258A and C267A. As demonstrated above in Fig. 4, inhibition of RA binding of DTNB-modified RAR␤ involves the modification of Cys 228 . Treatment of DTNB-modified Cys residues with NaCN results in the replacement of the bulky thiobis(2-nitrobenzoic acid) group with thiocyanide which is similar in size to the thiomethyl group transferred upon MMTS treatment (33,34). It is important to note that MMTS-treated C267A and DTNB-modified C267A treated with In a duplicate set of reactions the MMTS modified C267A was treated with either 10 mM DTT or 10 mM TBP as described in the legend to Fig. 1 (B). Following treatment with the sulfhydryl-modifying reagent and/or reducing reagents, RA binding was determined using 15 nM [ 3 H]RA as described in the legend to Fig. 1. The values presented are the mean Ϯ S.E. for at least three independent determinations performed in duplicate.
NaCN are virtually the same except for the presence of a thiomethyl group instead of a thiocyanide group on the modified Cys. Treatment of DTNB-modified C258A and C267A with NaCN results in a dose-dependent restoration of RA binding which plateaued at greater than 70% of the wild type RA binding at a concentration of 50 nM NaCN (data not shown). It is important to note that this level of RA binding is similar to the level of RA binding observed with MMTS-treated C228A and C267A. This suggests that modifications of Cys 228 which result in the transfer of small moieties such as thiocyanide and thiomethyl is not sufficient to fully inhibit RA binding. Taken together this suggests that loss of RA binding by MMTS treatment of RAR␤ is the result of the modification of both Cys 228 and Cys 267 . DISCUSSION In the current report we have extended the findings of Dallery et al. (20) to demonstrate that RA binding to all three RAR types is blocked similarly by specific sulfhydryl-modifying agents. The concentrations of DTNB and MMTS necessary to inhibit RA binding to RAR␤ and RAR␥ are similar to those which have been reported previously for the ligand binding domain of RAR␣ (20). The reversibility of this effect by reducing agents demonstrates the specificity of these reactions to sulfhydryl groups. Interestingly, unlike RAR␣ and RAR␤, RA binding by RAR␥ following MMTS treatment was only reversible to about 50% of control binding with the polar reducing agent DTT. However RA binding was restored to near unmodified levels using the hydrophobic reducing agent TBP. This suggests that there is at least one critical Cys residue responsible for inhibition of RA binding upon methanethiolation with MMTS which lies within a more hydrophobic region of RAR␥ than in RAR␤ and RAR␣. This region of greater hydrophobicity in the ligand binding pocket of RAR␥ compared with RAR␤ and RAR␣ is most likely one of several factors responsible for the ability of synthetic retinoids to display receptor selectivity.
The glucocorticoid receptor (GR), another member of the steroid/thyroid hormone superfamily of receptors, has also been shown to be sensitive to sulfhydryl modification (35). GR modified with MMTS exhibits a bimodal response in which inhibition of ligand binding is maximal both at low and high concentrations of the modifying reagent (36,37). It has been determined that this response is a result of intramolecular disulfide bond formation between two closely spaced Cys residues (vicinal dithiols) located in the ligand binding pocket of GR upon treatment with low concentrations of MMTS (36,37). We did not observe this biphasic pattern of inhibition of RA binding in any of the RARs treated with MMTS under similar conditions. In addition, our sodium arsenite results further support the conclusion that there are no reactive vicinal dithiols in any of the three RARs responsible for inhibition of RA binding.
The results of our protection-exchange experiment with RAR␤ demonstrating that RA binding can protect Cys residues from chemical modification suggests that these critical Cys residue(s) may be located in or near the ligand binding pocket. Protection may result from either the physical blocking of the modifying reagents by RA or a conformational change in the ligand binding domain upon RA binding which blocks the accessibility of the reactive Cys residues to the modification reagents. The latter is more likely based on the work of Renaud et al. (15) showing that the major difference in the crystal structure of apo-RXR␣ and holo-RAR␥ ligand binding domains is in the position of helix 12. In the apo-receptor the entrance to the RA binding site is open while in the holo-receptor helix 12 collapses over the entrance to the binding pocket blocking access to the site.
The loss of RA binding following treatment with MMTS and DTNB cannot be explained by the elimination of critical interactions between any of the Cys residues examined and RA since each of the three Cys mutants have an apparent K d for RA in the range of that of wild type RAR␤. Interestingly, the C228A mutant did display a 7-fold decrease in affinity for RA when compared with wild type RAR␤. This is not surprising since two amino acids of RAR␤, close in the primary sequence of Cys 228 (Ala 225 and Ile 232 ), have been demonstrated to be important for interaction with type-selective ligands (18). Furthermore, the crystal structure of holo-RAR␥ indicates that Cys 237 , the homologous residue to Cys 228 of RAR␤, lies within 4 Å of RA (15). It is possible that substitution of Cys 228 with an Ala removes an interaction between this Cys residue and RA which contributes to the overall stability of holo-RAR␤. On the other hand, it is also possible that this mutation has caused a small disruption of the local topography of the ligand binding pocket sufficient to result in this modest decrease in the affinity of C228A for RA.
Although inhibition of RA binding upon sulfhydryl modification of RAR␤ is a result of steric blocking of RA binding, the Cys residues whose modification is critical for this inhibition of RA binding differ depending on the reagent employed. Wild type RAR␤ displays similar sensitivity to DTNB and MMTS treatment; however, the C228A and C267A mutants represent novel receptors with unique sensitivity to these two sulfhydryl modifying reagents. Our finding of a differential response of these two mutants to reagents that transfer different size blocking groups is not unprecedented. It has been reported that specific sulfhydryl modification of rabbit muscle creatine kinase with blocking groups of increasing size resulted in the incremental decrease in enzymatic activity (38). Similar results have been found for the GR. Modification of critical Cys residues in GR with MMTS results in a greater amount of residual binding of ligand than modification by iodoacetamide which transfers a relatively large blocking group (36).
Our data demonstrate that both Cys 228 and Cys 267 are located within the ligand binding pocket of RAR␤ such that when modified they inhibit the binding of RA. However Cys 228 lies closer to the opening of the ligand binding pocket than Cys 267 because Cys 228 is accessible to modification by both large and small reagents and upon modification results in the inhibition of RA binding, whereas Cys 267 is only sensitive to small reagents. We have estimated the size of DTNB and MMTS by measuring the carbon center to carbon center distances on molecular models. The dimensions of MMTS, when viewed as a molecular structure, are 3.0 Å in height and 4.0 Å in width, whereas those of DTNB are 6.0 and 7.4 Å, respectively. Based on our sulfhydryl modification results, these measurements suggest that Cys 267 is located in a region of the pocket not accessible to molecules or side chains of molecules much larger than 4.0 Å in diameter. Since RA is no greater than 4.0 Å at its carboxylate end and at least 5.0 Å at the ␤-ionone ring, we can conclude that Cys 267 of RAR␤ is closest to the carboxylate end of RA. This conclusion is supported by the recently published crystal structure of the ligand binding domain of holo-RAR␥ which demonstrates that the homologous residue of Cys 228 (Cys 237 ) is within 4 Å of carbon 13 of RA (15). In addition, there is only one amino acid residue in the primary sequence between Cys 267 and Arg 269 of RAR␤ (Arg 278 of RAR␥). We have previously shown Arg 269 of RAR␤ to be important for RA binding and retinoid specificity most likely by interaction with the carboxyl group of RA (16,17), and the crystal structure of the ligand binding domain of RAR␥ places Arg 278 within 4 Å of the carboxylate oxygen 22 of RA (15). these studies. We also thank Dr. Charles Grubmeyer for many helpful discussions.