Helix 1/8 Interactions Influence the Activity of Nuclear Receptor Ligand-Binding Domains
Jason A. Holt,
Thomas G. Consler,
Shawn P. Williams,
Andrea H. Ayscue,
Lisa M. Leesnitzer,
G. Bruce Wisely and
Andrew N. Billin
Nuclear Receptor Discovery Research, GlaxoSmithKline Research and Development, Research Triangle Park, North Carolina 27709-3398
Address all correspondence and requests for reprints to: Andrew Billin, GlaxoSmithKline Research and Development, Research Triangle Park, North Carolina 27709-3398. E-mail: andrew.n.billin{at}gsk.com.
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ABSTRACT
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The ligand-binding domain (LBD) of apo-nuclear receptors in solution is thought to be a very dynamic structure with many possible conformations. Upon ligand binding, the structure is stabilized to a more rigid conformation. The dynamic stabilization assay is a LBD reassembly assay that takes advantage of the high specificity of the intramolecular interactions that comprise the ligand-bound LBD. Here, we demonstrate dynamic stabilization for the nuclear receptors peroxisome proliferator-activated receptor (PPAR)
and nerve growth factor inducible (NGFIB)ß and identify residues important for stabilization of the intramolecular interactions induced by PPAR
ligands. Site-directed mutagenesis studies identified residues in helices 1 and 8 required for LBD reassembly. Further, disrupting the helix 1/8 interaction in the context of the holo-LBD alters the response of the receptor in a compound-specific manner, suggesting that residues far from the ligand-binding pocket can influence the stability of the ligand-bound receptor. Thus, these results support and extend models of the apo-LBD of PPAR
as a dynamic structure.
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INTRODUCTION
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NUCLEAR RECEPTORS REGULATE gene transcription and are divided into three basic classes. The first two classes, the endocrine and adopted receptors, contain receptors with known physiological ligands such as hormones, fatty acids, cholesterol, and bile acids. The third class, the orphan receptors, are not regulated by any known small molecule (1). For the ligand-regulated nuclear receptors (NRs), binding of a small molecule provokes a change in its activity, either activating or repressing transcription. Thus, these receptors link flux in hormones and metabolites to gene expression. As a class, these receptors have been identified as key players in a variety of biological processes including the regulation of metabolism, reproduction, development, and apoptosis. Hence, they are excellent targets for drug development, and many of the receptors are already known targets for therapeutically useful compounds (1, 2). Identification of the precise structure/function relationships that govern the transcriptional activity of the NRs will help guide the development of highly selective and efficacious drugs.
Structural and functional studies of a number of NRs have led to a detailed mechanistic understanding of ligand-dependent transactivation (3). Ligand-dependent activation occurs through the approximately 250-amino-acid ligand-binding domain (LBD) at the carboxyl terminus of the protein. The overall structure of the NR LBD appears to be an antiparallel sandwich of between 11 and 13
-helices. This predominantly
-helical domain is multifunctional, acting as a ligand binding, dimerization, and transactivation domain (4). The ligand-binding pocket is a hydrophobic cavity in the interior of the LBD, whereas exposed on the surface of the LBD is the transcriptional activation function 2 (AF2) domain in the C-terminal
-helix. Agonist binding causes a conformational change that results in the AF2 being positioned for efficient recruitment of coactivators, primarily of the p160 and vitamin D receptor-interacting protein/thyroid receptor-associated protein family (3, 4). The extent of movement of the AF2 in response to ligand binding is thought to vary by receptor type. For example, the change in conformation of AF2 in RXR is thought to be large, whereas the change in conformation of AF2 in PPAR
and PPAR
is thought to be relatively subtle (5, 6, 7, 8, 9). Thus, conformational changes induced by ligand binding are conserved features of the NR transactivation mechanism.
Many structural techniques suggest that the transition from apo-receptor to ligand-bound receptor involves large-scale conformational changes. First, limited proteolysis of apo- and ligand-bound LBDs reveals differential cleavage patterns suggestive of widespread ligand-induced conformational changes (10, 11, 12, 13, 14, 15). Second, thermal denaturation studies have demonstrated large ligand-induced shifts in melting temperature, again suggestive of ligand-induced conformational changes throughout the LBD (16). Third, nuclear magnetic resonance studies of PPAR
and PPAR
both demonstrate global changes in the conformation of the receptor in response to ligand binding (17, 18). Fourth, many conformational changes were identified in the structure of the PPAR
LBD when bound to rosiglitazone as compared with the apo-receptor (7). Thus, ligand binding appears to alter the conformation of multiple locales in the LBD. The recently described dynamic stability (DS) assay examines the ability of ligand to reassemble a LBD split between helix 2 and the rest of the LBD (19, 20). Because this assay detects a ligand-dependent interaction between helices 12 and the remainder of the LBD, it is likely detecting conformational changes independent of the AF2. We have extended these initial findings to demonstrate the DS effect for PPAR
and nerve growth factor inducible (NGFIB)ß, receptors of the adopted and orphan class, respectively. We have also identified residues in helices 1 and 8 that are crucial for the reassociation of the split LBD in the DS assay. Further, we have determined that the interaction of helices 1 and 8 is crucial for the full activity of the ligand-bound PPAR
LBD. Our results support models of the apo-LBD of PPAR
as a dynamic structure.
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RESULTS
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We wished to determine whether the DS assay could be applied to a broader range of NRs as well as understand the molecular basis for the interactions that govern the stabilization of the LBD (see Fig. 1
for an overview of the structural features considered in this work). For this study, we chose to focus on the receptors PPAR
and NGFIBß. Previous results for the thyroid hormone receptor (TR) suggested that the region encompassing approximately 12 amino acids N terminal to the beginning of helix 1 and amino acids extending to just before helix 3 were sufficient to detect association of the separated receptor domains in mammalian two-hybrid experiments (19). We constructed a Gal4 fusion encompassing amino acids 197273 of PPAR
. Amino acid 197 is 12 residues N terminal of the start of helix 1 and amino acid 273 is three amino acids N terminal to the beginning of helix 3 in the crystal structure of the PPAR
receptor (7). The PPAR
herpes simplex virus protein 16 (VP16) fusion begins at amino acid 274 and continues to the last amino acid of the receptor. The ability of these proteins to associate was tested in the mammalian two-hybrid assay by holding the concentration of the transfected Gal4 PPAR
helix 12 plasmid constant, increasing the amount of transfected VP16 PPAR
helix 312 plasmid, and determining the effect on reporter gene expression. Figure 2A
shows that titration of increasing amounts of the PPAR
VP16 fusion plasmid resulted in increasing levels of reporter gene activity. With 4 ng of the Gal4 PPAR
helix 12 plasmid and 80 ng of the PPAR
VP16 helix 312 plasmid transfected, approximately 10-fold activation was observed. The experiment was performed in charcoal-stripped, delipidated serum and with no added synthetic ligand, and thus this interaction is likely the result of a mass action effect on the association of the two PPAR
LBD pieces.

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Fig. 1. Ribbon Diagram of the Human PPAR LBD
Helical segments involved in the DS effect are indicated. The AF-2 helix is in pink, and regions contacted by coactivators are indicated in yellow. Rosiglitazone is shown bound in the interior of the LBD.
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Fig. 2. Reassembly of the Split LBDs of PPAR and NGFIBß
A and B, CV-1 cells were transfected with the indicated quantities of the expression plasmids and the normalized luciferase values were plotted as fold activation over the basal expression of the reporter plasmid.
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Next we determined whether the DS interaction for PPAR
was sensitive to ligand. The mammalian two-hybrid plasmids were transfected at concentrations that did not produce a signal from the reporter gene in previous assays (see Fig. 2A
). Including a 1-µm concentration of the PPAR
ligand GW1929 (21) produced a robust activation of the reporter gene (Fig. 3A
), indicating ligand-dependent interaction of the two LBD fragments. Indeed, the signal produced from the ligand-treated cells was much more robust than that produced from cells transfected with the highest amount of PPAR
VP16 fusion plasmid (Fig. 2A
). Next we measured the dosage-dependent response of PPAR
to GW1929 in the DS assay. Strikingly, GW1929 was much less potent in the DS assay than in the wild-type PPAR
holo-LBD Gal4 fusion (Fig. 3B
). The EC50 for GW1929 with wild-type PPAR
is approximately 4 nM, whereas the EC50 for GW1929 in the PPAR
DS assay is approximately 118 nM, nearly a 30-fold drop in potency. To determine whether other synthetic ligands also display altered potencies in the DS assay, we tested a representative thiazolidinedione (TZD) pioglitazone, and the non-TZD PPAR
agonist GW0072 (22). The EC50 for pioglitazone is approximately 300 nM in the wild-type PPAR
assay and approximately 7100 nM in the DS assay, an approximately 24-fold reduction in the apparent potency. Similarly, GW0072 is approximately 28-fold less potent with an EC50 of 119 nM in the wild-type PPAR
assay and an EC50 of approximately 3300 nM in the DS assay. These data demonstrate that the DS PPAR
assay is sensitive to both agonists (GW1929, pioglitazone) and modulator ligands (GW0072). However, the potency of the compounds is 20- to 30-fold lower in the PPAR
DS assay than in the holo-LBD assay.
To define amino acid residues required for the interaction of the two fragments, we examined the crystal structure of PPAR
for possible intramolecular interactions that may mediate the interaction of helix 1 with the rest of the LBD. Inspection of the crystal structure of the PPAR
LBD (7) revealed that helix 1 appears to have amphipathic characteristics with hydrophobic residues packed against helix 8 (Fig. 1
). The side chain of L218 in helix 1 and I386 in helix 8 are approximately 4 Å apart and appear to make particularly strong hydrophobic contacts. We reasoned that mutation of L218 or I386 would likely have a profound effect on the interaction of helices 1 and 8. However, changing the amino acid composition of a helical structure may impair its ability to form an
-helix. Thus, we first determined whether mutating L218 and I386 to alanine or glutamate would compromise the ability of helix 1 or 8 to form an
-helix and compromise their secondary structure.
Peptides encompassing helix 1 or 8 were synthesized either as the wild-type sequence or as the mutants L218A, L218E, I386A, and I386E. Circular dichroism spectroscopy was used to measure the helical content of the peptides and discern whether the mutants deviated significantly in their propensity to form helical structures from the wild type. Short peptides usually do not form secondary structure in aqueous solution because of competition from the solvent for structure stabilizing intramolecular hydrogen bonds. TFE is commonly used to stabilize
-helical structure in peptides with inherent helical propensity (23) and was used here to determine the helical propensity of the PPAR
peptides. In 0% TFE, the helix 1 peptides displayed between 9 and 13%
-helical content (Table 1
). In 16.6% TFE, the peptides displayed increased
-helical content: wild-type 38%, L218A 47%, L218E 33% (Table 1
). Thus, addition of TFE increased the helical content of the peptides, and this is consistent with the structure of helix 1 derived from the crystal structure. The L218A mutant had slightly greater helical propensity than the wild-type peptide. The helical content of the glutamate substitution for helix 1 is slightly less than that of the wild-type peptide but still displays intrinsic helical propensity (wild-type 38% vs. L218E 33%). Similarly, the helix 8 peptides (I386, I386A, I386E) were analyzed for
-helical propensity by circular dichroism (Table 1
). In 0% TFE all three peptides had
-helical content: wild type, 41%, I386A 38%, I386E 24%. In 16.6% TFE, the wild-type peptide was relatively unchanged but both the mutants displayed increased helical content: 79% for I386A and 98% for I386E. Thus, the mutations do not have a deleterious effect on the structure of the peptides and even appear to increase the
-helical propensity of the helix 8 peptides. The reason for the wild-type helix 8 peptide not increasing in helical propensity in 16.6% TFE is not known, but may have to do with the additional amino acids incorporated into this peptide (see Materials and Methods). These numbers compare favorably with other helical regions as isolated peptides. In fact, the PPAR
peptides have a stronger helical propensity than the Sin3A interaction domain of Mad1, a known
-helix (24, 25).
To determine the contribution of the predicted interactions between helices 1 and 8 to the DS effect, L218 and I386 were changed to alanine or glutamate in the context of the appropriate chimeric fusion, and the resultant mutants were tested for function in the DS assay. The mutations did not appear to drastically alter the expression levels of the proteins (Fig. 4A
). L218A was inactive when tested with wild-type PPAR
helix312VP16 regardless of the inclusion of GW1929 (Fig. 4B
). Thus, replacement of the hydrophobic leucine residue with the small side chain of alanine has severe consequences for the productive interaction of the LBD fragments in the DS assay. Because the replacement is unlikely to disrupt the helical propensity of the helix 1 peptide, this effect is likely to be a direct consequence of disrupting hydrophobic interactions mediated by helix 1. Replacement of L218 with glutamate also abolished the interaction of the two fragments regardless of the inclusion of ligand in the assay (Fig. 4C
). Due to the very different chemical properties of leucine and glutamate side chains, this mutation is likely to have drastic effects on the hydrophobic interactions of helices 1 and 8. In addition, this mutation slightly reduces the helical propensity of the helix 1 peptide and this could also contribute to disrupting the interaction.

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Fig. 4. Effect of Mutations in Helices 1 and 8 of PPAR on LBD Reassembly
A, Western blot of tansfected cells showing relative expression levels of the wild-type and mutant chimeric proteins. B, Alanine substitutions in helix 1 (L218A) or 8 (I386A) of PPAR were tested for LBD DS by transfecting the indicated plasmids into CV-1 cells and determining the activity of the reporter gene. Where appropriate, GW1929 was added to 1 µM. C, Glutamate substitutions in helix 1 (L218E) or helix 8 (I386E) were tested as in B.
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Mutations in helix 8 also abolished DS of the PPAR
LBD. The I386A mutation prevented a productive interaction in the DS assay in the absence of ligand, even though with the same quantity of input plasmids the wild-type fragments produce a readily detectable signal in the assay (Fig. 4B
). Upon addition of GW1929, an approximately 5-fold increase was seen with wild-type helix 12 and I386A helix 312. This increase was approximately 4-fold lower than that seen with the wild-type fragments. Thus, this mutation attenuates the ability of the two fragments to interact. Similar to the results obtained with the L218E mutant, the I386E mutation abolished the interaction of the two LBD fragments in the DS assay (Fig. 4C
). Together, these data suggest that the hydrophobic contact between L218 and I386 is crucial for the interaction of helix 1 with the remainder of the LBD.
To determine whether the intramolecular interaction between L218 and I386 is significant in the context of the holo-LBD, we introduced the same substitutions (L218A, L218E, I386A, I386E) into the PPAR
LBD as a chimeric molecule with the GAL4 DNA-binding domain. The mutants were tested in dose-response, transient transfection assays with GW1929, pioglitazone, and GW0072 (Table 2
). For GW1929 the EC50 for the wild-type receptor was 4 nM as was the EC50 for L218A and I386A. Thus, this set of mutations did not alter the response of the receptor to GW1929. However, for the L218E and I386E mutations the results were pronounced. The EC50 for GW1929 on L218E was 1200 nM and on I386E the EC50 was approximately 273 nM. The EC50 for pioglitazone on the wild-type receptor was approximately 300 nM. The EC50 for the L218A mutant was approximately 2200 nM and for the I386A mutant was approximately 873 nM. For the L218E and I386E mutants, pioglitazone failed to activate the receptor. Thus, for this compound, transcriptional activity of the receptor is severely impaired when either of the two residues is mutated. For the partial agonist GW0072, the wild-type receptor EC50 was approximately 119 nM. The L218A receptor EC50 was greater than 1000 nM. The EC50 for the I386A mutation was approximately 640 nM. For the L218E and the I386E mutation, the EC50s were also greater than 1000 nM. Taken together, the results suggest that structural features not involved in direct ligand binding or transcriptional activation can alter the transcriptional response of the receptor.
To determine whether ligand binding is altered indirectly by disrupting helix 1/8 interactions, we expressed the PPAR
mutants as glutathione-S-transferase and 6X histidine fusion proteins and tested them for ligand binding. All of the constructs expressed soluble protein. Only the wild-type and I386A mutant retained binding activity for radiolabeled rosiglitazone (data not shown). This was unexpected because all of the mutants retained some transcriptional activity in the cell based assays (Table 2
). To determine whether the I386A mutant alters the binding affinity of the receptor for ligand, we performed a competition binding assay with biotinylated wild-type PPAR
bound to SPA (scintillation proximity assay) beads, radiolabeled rosiglitazone, and unbiotinylated wild-type PPAR
or the I386A mutant as a competitor. As can be seen in Fig. 5
the inhibition constant for the wild-type receptor is approximately 5.2 nM, and the inhibition constant for the I386A mutant is approximately 28 nM. Thus, the mutant is impaired for binding rosiglitazone. Therefore, disrupting the helix 1/8 interaction can alter the binding characteristics of the ligand-binding pocket. Further, not all compounds behave similarly with the mutant receptors. GW1929 has wild-type transactivation characteristics on the I386A mutant, whereas the EC50 for pioglitazone (structurally related to rosiglitazone) is about 3-fold higher on I386A than on the wild-type receptor. Thus, these mutant receptors are most likely detecting differences in the ability of the compounds to bind and stabilize the LBD and this activity may be related to the potency of the compound.
As a representative of another class of orphan receptors, we determined whether the appropriate pieces of the LBD of NGFIBß could be stimulated to associate in a ligand-independent manner. We constructed a Gal4 fusion encompassing amino acids 346 to 399 of NGFIBß. Amino acid 346 is 12 residues N terminal of the putative start of helix 1 and amino acid 399 is the last amino acid just before the predicted beginning of helix 3. The NGFIBß VP16 fusion begins at the presumed first amino acid of helix 3 and continues to the last amino acid of the receptor. The ability of the NGFIBß fusion proteins to associate was tested in the mammalian two-hybrid assay by holding the concentration of the Gal4 NGFIBß helix 12 plasmid constant, increasing the amount of NGFIBß VP16 helix 312 plasmid, and determining the effect on reporter gene expression. Figure 2B
shows that titration of increasing amounts of the NGFIBß VP16 fusion plasmid resulted in increasing levels of reporter gene activity. Again, this experiment was performed in charcoal-stripped, delipidated serum, and therefore the observed interaction is likely to be due to the effect of mass action on the association of the two LBD pieces. Thus, independent of added synthetic ligands, receptors from very different NR subfamilies display LBD reassembly indicative of DS.
To determine whether the hydrophobic interactions between helices 1 and 8 observed with PPAR
are also responsible for the association of the NGFIBß fusion proteins, we mutated the homologous amino acid residues (L368 in helix 1 and I506 in helix 8). The mutations did not significantly alter the levels of expressed proteins (Fig. 6A
). Changing L386 in helix 1 or I506 in helix 8 to alanine completely blocked the DS interaction for NGFIBß (Fig. 6B
). Similarly, glutamate substitution also prevented reassembly of the NGFIBß LBD (Fig. 6C
). Thus, the structural determinants of helix 1/8 interactions are conserved between distinct members of the NR superfamily.

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Fig. 6. Effect of Mutations in Helices 1 and 8 of NGFIBß on LBD Reassembly
A, Western blot of transfected cells showing relative expression levels of the wild-type (WT) and mutant (MT)chimeric proteins. B, Alanine substitutions in helix 1 (L368A) or 8 (I506A) of NGFIBß were tested for LBD DS by transfecting the indicated plasimds into CV-1 cells and determining the activity of the reporter gene. C, Glutamate substitutions in helix 1 (L386E) or 8 (I506E) were tested as in B.
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DISCUSSION
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The DS effect was previously demonstrated for members of the classical endocrine receptors and the retinoid X receptor (19, 20). We have extended these findings in the following ways. First, we determined that the DS effect is also a property of the adopted orphan PPAR
and the orphan receptor NGFIBß. Second, we determined that the sensitivity of the DS assay and the Gal4/NR LBD chimera assay are markedly different. Our results show that the three distinct PPAR
ligands tested here profile as being significantly less potent in the DS assay as compared with the Gal4LBD chimera assay. Third, we demonstrated by site-directed mutagenesis that the hydrophobic surface between helices 1 and 8 is crucial for the association observed in the DS assay. The helix 8 mutation also demonstrates that the interaction between the helix 1 fusion protein and the rest of the receptor represents a native interaction and not a fortuitous interaction with another part of the protein (e.g. the coactivator groove). Further, we demonstrated that the helix 1/8 interaction is conserved in a distinct NR family member, NGFIBß. Fourth, we show that the interaction between helices 1 and 8 is critical for the wild-type response of the PPAR
LBD to pioglitazone and GW0072, but not GW1929. Thus, LBDs harboring mutations in L218 of helix 1 or I386 of helix 8 display novel compound specific alterations in their response to ligand. This result is consistent with a model of receptor activation whereby ligand binding stabilizes the entire LBD. Further, it suggests that the stability of regions far from the known cofactor interaction surfaces is critical to promote efficient transactivation.
We have demonstrated that the DS assay can be used to reassemble the LBDs of PPAR
and NGFIBß. In the case of both of these receptors, reassembly was first demonstrated in a mammalian two-hybrid (M2H) assay, in the absence of added exogenous ligand, via overexpression of the helix 312 VP16 fusion. This result suggests that the DS assay may be especially useful in screening orphan receptors because the "functionality" of the assay can be determined without the use of a previously characterized ligand. We next showed that PPAR
ligands could stimulate the interaction of the two LBD fragments even when no basal interaction could be detected in the DS assay. Thus, as for other receptors in this assay, ligand acts to greatly facilitate the interaction of the LBD fragments. However, when tested in a dose-response experiment for their efficacy and potency in the DS assay, the PPAR
ligands were uniformly less potent than in the Gal4-LBD chimera assay. Why the disparity? The two assays measure different molecular events. The Gal4 holo-LBD chimera assay measures the transcriptional activity of the AF2 helix in response to ligand binding, whereas the DS assay is measuring the increased affinity of the large LBD fragment for the helix 12 fragment in response to ligand. Also, the ligand affinity to a truncated receptor in the DS assay may be different from that of the native holo-LBD tested in the Gal4-LBD assay. This result suggests that the DS assay is not as sensitive as the Gal4-LBD assay and ligands of low potency [such as some fatty acids in the case of the PPARs (26)] may not be detected in DS screens. Despite this note of caution, our results with PPAR
and NGFIBß, along with previously published results, indicate that the DS effect is likely to be useful in ligand screening experiments where predictive cofactor interactions are not known.
To determine the molecular basis for the helix 1/LBD interaction in PPAR
, we used the crystal structure of the rosiglitazone bound LBD to identify candidate amino acids in helices 1 and 8. Leucine 218 (helix 1) and isoleucine 386 (helix 8) are approximately 4 Å apart and are likely to make hydrophobic contacts. Our mutational analysis demonstrates that these two residues are critical for the association of helices 1 and 8. Mutation of either one of the residues to alanine or glutamate had drastic effects on the ability of the two fragments to associate in the M2H assay regardless of the presence of ligand. Mutating the homologous residues in NGFIBß (L386 and I506) to alanine or glutamate abolished the DS interaction for this receptor. Thus, our data also support a model in which the critical hydrophobic residues identified in helices 1 and 8 of PPAR
mediate the interaction of helices 1 and 8 in other NRs. Because most of the NRs have a hydrophobic residue at the positions homologous to L218 and I386 of PPAR
(data not shown), this interaction is probably an evolutionarily conserved feature of NR structure.
We next determined the role of helices 1 and 8 in the activity of the holo-LBD of PPAR
by separately introducing the L218A and I386A mutations into the PPAR
LBD/Gal4 chimera and examining its activity in a transient transfection assay. The dose response of the L218A and I386A receptor when compared with wild type was normal for GW1929. This result demonstrates that the L218A and I386A mutant receptors are capable of full activity. In contrast to GW1929, the potency of pioglitazone and GW0072 was greatly reduced. These results show that mutations in regions of the receptor far from the ligand-binding pocket and AF2 can have profound effects on the transcriptional activity of the receptor. The mutations are unlikely to have a direct effect on the conformation of the AF2 helix because helices 1 and 8 are very far from this structure. Another argument that the conformation of the AF2 helix is not directly altered in the mutants comes from the mode of binding of the compounds themselves. The acid head group of the glitazone class of compounds and of GW1929 is known to directly contact the AF2 helix via hydrogen bonds to the same group of amino acids (S289, H323, H449, and Y473) (7, 27). Because the transcriptional response of the L218A and I386A mutants to GW1929 is not altered relative to wild type, but their response to pioglitazone is altered, it is unlikely that the ability of the compounds to contact AF2 is altered in the mutants. Also, GW0072 does not make contacts with the AF2 helix (22) and it is less potent on both of the alanine substitution mutants. Thus, the ability of the ligands to contact the AF2 helix is not strictly correlated with their activity. Some other property of the mutant receptors is the likely source of the ligand-specific activities.
One possibility is that ligand binding itself is altered by disruption of the interaction between helices 1 and 8. Only the Escherichia coli produced wild-type protein and the I386A mutant were capable of binding radiolabeled rosiglitazone in saturation binding assays. This observation was unexpected for the alanine mutants because all of these mutants functioned to some degree in the transient transfection assay with pioglitazone (Table 2
). However, this observation is compatible with the loss of pioglitazone responsiveness seen for the glutamate substitutions. To test the hypothesis that NR cofactors may help stabilize the mutant LBD in mammalian cells, we expressed the mutant PPAR
LBDs as glutathione-S-transferase fusions in an E. coli strain that coexpresses the NR interaction domain of the coactivator steroid receptor coactivator-1. Despite the presence of large amounts of the coactivator we did not detect increased ligand-binding activity for any of the mutants (data not shown). We do not currently understand why the mutant receptors retain activity in the mammalian cell-based assays but not when expressed in E. coli.
In a competition binding assay with wild-type PPAR
LBD, the 6X histidine I386A mutant binds rosiglitazone 5- to 6-fold less well than wild-type PPAR
. This correlates well with the approximately 3-fold right shift in the dose response curve for pioglitazone in the transient transfection assay for this mutant. Thus, disrupting normal helix 1/8 interactions may alter the affinity of the receptor for ligand. This is an unexpected and novel finding because these two helices are on the surface of the LBD and do not form part of the hydrophobic core of the LBD. We speculate that the helices 1 and 8 mutations may have a destabilizing effect on the structure of the LBD that results in a lower affinity for ligand. GW1929 is much more potent than pioglitazone, and the I386A mutant behaves as the wild-type receptor in the transactivation assay with GW1929. This compound may be able to overcome the altered binding properties of the I386A ligand-binding pocket.
Because the packing of the interior of the receptor is mostly driven by hydrophobic interactions, the relative lipophilicity of the ligand and the extent of its hydrophobic interactions with the binding pocket may determine the potencies observed with the mutants in the holo-LBD transient transfection assays. Indeed, the 50-fold higher binding affinity of farglitazar (GI 262570) over rosiglitazone for PPAR
has been attributed to the extensive hydrophobic interactions made by farglitazar with the binding pocket that are not possible with the TZD class of ligands (27). The response of the alanine mutations to GW1929 is identical to that of the wild-type receptor, even though the potency of pioglitazone and GW0072 is reduced on the alanine substitution mutants. Because GW1929 belongs to the same chemical series of tyrosine analogs as farglitazar, it is possible that the hydrophobic contacts made by this compound can overcome the destabilizing effects of the alanine substitutions and stabilize the LBD sufficiently to lock it in the active conformation.
The L218E and I386E mutants had drastic effects on the transcriptional activity of the PPAR
Gal4LBD fusions. Again, this is likely due to a reduction in the affinity of ligand for the receptor. Pioglitazone was completely inactive, GW1929 was much less potent, and GW0072 had reduced potency similar to that observed with the alanine substitutions. Because the glutamate substitution is likely to have a more drastic effect on the interaction of helices 1 and 8 than the alanine substitutions, it is not surprising that the glutamate mutants are more severely attenuated in their ability to respond to ligand. GW1929 is still active, especially on the I386E mutant, and this is consistent with the hypothesis that the greater degree of lipophilicity of this ligand can overcome some of the stabilization lost due to the mutations in helix 1 or 8.
In summary, we have shown that DS is a conserved feature of NRs as diverse as PPAR
and NGFIBß. Further, we have used structure-based predictions to identify specific residues required for the interaction of helices 1 and 8. These two residues are required both for the DS effect, and for efficient transcriptional activation in the holo-LBD. We conclude that the interaction between helices 1 and 8 is required to maintain the conformation of the receptor. Disrupting the helix 1/8 interaction likely alters the ability of the LBD to bind ligand, and thus these surface interactions contribute to the stability of the ligand-binding core of the receptor. We predict that this interaction is a common feature of conformational stabilization because hydrophobic residues are found in homologous positions in most NRs. Further, we have identified a ligand-dependent interaction that is independent of receptor cofactors, but that is required for full receptor activity.
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MATERIALS AND METHODS
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Cell Culture
CV-1 cells were maintained for growth in DMEM (GIBCO/Invitrogen life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Irvine Scientific, Santa Ana, CA), 100 U/ml penicillin G sodium/100 µg/ml streptomycin sulfate (GIBCO/Invitrogen Life Technologies), and 2 mM L-glutamine (GIBCO/Invitrogen Life Technologies). Passaging was performed every 34 d at a 1:10 dilution. For experiments CV-1 cells were plated in 96-well plates at a density of 24,000 cells/well in phenol red-free DMEM:Nutrient Mixture F-12 (Ham) 1:1 (GIBCO/Invitrogen Life Technologies) supplemented with 10% charcoal/dextran-treated fetal bovine serum (Hyclone, Logan, UT), 100 U/ml penicillin G sodium/100 µg/ml streptomycin sulfate, and 2 mM L-glutamine.
Cell Transfection and Drug Treatments
Transfections were performed 24 h after cells were plated. LipofectaMINE Reagent (Invitrogen Life Technologies, Carlsbad, CA) was used for transfecting cells and performed essentially according to manufacturers instructions. For M2H assays 8 ng upstream activating sequence-thymidine kinase-Luciferase reporter, 8 ng ß-actin secreted placetal alkaline phosphastase, and varying amounts of pBluescript carrier plasmid, pVP16 fusion constructs and Gal4 fusion constructs were used. For Gal4 holo-LBD assays 8 ng ß-actin secreted placental alkaline phosphatase, 8 ng upstream activating sequence-thymidine kinase-Luciferase, 56 ng pBluescript carrier plasmid, and 8 ng Gal4 PPAR
LBD wild type or mutant was transfected into each well. Drug dilutions were prepared in phenol red-free DMEM:nutrient mixture F-12 (Ham) 1:1 (GIBCO/Invitrogen Life Technologies) supplemented with 10% charcoal-stripped, delipidated, heat-inactivated calf serum (Sigma, St. Louis, MO), 100 U/ml penicillin G sodium/100 µg/ml streptomycin sulfate, and 2 mM L-glutamine. Treatments included the PPAR
full agonists GW1929 (21) and pioglitazone and the PPAR
partial agonist GW0072 (22). Cells were incubated for 24 h in the presence of drugs, after which the medium was sampled and assayed for alkaline phosphatase activity. Luciferase reporter activity was measured using the LucLite assay system (Packard Instrument Co., Meriden, CT).
Construction of NR Constructs
Gal4 human PPAR
(hPPAR
) 197273 (helix 12) (accession no. L40904), Gal4 hPPAR
holo-LBD, and Gal4 hNGFIBß346399 (helix 12) (accession no. X75918) were generated by PCR. The cDNAs encoding amino acids 197273, 274-STOP, and 346399, respectively, were fused to amino acids 1147 of the yeast transcription factor Gal4 and inserted into the pSG5 expression vector (Stratagene, La Jolla, CA). VP16 hPPAR
274-STOP (helix 312) and VP16 NGFIBß 400-STOP (helix 312) were generated by PCR and the cDNAs encoding amino acids 274-STOP and 400-STOP, respectively, were fused to the VP16 activation domain in the pVP16 vector (CLONTECH, Palo Alto, CA). Because no crystal structure is available for NGFIBß, residues were identified for mutation by alignment and visual inspection of the putative helices 1 and 8 regions. All mutations were created by using the QuikChange Site Directed Mutagenesis Kit (Stratagene) and were performed essentially to manufacturers protocol. All constructs were verified by nucleotide sequencing.
Antibodies and Western Blot Analysis
Whole cell extracts were made in 3x sodium dodecyl sulfate loading buffer and resolved on 420% SDS-PAGE gels. Western blotting was by standard techniques. The following antibodies were used: anti-Gal4 (Santa Cruz Biotechnology, Santa Cruz, CA; SC-510), anti-NGFIBß/Nurr1 (Santa Cruz Biotechnology, SC-990).
PPAR
Binding Assays
PPAR
binding assays were performed essentially as described (28).
Peptide Synthesis
Peptides representing wild-type and mutant helices 1 and 8 from human PPAR
were synthesized (SynPep Corp., Dublin, CA) as follows: wild-type helix 1, PESADLRALAKHLYDSIKSFPLT; L218A, PESADLRALAKHAYDSIKSFPLT; L218E, PESADLRALAKHEYDSIKSFPLT. Wild-type helix 8, NALELLDDSDLAIFIAVIILLSGDR; I386, LELLDDSDLAAFIAVIILLSGD; I386E, LELLDDSDLAEFIAVIILLSGD. Note that the wild-type helix 8 peptide is two residues longer on the N-terminal side and one residue longer on the C-terminal side than the two mutants. This was necessitated by difficulties in synthesizing the shorter wild-type peptide.
Circular Dichroism Experiments
All peptides were solubilized in PBS [100 mM sodium phosphate (pH 8), 150 mM sodium chloride], at a nominal concentration of 50 µM (by total peptide weight, assuming a 70% peptide content). The concentration of each peptide solution was subsequently determined by quantitative amino acid analysis and these values were used in the data conversion to mean residue ellipticity. The actual peptide concentrations ranged from 3062 µM (peptide content ranged from 4386%). Trifluoroethanol was added directly to the peptide solutions and the solution was allowed to equilibrate at 25 C for 30 min before spectroscopy. Circular dichroism spectra were measured in 1-mm pathlength cuvettes in PBS at 25 C using an Aviv 62DS spectropolarimeter (Aviv Associates, Lakewood, NJ). Mean residue ellipticity was calculated by dividing the raw ellipticity by the product of the pathlength (0.1 cm) and the mean residue concentration in decimolar (10 x n x molar concentration, where n is the number of residues in the peptide). The percent
-helix was calculated based upon 0% and 100% helix representing a mean residue ellipticity at 222 nm of -2,000 deg cm2 dmol-1 and -28,400 deg cm2 dmol-1, respectively (29).
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ACKNOWLEDGMENTS
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We thank Tim M. Willson and John T. Moore for critical reading of the manuscript.
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FOOTNOTES
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Abbreviations: AF, Activation function; DS, dynamic stability; hPPAR
, human PPAR
; LBD, ligand-binding domain; M2H, mammalian two-hybrid; NGFIB, nerve growth factor inducible; NRs, nuclear receptors; PPAR, peroxisome proliferator-activated protein; SPA, scintillation proximity assay; TR, thyroid hormone receptor; TZD, thiazolidinedione.
Received for publication January 2, 2003.
Accepted for publication June 11, 2003.
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REFERENCES
|
---|
- Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ 2001 Nuclear receptors and lipid physiology: opening the X-files. Science 294:18661870[Abstract/Free Full Text]
- Willson TM, Brown PJ, Sternbach DD, Henke BR 2000 The PPARs: from orphan receptors to drug discovery J Med Chem 43:527550[CrossRef][Medline]
- Rosenfeld MG, Glass CK 2001 Coregulator codes of transcriptional regulation by nuclear receptors. J Biol Chem 276:3686536868[Free Full Text]
- Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835839[Medline]
- Bourguet W, Ruff M, Chambon P, Gronemeyer H, Moras D 1995 Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-
. Nature 375:377382[CrossRef][Medline]
- Egea PF, Mitschler A, Rochel N, Ruff M, Chambon P, Moras D 2000 Crystal structure of the human RXR
ligand-binding domain bound to its natural ligand: 9-cis retinoic acid. EMBO J 19:2592601[Abstract/Free Full Text]
- Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH, Kurokawa R, Rosenfeld MG, Willson TM, Glass CK, Milburn MV 1998 Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-
. Nature 395:137143[CrossRef][Medline]
- Uppenberg J, Svensson C, Jaki M, Bertilsson G, Jendeberg L, Berkenstam A 1998 Crystal structure of the ligand binding domain of the human nuclear receptor PPAR
. J Biol Chem 273:3110831112[Abstract/Free Full Text]
- Xu HE, Lambert MH, Montana VG, Parks DJ, Blanchard SG, Brown PJ, Sternbach DD, Lehmann JM, Wisely GB, Willson TM, Kliewer SA, Milburn MV 1999 Molecular recognition of fatty acids by peroxisome proliferator-activated receptors. Mol Cell 3:397403[Medline]
- Attardi B, Happe HK 1986 Comparison of the physicochemical properties of uterine nuclear estrogen receptors bound to estradiol or 4-hydroxytamoxifen. Endocrinology 119:904915[Abstract]
- Vaisanen S, Juntunen K, Itkonen A, Vihko P, Maenpaa PH 1997 Conformational studies of human vitamin-D receptor by antipeptide antibodies, partial proteolytic digestion and ligand binding. Eur J Biochem 248:156162[Abstract]
- Minucci S, Leid M, Toyama R, Saint-Jeannet JP, Peterson VJ, Horn V, Ishmael JE, Bhattacharyya N, Dey A, Dawid IB, Ozato K 1997 Retinoid X receptor (RXR) within the RXR-retinoic acid receptor heterodimer binds its ligand and enhances retinoid-dependent gene expression. Mol Cell Biol 17:644655[Abstract]
- Couette B, Fagart J, Jalaguier S, Lombes M, Souque A, Rafestin-Oblin ME 1996 Ligand-induced conformational change in the human mineralocorticoid receptor occurs within its hetero-oligomeric structure. Biochem J 315:421427[Medline]
- Kuil CW, Berrevoets CA, Mulder E 1995 Ligand-induced conformational alterations of the androgen receptor analyzed by limited trypsinization. Studies on the mechanism of antiandrogen action. J Biol Chem 270:2756927576[Abstract/Free Full Text]
- Seielstad DA, Carlson KE, Kushner PJ, Greene GL, Katzenellenbogen JA 1995 Analysis of the structural core of the human estrogen receptor ligand binding domain by selective proteolysis/mass spectrometric analysis. Biochemistry 34:1260512615[Medline]
- Greenfield N, Vijayanathan V, Thomas TJ, Gallo MA, Thomas T 2001 Increase in the stability and helical content of estrogen receptor
in the presence of the estrogen response element: analysis by circular dichroism spectroscopy. Biochemistry 40:66466652[CrossRef][Medline]
- Cronet P, Petersen JF, Folmer R, Blomberg N, Sjoblom K, Karlsson U, Lindstedt EL, Bamberg K 2001 Structure of the PPAR
and -
ligand binding domain in complex with AZ 242; ligand selectivity and agonist activation in the PPAR family. Structure (Camb) 9:699706[CrossRef][Medline]
- Johnson BA, Wilson EM, Li Y, Moller DE, Smith RG, Zhou G2000 Ligand-induced stabilization of PPAR
monitored by NMR spectroscopy: implications for nuclear receptor activation. J Mol Biol 298:187194
- Pissios P, Tzameli I, Kushner P, Moore DD 2000 Dynamic stabilization of nuclear receptor ligand binding domains by hormone or corepressor binding. Mol Cell 6:245253[Medline]
- Pissios P, Tzameli I, Moore DD 2001 New insights into receptor ligand binding domains from a novel assembly assay. J Steroid Biochem Mol Biol 76:37[CrossRef][Medline]
- Way JM, Harrington WW, Brown KK, Gottschalk WK, Sundseth SS, Mansfield TA, Ramachandran RK, Willson TM, Kliewer SA 2001 Comprehensive messenger ribonucleic acid profiling reveals that peroxisome proliferator-activated receptor
activation has coordinate effects on gene expression in multiple insulin-sensitive tissues. Endocrinology 142:12691277[Abstract/Free Full Text]
- Oberfield JL, Collins JL, Holmes CP, Goreham DM, Cooper JP, Cobb JE, Lenhard JM, Hull-Ryde EA, Mohr CP, Blanchard SG, Parks DJ, Moore LB, Lehmann JM, Plunket K, Miller AB, Milburn MV, Kliewer SA, Willson TM 1999 A peroxisome proliferator-activated receptor
ligand inhibits adipocyte differentiation. Proc Natl Acad Sci USA 96:61026106[Abstract/Free Full Text]
- Kentsis A, Sosnick TR 1998 Trifluoroethanol promotes helix formation by destabilizing backbone exposure: desolvation rather than native hydrogen bonding defines the kinetic pathway of dimeric coiled coil folding. Biochemistry 37:1461314622[CrossRef][Medline]
- Eilers AL, Billin AN, Liu J, Ayer DE 1999 A 13-amino acid amphipathic
-helix is required for the functional interaction between the transcriptional repressor Mad1 and mSin3A. J Biol Chem 274:3275032756[Abstract/Free Full Text]
- Brubaker K, Cowley SM, Huang K, Loo L, Yochum GS, Ayer DE, Eisenman RN, Radhakrishnan I 2000 Solution structure of the interacting domains of the Mad-Sin3 complex: implications for recruitment of a chromatin-modifying complex. Cell 103:655665[Medline]
- Kliewer SA, Sundseth SS, Jones SA, Brown PJ, Wisely GB, Koble CS, Devchand P, Wahli W, Willson TM, Lenhard JM, Lehmann JM 1997 Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors
and
. Proc Natl Acad Sci USA 94:431823[Abstract/Free Full Text]
- Gampe Jr RT, Montana VG, Lambert MH, Miller AB, Bledsoe RK, Milburn MV, Kliewer SA, Willson TM, Xu HE 2000 Asymmetry in the PPAR
/RXR
crystal structure reveals the molecular basis of heterodimerization among nuclear receptors. Mol Cell 5:545555[Medline]
- Leesnitzer LM, Parks DJ, Bledsoe RK, Cobb JE, Collins JL, Consler TG, Davis RG, Hull-Ryde EA, Lenhard JM, Patel L, Plunket KD, Shenk JL, Stimmel JB, Therapontos C, Willson TM, Blanchard SG 2002 Functional consequences of cysteine modification in the ligand binding sites of peroxisome proliferator activated receptors by GW9662. Biochemistry 41:66406650[CrossRef][Medline]
- Wu CS, Ikeda K, Yang JT 1981 Ordered conformation of polypeptides and proteins in acidic dodecyl sulfate solution. Biochemistry 20:566570[Medline]