Title: Peroxisome Proliferator-activated Receptor α Interacts with High Affinity and Is Conformationally Responsive to Endogenous Ligands
Abstract: Although the peroxisome proliferator-activated receptor (PPARα) binds and is activated by a variety of synthetic xenobiotics, the identity of the high affinity endogenous ligand(s) is incompletely resolved. Likewise, it is not known how putative endogenous ligands alter PPARα conformation in order to affect transcriptional regulation. Direct fluorescence binding and fluorescence displacement assays showed for the first time that PPARα exhibits high affinity (1–14 nm Kd values) for unsaturated long chain fatty acyl-CoAs as well as unsaturated long chain fatty acids commonly found in mammalian cells. Fluorescence resonance energy transfer between PPARα aromatic amino acids and bound corresponding naturally occurring fluorescent ligands (i.e. cis-parinaroyl-CoA, trans-parinaric acid) yielded intermolecular distances of 25–29 Å, confirming close molecular interaction. Interestingly, although PPARα also exhibited high affinity for saturated long chain fatty acyl-CoAs, regardless of chain length (1–13 nm Kd values), saturated long chain fatty acids were not significantly bound. In contrast to the similar affinities of PPARα for fatty acyl-CoAs and unsaturated fatty acids, CoA thioesters of peroxisome proliferator drugs were bound with 5–6-fold higher affinities than their free acid forms. Circular dichroism demonstrated that high affinity ligands (long chain fatty acyl-CoAs, unsaturated fatty acids), but not weak affinity ligands (saturated fatty acids), elicited conformational changes in PPARα structure, a hallmark of ligand-activated nuclear receptors. Finally, these ligand specificities and induced conformational changes correlated functionally with co-activator binding. In summary, since nuclear concentrations of these ligands are in the nanomolar range, long chain fatty acyl-CoAs and unsaturated fatty acids may both represent endogenous PPARα ligands. Furthermore, the finding that saturated fatty acyl-CoAs, rather than saturated fatty acids, are high affinity PPARα ligands provides a mechanism accounting for saturated fatty acid transactivation in cell-based assays. Although the peroxisome proliferator-activated receptor (PPARα) binds and is activated by a variety of synthetic xenobiotics, the identity of the high affinity endogenous ligand(s) is incompletely resolved. Likewise, it is not known how putative endogenous ligands alter PPARα conformation in order to affect transcriptional regulation. Direct fluorescence binding and fluorescence displacement assays showed for the first time that PPARα exhibits high affinity (1–14 nm Kd values) for unsaturated long chain fatty acyl-CoAs as well as unsaturated long chain fatty acids commonly found in mammalian cells. Fluorescence resonance energy transfer between PPARα aromatic amino acids and bound corresponding naturally occurring fluorescent ligands (i.e. cis-parinaroyl-CoA, trans-parinaric acid) yielded intermolecular distances of 25–29 Å, confirming close molecular interaction. Interestingly, although PPARα also exhibited high affinity for saturated long chain fatty acyl-CoAs, regardless of chain length (1–13 nm Kd values), saturated long chain fatty acids were not significantly bound. In contrast to the similar affinities of PPARα for fatty acyl-CoAs and unsaturated fatty acids, CoA thioesters of peroxisome proliferator drugs were bound with 5–6-fold higher affinities than their free acid forms. Circular dichroism demonstrated that high affinity ligands (long chain fatty acyl-CoAs, unsaturated fatty acids), but not weak affinity ligands (saturated fatty acids), elicited conformational changes in PPARα structure, a hallmark of ligand-activated nuclear receptors. Finally, these ligand specificities and induced conformational changes correlated functionally with co-activator binding. In summary, since nuclear concentrations of these ligands are in the nanomolar range, long chain fatty acyl-CoAs and unsaturated fatty acids may both represent endogenous PPARα ligands. Furthermore, the finding that saturated fatty acyl-CoAs, rather than saturated fatty acids, are high affinity PPARα ligands provides a mechanism accounting for saturated fatty acid transactivation in cell-based assays. Peroxisome proliferator-activated receptors (PPARs) 1The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; PPARαΔAB, peroxisome proliferator-activated receptor α composed of amino acids 101–468 (i.e. missing only the amino-terminal A/B domain); LBD, ligand binding domain; LCFA, long chain fatty acid; LCFA-CoA, long chain fatty acyl-CoA; FRET, fluorescence resonance energy transfer; L-FABP, liver fatty acid-binding protein; SCP-2, sterol carrier protein-2; cis-parinaric acid, (9Z,11E,13E,15Z)-octadecatetraenoic acid; trans-parinaric acid, (9E,11E,13E,15E)-octadecatetraenoic acid; cis-parinaroyl-CoA, (9Z,11E,13E,15Z)-octadecatetraenoyl coenzyme A; NBD-chloride, 7-nitrobenz-2-oxa-1,3-diaxol-chloride; NBD-stearic acid, 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-octadecanoic acid; bezafibrate, 2-(4-(chlorobenzamidoethyl)phenoy)-2-methylpropionic acid; Medica 16, β,β′-tetramethylhexadecane dioic acid; SRC-1, steroid receptor coactivator-1.1The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; PPARαΔAB, peroxisome proliferator-activated receptor α composed of amino acids 101–468 (i.e. missing only the amino-terminal A/B domain); LBD, ligand binding domain; LCFA, long chain fatty acid; LCFA-CoA, long chain fatty acyl-CoA; FRET, fluorescence resonance energy transfer; L-FABP, liver fatty acid-binding protein; SCP-2, sterol carrier protein-2; cis-parinaric acid, (9Z,11E,13E,15Z)-octadecatetraenoic acid; trans-parinaric acid, (9E,11E,13E,15E)-octadecatetraenoic acid; cis-parinaroyl-CoA, (9Z,11E,13E,15Z)-octadecatetraenoyl coenzyme A; NBD-chloride, 7-nitrobenz-2-oxa-1,3-diaxol-chloride; NBD-stearic acid, 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-octadecanoic acid; bezafibrate, 2-(4-(chlorobenzamidoethyl)phenoy)-2-methylpropionic acid; Medica 16, β,β′-tetramethylhexadecane dioic acid; SRC-1, steroid receptor coactivator-1. are crucial nuclear receptors controlling transcription of a variety of genes involved in fatty acid oxidation and cell differentiation (1Francis G.A. Fayard E. Picard F. Auwerx J. Annu. Rev. Physiol. 2003; 65: 261-311Crossref PubMed Scopus (495) Google Scholar). Abnormal PPAR activation contributes to lipotoxicity associated with obesity, insulin resistance, type 2 diabetes, and hyperlipidemia (1Francis G.A. Fayard E. Picard F. Auwerx J. Annu. Rev. Physiol. 2003; 65: 261-311Crossref PubMed Scopus (495) Google Scholar, 2Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2805) Google Scholar). PPARs have been identified in several species, with at least three isotypes recognized (α, β/δ, and γ; NR1C1, NR1C2, and NR1C3, respectively). These isotypes are differentially expressed in select tissues, with expression levels depending on cellular processes (reviewed in Ref. 3Hihi A.K. Michalik L. Wahli W. Cell. Mol. Life Sci. 2002; 59: 645-650Crossref Scopus (267) Google Scholar). The primary amino acid sequence of PPARs is similarly organized as that of other nuclear receptors, with an N-terminal A/B domain containing a ligand-independent transactivation function, the DNA-binding domain (C), a hinge region (D), the ligand-binding domain (LBD) containing a ligand-dependent transactivation function (E), and a C-terminal F domain (4Blanquart C. Barbier O. Fruchart J.C. Staels B. Glineur C. J. Steroid Biochem. Mol. Biol. 2003; 85: 267-273Crossref PubMed Scopus (254) Google Scholar). Because of their multiple roles in regulating fatty acid metabolism and cell differentiation as well as disease (diabetes, obesity, cancer), much attention has focused on the specificities of the PPAR family for xenobiotics/therapeutic agents that bind and regulate the transcriptional activity of PPARα (5Gottlicher M. Widmark E. Li Q. Gustafsson J.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4653-4657Crossref PubMed Scopus (795) Google Scholar, 6Issemann I. Green S. Nature. 1990; 347: 645-650Crossref PubMed Scopus (3011) Google Scholar). Although once believed to be orphan receptors, PPARs are now recognized to be ligand-activated members of the steroid/thyroid nuclear hormone receptor superfamily. Each isotype is encoded by a different gene and exhibits unique binding specificities for a broad variety of xenobiotics (reviewed in Refs. 1Francis G.A. Fayard E. Picard F. Auwerx J. Annu. Rev. Physiol. 2003; 65: 261-311Crossref PubMed Scopus (495) Google Scholar and 3Hihi A.K. Michalik L. Wahli W. Cell. Mol. Life Sci. 2002; 59: 645-650Crossref Scopus (267) Google Scholar). PPARα is now recognized to promiscuously bind with high affinity xenobiotic substances such as hypolipidemic agents, plasticizers, herbicides, and dietary factors (reviewed in Refs. 7Green S. Wahli W. Mol. Cell. Endocrinol. 1994; 100: 149-153Crossref PubMed Scopus (157) Google Scholar, 8Lemberger T. Desvergne B. Wahli W. Annu. Rev. Cell Dev. Biol. 1996; 12: 335-363Crossref PubMed Scopus (630) Google Scholar, 9Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 94: 4312-4317Crossref Scopus (1844) Google Scholar, 10Wahli W. Devchand P.R. Ijpenberg A. Desvergne B. Nigam S. Pace-Asciak C.R. Lipoxygenases and Their Metabolites. Plenum Press, New York1999: 199-209Google Scholar). The acceptance of such a large variety of structurally diverse, xenobiotic ligands is believed to be due to the large pocket comprising the binding site of the LBD (11Xu H.E. Lambert M.H. Montana V.G. Plunket K.D. Moore L.B. Collins J.L. Oplinger J.A. Kliewer S.A. Gampe R.T. McKee D.D. Moore J.T. Willson T.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13919-13924Crossref PubMed Scopus (456) Google Scholar). Although the above studies demonstrate high affinity binding of multiple xenobiotics, investigations attempting to identify the endogenous high affinity ligand(s) of PPARα are less clear. First, although long chain fatty acids (LCFAs) are thought to be putative endogenous PPARα ligands, most radioligand binding studies suggest that PPARα exhibits only weak affinities for LCFAs. Such radioligand binding assays demonstrate that PPARα binds unsaturated LCFAs (arachidonic, petroselenic, linolenic, linoleic, and oleic acids) with only weak affinities (Kd values in the micromolar range) and saturated LCFAs (lauric and palmitic acids) are bound even less well (9Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 94: 4312-4317Crossref Scopus (1844) Google Scholar, 12Keller H. Dreyer C. Medin J. Mahfoudi A. Ozato K. Wahli W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2160-2164Crossref PubMed Scopus (846) Google Scholar, 13Kliewer S.A. Sundseth S.S. Jones S.A. Brown P.J. Wisely G.B. Koble C.S. Devchand P. Wahli W. Willson T.M. Lenhard J.M. Lehmann J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4318-4323Crossref PubMed Scopus (1859) Google Scholar, 14Isseman I. Prince R.A. J. Mol. Endocrinol. 1993; 11: 37-47Crossref PubMed Scopus (282) Google Scholar). These radioligand-based affinities for LCFAs are several orders of magnitude weaker than PPARα exhibits for synthetic xenobiotics. Further, recent confocal fluorescence imaging of nonmetabolizable fluorescent LCFAs in living cells shows that nucleoplasmic LCFA concentrations are in the range of 39–68 nm (15Huang H. Starodub O. McIntosh A. Kier A.B. Schroeder F. J. Biol. Chem. 2002; 277: 29139-29151Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 16Huang H. Starodub O. McIntosh A. Atshaves B.P. Woldegiorgis G. Kier A.B. Schroeder F. Biochemistry. 2004; 43: 2484-2500Crossref PubMed Scopus (88) Google Scholar). Thus, based on radioligand binding assays, it would appear unlikely that long chain fatty acids are physiologically significant endogenous ligands for PPARα. However, it is known that such radioligand binding assays underestimate the affinities of other fatty acid binding proteins (liver fatty acid binding protein (L-FABP); sterol carrier protein-2 (SCP-2)) by several orders of magnitude (reviewed in Refs. 17McArthur M.J. Atshaves B.P. Frolov A. Foxworth W.D. Kier A.B. Schroeder F. J. Lipid Res. 1999; 40: 1371-1383Abstract Full Text Full Text PDF PubMed Google Scholar, 18Frolov A. Cho T.H. Murphy E.J. Schroeder F. Biochemistry. 1997; 36: 6545-6555Crossref PubMed Scopus (93) Google Scholar, 19Rolf B. Oudenampsen-Kruger E. Borchers T. Faergeman N.J. Knudsen J. Lezius A. Spener F. Biochim. Biophys. Acta. 1995; 1259: 245-253Crossref PubMed Scopus (87) Google Scholar). Interestingly, more recent data with a direct fluorescent ligand binding assay determined that mouse PPARα binds a naturally occurring fluorescent LCFA, trans-parinaric acid, with high affinity as shown by a Kd of 30 nm (20Lin Q. Ruuska S.E. Shaw N.S. Dong D. Noy N. Biochemistry. 1999; 38: 185-190Crossref PubMed Scopus (233) Google Scholar, 21Ellinghaus P. Wolfrum C. Assmann G. Spener F. Seedorf U. J. Biol. Chem. 1999; 274: 2766-2772Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Furthermore, displacement of this fluorescent ligand by nonfluorescent LCFAs yielded nanomolar Ki values for a variety of naturally occurring, nonfluorescent unsaturated LCFAs (arachidonic, linolenic, linoleic, and oleic acids) but not saturated LCFAs (stearic and palmitic acids) (20Lin Q. Ruuska S.E. Shaw N.S. Dong D. Noy N. Biochemistry. 1999; 38: 185-190Crossref PubMed Scopus (233) Google Scholar). Whether these high affinities reflect a unique property of the fluorescent ligand, trans-parinaric acid, or whether radioligand binding assays underestimate PPARα affinities remains to be determined. Resolving these issues is important, because the concentration of LCFAs in the nucleoplasm is in the nanomolar rather than micromolar range (15Huang H. Starodub O. McIntosh A. Kier A.B. Schroeder F. J. Biol. Chem. 2002; 277: 29139-29151Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Second, it is unclear whether only unsaturated but not saturated LCFAs represent physiologically significant endogenous PPARα ligands. In transactivation assays wherein cultured cells are supplemented with exogenous LCFAs, both saturated (palmitic) and unsaturated (arachidonic, linoleic, linolenic, and oleic) fatty acids enhance PPARα transactivation nearly equally well (5Gottlicher M. Widmark E. Li Q. Gustafsson J.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4653-4657Crossref PubMed Scopus (795) Google Scholar, 9Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 94: 4312-4317Crossref Scopus (1844) Google Scholar, 12Keller H. Dreyer C. Medin J. Mahfoudi A. Ozato K. Wahli W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2160-2164Crossref PubMed Scopus (846) Google Scholar, 13Kliewer S.A. Sundseth S.S. Jones S.A. Brown P.J. Wisely G.B. Koble C.S. Devchand P. Wahli W. Willson T.M. Lenhard J.M. Lehmann J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4318-4323Crossref PubMed Scopus (1859) Google Scholar, 22Wolfrum C. Ellinghaus P. Fobker M. Seedorf U. Assmann G. Borchers T. Spener F. J. Lipid Res. 1999; 40: 708-714Abstract Full Text Full Text PDF PubMed Google Scholar, 23Krey G. Braissant O. L'Horset F. Kalkhoven E. Perroud M. Parker M.G. Wahli W. Mol. Endocrinol. 1997; 11: 779-791Crossref PubMed Scopus (908) Google Scholar, 24Banner C.D. Gottlicher M. Widmark E. Sjovall J. Rafter J.J. Gustafsson J. J. Lipid Res. 1993; 34: 1583-1591Abstract Full Text PDF PubMed Google Scholar). In dietary studies wherein rats are fed high fat diets, PPARα-activated gene expression is increased, regardless of whether the dietary lipid composition is mostly polyunsaturated, monounsaturated, or saturated LCFA (25Bonilla S. Redonnet A. Noel-Suberville C. Pallet V. Garcin H. Higueret P. Br. J. Nutr. 2000; 83: 665-671Crossref PubMed Google Scholar). If LCFAs are the exclusive endogenous ligand for PPARα, it is difficult to reconcile these PPARα transactivation and activation data with the ligand binding data indicating that PPARα binds well only to the unsaturated, but not saturated, LCFAs (9Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 94: 4312-4317Crossref Scopus (1844) Google Scholar, 12Keller H. Dreyer C. Medin J. Mahfoudi A. Ozato K. Wahli W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2160-2164Crossref PubMed Scopus (846) Google Scholar, 13Kliewer S.A. Sundseth S.S. Jones S.A. Brown P.J. Wisely G.B. Koble C.S. Devchand P. Wahli W. Willson T.M. Lenhard J.M. Lehmann J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4318-4323Crossref PubMed Scopus (1859) Google Scholar, 14Isseman I. Prince R.A. J. Mol. Endocrinol. 1993; 11: 37-47Crossref PubMed Scopus (282) Google Scholar, 20Lin Q. Ruuska S.E. Shaw N.S. Dong D. Noy N. Biochemistry. 1999; 38: 185-190Crossref PubMed Scopus (233) Google Scholar). Taken together, these studies would suggest that, especially in the case of saturated LCFAs, a metabolite rather than the saturated LCFA itself might be the active endogenous PPARα ligand (24Banner C.D. Gottlicher M. Widmark E. Sjovall J. Rafter J.J. Gustafsson J. J. Lipid Res. 1993; 34: 1583-1591Abstract Full Text PDF PubMed Google Scholar). Third, increasing data indicate that a LCFA metabolite such as LCFA-CoA may represent active endogenous high affinity PPARα ligand(s). Early radioligand competition studies show that both unsaturated and saturated LCFA-CoAs are weak PPARα ligands. Displacement of a bound radiolabeled substrate by LCFA-CoAs (palmitoyl-CoA, oleoyl-CoA, and linoleoyl-CoA) yields micromolar Ki values comparable with those obtained with the corresponding LCFAs in the same radioligand binding assay (i.e. Ki < 5 μm) (26Murakami K. Ide T. Nakazawa T. Okazaki T. Mochizuki T. Kadowaki T. Biochem. J. 2001; 353: 231-238Crossref PubMed Scopus (38) Google Scholar). Since nucleoplasmic levels of LCFA-CoAs are very low, in the 3 nm range (16Huang H. Starodub O. McIntosh A. Atshaves B.P. Woldegiorgis G. Kier A.B. Schroeder F. Biochemistry. 2004; 43: 2484-2500Crossref PubMed Scopus (88) Google Scholar), based on radioligand binding data it would appear unlikely that LCFA-CoAs represent physiologically important endogenous ligands for PPARα. In contrast, other data suggest that LCFA-CoAs compete with LCFAs for binding to PPARα and antagonize PPARα transcriptional activation (26Murakami K. Ide T. Nakazawa T. Okazaki T. Mochizuki T. Kadowaki T. Biochem. J. 2001; 353: 231-238Crossref PubMed Scopus (38) Google Scholar, 27Elholm M. Dam I. Jorgenesen C. Krogsdam A.-M. Holst D. Kratchamarova I. Gottlicher M. Gustafsson J.A. Berge R.K. Flatmark T. Knudsen J. Mandrup S. Kristiansen K. J. Biol. Chem. 2001; 276: 21410-21416Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). 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Thus, the possibility that the radioligand binding assay significantly underestimates the affinity of PPARα for LCFA-CoAs must be considered. Fourth, although it is accepted that ligand-induced conformational changes are associated with the transcriptional activation of ligand-activated nuclear receptors (reviewed in Refs. 1Francis G.A. Fayard E. Picard F. Auwerx J. Annu. Rev. Physiol. 2003; 65: 261-311Crossref PubMed Scopus (495) Google Scholar and 31Xu J. Nawaz Z. Tsai S.Y. Tsai M.-J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12195-12199Crossref PubMed Scopus (79) Google Scholar, 32Escher P. Wahli W. Mutat. Res. 2000; 448: 121-138Crossref PubMed Scopus (401) Google Scholar, 33Petrescu A.D. Hertz R. Bar-Tana J. Schroeder F. Kier A.B. J. Biol. Chem. 2002; 277: 23988-23999Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 34Svensson S. Osteberg T. Jacobsson M. Norstrom C. Stefansson K. Hallen D. Johansson I.C. Zachrisson K. Ogg D. Jendeberg L. 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However, other PPAR isoforms demonstrate such ligand-induced alterations in structure as evidenced by x-ray crystallography of PPARγ and PPARβ, where LCFA (i.e. eicosapentaenoic acid) or xenobiotic (e.g. glitazones) binding alters the structure/conformation of these isoforms (reviewed in Ref. 32Escher P. Wahli W. Mutat. Res. 2000; 448: 121-138Crossref PubMed Scopus (401) Google Scholar). Finally, recent experiments demonstrate that binding of a nonhydrolyzable LCFA-CoA analogue changes PPARα sensitivity to protease digestion and alters its ability to bind to co-activators, suggesting a conformational change (27Elholm M. Dam I. Jorgenesen C. Krogsdam A.-M. Holst D. Kratchamarova I. Gottlicher M. Gustafsson J.A. Berge R.K. Flatmark T. Knudsen J. Mandrup S. Kristiansen K. J. Biol. Chem. 2001; 276: 21410-21416Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Unfortunately, these experiments looked at pure recombinant proteins in the presence of high levels of free ligands, so the effect of ligands on native proteins in the presence of other factors is still unknown. Such ligand-induced conformational changes are a hallmark of ligand-activated nuclear receptors (reviewed in Refs. 1Francis G.A. Fayard E. Picard F. Auwerx J. Annu. Rev. Physiol. 2003; 65: 261-311Crossref PubMed Scopus (495) Google Scholar and 31Xu J. Nawaz Z. Tsai S.Y. Tsai M.-J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12195-12199Crossref PubMed Scopus (79) Google Scholar, 32Escher P. Wahli W. Mutat. Res. 2000; 448: 121-138Crossref PubMed Scopus (401) Google Scholar, 33Petrescu A.D. Hertz R. Bar-Tana J. Schroeder F. Kier A.B. J. Biol. Chem. 2002; 277: 23988-23999Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 34Svensson S. Osteberg T. Jacobsson M. Norstrom C. Stefansson K. Hallen D. Johansson I.C. Zachrisson K. Ogg D. Jendeberg L. EMBO J. 2003; 22: 4625-4633Crossref PubMed Scopus (228) Google Scholar). Thus, if LCFAs and/or LCFA-CoAs represent functional ligands for PPARα, it is essential to demonstrate that these endogenous ligands physically alter the structure/conformation of the PPARα isoform. The objective of the present study was to begin to resolve these issues through use of (i) direct ligand binding assays that do not suffer from the limitations of radioligand binding assays (i.e. fluorescent ligand binding assays, nonfluorescent ligand binding assays based on quenching of tyrosine emission, and displacement of bound fluorescent ligand), (ii) fluorescence resonance energy transfer (FRET) between PPARα aromatic amino acids and bound fluorescent ligand (trans-parinaric acid and cis-parinaroyl-CoA) to calculate the intermolecular distance, (iii) circular dichroism to characterize potential ligand-induced changes in PPARα secondary structure, and (iv) co-immunoprecipitation to determine whether ligands that bind and alter PPARα secondary structure also influence the ability of PPARα to interact with co-activators. Chemicals—cis-Parinaric acid, trans-parinaric acid, NBD-chloride, and NBD-stearic acid were purchased from Molecular Probes, Inc. (Eugene, OR). cis-Parinaroyl-CoA was synthesized as previously described (33Petrescu A.D. Hertz R. Bar-Tana J. Schroeder F. Kier A.B. J. Biol. Chem. 2002; 277: 23988-23999Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) and purified by high performance liquid chromatography (37Hubbell T. Behnke W.D. Woodford J.K. Schroeder F. Biochemistry. 1994; 33: 3327-3334Crossref PubMed Scopus (57) Google Scholar). Coenzyme A, palmitic acid, palmitoleic acid, oleic acid, myristoyl-CoA, palmitoyl-CoA, stearoyl-CoA, palmitoleoyl-CoA, oleoyl-CoA, linoleoyl-CoA, arachidonoyl-CoA, and bezafibrate were from Sigma. Medica 16, as well as the coenzyme A thioesters of Medica 16 and bezafibrate, were chemically synthesized as described (38Bar-Tana J. Kahn-Rose G. Srebnik B. J. Biol. Chem. 1985; 260: 8404-8410Abstract Full Text PDF PubMed Google Scholar, 39Kawaguchi A. Yohmura T. Okuda S. J. Biochem. (Tokyo). 1981; 89: 337-339Crossref PubMed Scopus (145) Google Scholar) and kindly provided along with the glitazones and S-hexadecyl-CoA by Dr. J. Bar-Tana (Hebrew University, Israel). All CoA thioesters, whether freshly synthesized or obtained commercially, were >95% undegraded. Mouse PPARα monoclonal, rabbit PPARα polyclonal, mouse steroid receptor coactivator-1 (SRC-1) monoclonal, and rabbit SRC-1 polyclonal antibodies were from Affinity BioReagents (Golden, CO). Anti-rabbit IgG secondary antibodies were from Sigma. Mammalian co-immunoprecipitation kit, chemiluminescent substrate, and film were from Pierce. Recombinant Mouse PPARα Protein—In the present investigation, PPARαΔAB (amino acids 101–468) was utilized for all ligand binding and structure determinations. This choice, rather than the much smaller LBD, was based on recent studies demonstrating that deletion of additional parts of a nuclear transcription factor can significantly alter ligand binding affinity and specificity. 2Petrescu, A. D., Hertz, R., Bar-Tana, J., Schroeder, F., and Kier, A. B. (2005) J. Biol. Chem. 280, in press. The cDNA encoding mouse PPARα with a deletion of the amino-terminal A/B domain (i.e. encoding PPARα amino acids 101–468) was cloned into a His6-tagged bacterial expression vector (pET-PPARαΔAB) and was a generous gift from Dr. N. Noy (Cornell University) (20Lin Q. Ruuska S.E. Shaw N.S. Dong D. Noy N. Biochemistry. 1999; 38: 185-190Crossref PubMed Scopus (233) Google Scholar). The recombinant protein was expressed in the BL21(DE3)pLysS strain of Escherichia coli as described (20Lin Q. Ruuska S.E. Shaw N.S. Dong D. Noy N. Biochemistry. 1999; 38: 185-190Crossref PubMed Scopus (233) Google Scholar) and purified by affinity chromatography with cobalt resin (BD Biosciences Clontech). Purified protein was dialyzed against a buffer containing 10 mm Hepes (pH 8.0), 0.1 mm EDTA, 1 mm dithiothreitol, 400 mm KCl, and 10% glycerol and stored at –80 °C in 25% glycerol. Protein concentration was determined by Bradford assay. Protein purity was assessed by SDS-PAGE and Western blotting. SDS-PAGE and Coomassie Blue staining detected a single intense band of ∼50 kDa (not shown). Western blotting with rabbit anti-mouse PPARα monoclonal antibody followed by goat anti-rabbit IgG-alkaline phosphatase conjugate was performed as described (41Martin G.G. Huang H. Atshaves B.P. Binas B. Schroeder F. Biochemistry. 2003; 42: 11520-11532Crossref PubMed Scopus (56) Google Scholar). Western blotting with rabbit anti-mouse PPARα monoclonal antibody also resulted in a single band at 50 kDa (not shown). Both SDS-PAGE/Coomassie Blue protein staining and Western blotting confirmed the presence of the PPARαΔAB protein in monomeric form. Direct Fluorescent Ligand Binding Assays—Direct fluorescent ligand (NBD-stearic acid, NBD-chloride, cis-parinaric acid, trans-parinaric acid, and cis-parinaroyl-CoA) binding measurements were performed as described earlier (29Frolov A.A. Schroeder F. J. Biol. Chem. 1998; 273: 11049-11055Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 33Petrescu A.D. Hertz R. Bar-Tana J. Schroeder F. Kier A.B. J. Biol. Chem. 2002; 277: 23988-23999Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 37Hubbell T. Behnke W.D. Woodford J.K. Schroeder F. Biochemistry. 1994; 33: 3327-3334Crossref PubMed Scopus (57) Google Scholar, 42Nemecz G. Hubbell T. Jefferson J.R. Lowe J.B. Schroeder F. Arch. Biochem. Biophys. 1991; 286: 300-309Crossref PubMed Scopus (105) Google Scholar). Briefly, increasing quantities of fluorescent ligand (5–2000 nm) were added from a concentrated stock to 0.1 μm PPARαΔAB in 2 ml of phosphate-buffered saline (pH 7.4). Excitation was carried out at 466 nm for NBD-stearate and at 310 nm for parinaric acid or parinaroyl-CoA. Fluorescence emission spectra were obtained at 24 °C with a PC1 photon-counting spectrofluorometer (ISS Inc., Champaign, IL) and corrected for background (protein only and fluorescent ligand only), and maximal intensities were measured. The dissociation constant (Kd) and the number of binding sites (n) were obtained by a reciprocal plot of 1/(1 – F/Fmax) and CL/F/Fmax according to Equation 1 as previously desc