title: Monitoring Solution Structures of Peroxisome Proliferator-Activated Receptor β/δ upon Ligand Binding.
abstract: Peroxisome proliferator-activated receptors (PPARs) have been intensively studied as drug targets to treat type 2 diabetes, lipid disorders, and metabolic syndrome. This study is part of our ongoing efforts to map conformational changes in PPARs in solution by a combination of chemical cross-linking and mass spectrometry (MS). To our best knowledge, we performed the first studies addressing solution structures of full-length PPAR-β/δ. We monitored the conformations of the ligand-binding domain (LBD) as well as full-length PPAR-β/δ upon binding of two agonists. (Photo-) cross-linking relied on (i) a variety of externally introduced amine- and carboxyl-reactive linkers and (ii) the incorporation of the photo-reactive amino acid p-benzoylphenylalanine (Bpa) into PPAR-β/δ by genetic engineering. The distances derived from cross-linking experiments allowed us to monitor conformational changes in PPAR-β/δ upon ligand binding. The cross-linking/MS approach proved highly advantageous to study nuclear receptors, such as PPARs, and revealed the interplay between DBD (DNA-binding domain) and LDB in PPAR-β/δ. Our results indicate the stabilization of a specific conformation through ligand binding in PPAR-β/δ LBD as well as full-length PPAR-β/δ. Moreover, our results suggest a close distance between the N- and C-terminal regions of full-length PPAR-β/δ in the presence of GW1516. Chemical cross-linking/MS allowed us gaining detailed insights into conformational changes that are induced in PPARs when activating ligands are present. Thus, cross-linking/MS should be added to the arsenal of structural methods available for studying nuclear receptors.
authors.affiliation: Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, D-06120, Halle/Saale, Germany.
authors.forename: Rico
authors.initials: R
authors.lastname: Schwarz
authors.affiliation: Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, D-06120, Halle/Saale, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Tänzler
authors.affiliation: Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, D-06120, Halle/Saale, Germany.
authors.forename: Christian H
authors.initials: CH
authors.lastname: Ihling
authors.affiliation: Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, D-06120, Halle/Saale, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Sinz
mesh_terms: D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D006801:Humans; D008024:Ligands; D047494:PPAR delta / Q000819:agonists / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D048068:PPAR-beta / Q000819:agonists / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012996:Solutions; D053719:Tandem Mass Spectrometry; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism; D010636:Phenols; D013438:Sulfhydryl Compounds
chemical_list: D003432:Cross-Linking Reagents; D004247:DNA; D008024:Ligands; D047494:PPAR delta; D048068:PPAR-beta; D012996:Solutions; D013844:Thiazoles; C425931:GW 501516; C479979:(4-(((2-(3-fluoro-4-(trifluoromethyl)phenyl)-4-methyl-1,3-thiazol-5-yl)methyl)sulfanyl)-2-methylphenoxy)acetic acid; D010636:Phenols; D013438:Sulfhydryl Compounds