title: Helix 11 dynamics is critical for constitutive androstane receptor activity.
abstract: The constitutive androstane receptor (CAR) transactivation can occur in the absence of exogenous ligand and this activity is enhanced by agonists TCPOBOP and meclizine. We use biophysical and cell-based assays to show that increased activity of CAR(TCPOBOP) relative to CAR(meclizine) corresponds to a higher affinity of CAR(TCPOBOP) for the steroid receptor coactivator-1. Additionally, steady-state fluorescence spectra suggest conformational differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Hydrogen/deuterium exchange (HDX) data indicate that the CAR activation function 2 (AF-2) is more stable in CAR(TCPOBOP):RXR and CAR(meclizine):RXR than in CAR:RXR. HDX kinetics also show significant differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Unlike CAR(meclizine):RXR, CAR(TCPOBOP):RXR shows a higher overall stabilization that extends into RXR. We identify residues 339-345 in CAR as an allosteric regulatory site with a greater magnitude reduction in exchange kinetics in CAR(TCPOBOP):RXR than CAR(meclizine):RXR. Accordingly, assays with mutations on CAR at leucine-340 and leucine-343 confirm this region as an important determinant of CAR activity.
authors.affiliation: Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA.
authors.forename: Edward
authors.initials: E
authors.lastname: Wright
authors.forename: Scott A
authors.initials: SA
authors.lastname: Busby
authors.forename: Sarah
authors.initials: S
authors.lastname: Wisecarver
authors.forename: Jeremy
authors.initials: J
authors.lastname: Vincent
authors.forename: Patrick R
authors.initials: PR
authors.lastname: Griffin
authors.forename: Elias J
authors.initials: EJ
authors.lastname: Fernandez
mesh_terms: D000495:Allosteric Site; D000818:Animals; D001665:Binding Sites; D000090702:Constitutive Androstane Receptor; D006801:Humans; D008468:Meclizine / Q000494:pharmacology*; D051379:Mice; D008958:Models, Molecular; D056920:Nuclear Receptor Coactivator 1 / Q000737:chemistry; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011725:Pyridines / Q000494:pharmacology*; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D047488:Retinoid X Receptors / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics; D015533:Transcriptional Activation
chemical_list: D000090702:Constitutive Androstane Receptor; D011725:Pyridines; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D047488:Retinoid X Receptors; D008468:Meclizine; C028474:1,4-bis(2-(3,5-dichloropyridyloxy))benzene; D056920:Nuclear Receptor Coactivator 1