Molecular Dynamics Simulations for Human CAR Inverse Agonists Johanna Jyrkka ̈ rinne,* Jenni Kü blbeck, Juha Pulkkinen, Paavo Honkakoski, Reino Laatikainen, Antti Poso, and Tuomo Laitinen* University of Eastern Finland, Faculty of Health Sciences, School of Pharmacy, P.O. Box 1627, FI-70211 Kuopio, Finland * S Supporting Information ABSTRACT: Constitutive androstane receptor (CAR), along with pregnane x receptor (PXR), is an important metabolic sensor in the hepatocytes. Like all other nuclear receptors (NRs), CAR works in concert with coregulator proteins, coactivators, and corepressors which bind to the NRs. The main basis for the receptor to distinguish between coactivators and corepressors is the position of the C-terminal helix 12 (H12), which is determined by the bound NR ligand. CAR, having constitutive activity, can be repressed or further activated by its ligands. Crystal structure of human CAR bound to an agonist and a coactivator peptide is available, but no structural information on an inverse agonist-bound human CAR and a corepressor exists. In our previous molecular dynamics (MD) studies, no corepressor peptide was included. Therefore, probably due to the strong interactions which keep the relatively short H12 of CAR in the active position, the structural changes elicited by inverse agonists were very subtle, and H12 of CAR seemed to more or less retain its active conformation. Here, we have run a series of MD simulations to study the movement of H12 in the presence of both activating and repressing ligands as well as a corepressor peptide. The presence of the corepressor on the coregulator surface of CAR induced a clear shift of H12 of the inverse agonists-bound CAR. In general, H12 moved toward H10 and not away from the ligand binding domain, as seen in some other NRs. However, H12 of CAR is short enough that this movement seems to be adequate to accommodate the binding of the corepressor. ■ INTRODUCTION Nuclear receptors (NRs) are ligand-dependent transcription factors that can bind to specific DNA elements on the pro- moter sites of their target genes and thereby regulate gene expression. The constitutive androstane receptor (CAR) is one of several NRs that act as metabolic sensors. CAR has a large and diverse set of endogenous and exogenous ligands that include environmental pollutants, many prescription drugs, and steroid hormones. 1 CAR’s target genes encode both the meta- bolizing enzymes and transport proteins that are important for the elimination of both xenobiotic and endogenous com- pounds. In addition to xenobiotic metabolism, it has become evident that CAR also has a role in several physiological processes, such as energy metabolism and the metabolic conversion of heme, bilirubin, bile acids, and thyroid hormone. 2 To be active, NRs need the help of different coregulator proteins, termed coactivators and corepressors. 3 Upon ligand binding, the conformation of the NR ligand binding domain (LBD) changes, the most important switch for activation/ inactivation being the position of C-terminal helix, H12. In its active position, H12 forms a groove together with residues from helices 3 and 4, where the coactivators bind with their specific amino acid motif resembling LxxLL. Co-repressors have an overlapping binding site with coactivators; however, they have a longer binding motif (LxxI/HxxxI/L) than coactivators. Therefore, displacement of H12 is a prerequisite for corepressor binding. 3 Pure antagonists and inverse agonists usually have a bulky side chain that sterically pushes H12 away from its active position, thereby preventing coactivator binding. Some antagonists/inverse agonists (so-called passive antago- nists) lack the bulky side chain, yet they fail to stabilize the active conformation of H12. 3,4 CAR has quite a large ligand binding pocket (LBP) and, thus, can recognize a wide spectrum of ligands. 1 Another special feature of CAR is its constitutive activity: H12 can maintain its active position in the absence of any ligand. Therefore, ligand binding to CAR may result in agonism or inverse agonism. Furthermore, the same ligand may be able to recruit either coactivators or corepressors, and the net CAR activity depends on the pool of coregulators available in the cell. 5 The exact mechanism of inverse agonism for CAR remains partially unclear. Only two agonist-bound but no inverse agonist-bound human CAR (hCAR) crystal structures exist. 6 For mouse CAR (mCAR), one inverse agonist-bound crystal structure with androstenol ligand exists, 7 but no coregulator peptides are included in it. Due to the species differences, 1 direct extrapolation of the information obtained from that crystal structure to human CAR is not possible because the identity in the human and mouse CAR LBD sequences is only 72%. Many of these differences are in the LBP, making it approximately 100 Å 3 smaller in mCAR than in hCAR and causing considerable differences in the ligand specificity. Also, the interactions between H12 and LBD are different. We have previously reported on novel agonists and inverse agonists for CAR and studied their molecular mechanisms by Received: September 13, 2011 Published: January 11, 2012 Article pubs.acs.org/jcim © 2012 American Chemical Society 457 dx.doi.org/10.1021/ci200432k | J. Chem. Inf. Model. 2012, 52, 457-464