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Kinetic and Thermodynamic Characterization of Dihydrotestosterone-Induced Conformational Perturbations in Androgen Receptor Ligand-Binding Domain

Ravi Jasuja, Jagadish Ulloor, Christopher M. Yengo, Karen Choong, Andrei Y. Istomin, Dennis R. Livesay, Donald J. Jacobs, Ronald S. Swerdloff, Jaroslava Mikovská, Randy W. Larsen and Shalender Bhasin

Section of Endocrinology, Diabetes, and Nutrition (R.J., J.U., K.C., S.B.), Boston University School of Medicine, Boston, Massachusetts 02199; Division of Endocrinology (R.J.), Charles R. Drew University of Medicine and Science, Los Angeles, California 90059; Departments of Biology (C.M.Y.), Physics and Optical Sciences (A.Y.I., D.J.J.), and Computer Science (A.Y.I., D.R.L.) and Bioinformatics Research Center (A.Y.I., D.R.L.), University of North Carolina, Charlotte, North Carolina 28223; Division of Endocrinology (R.S.S.), Harbor-UCLA Medical Center, Los Angeles Biomedical Research Institute, Torrance, California 90502; Department of Chemistry and Biochemistry (J.M.), Florida International University, Miami, Florida 33181; and Department of Chemistry (R.W.L.), University of South Florida, Tampa, Florida 33620

Address all correspondence and requests for reprints to: Dr. Ravi Jasuja, Muscle and Aging Research Unit, Boston University School of Medicine, Section of Endocrinology, Diabetes, and Nutrition, Boston, Massachusetts 02199. E-mail: jasuja{at}bu.edu.

Ligand-induced conformational perturbations in androgen receptor (AR) are important in coactivator recruitment and transactivation. However, molecular rearrangements in AR ligand-binding domain (AR-LBD) associated with agonist binding and their kinetic and thermodynamic parameters are poorly understood. We used steady-state second-derivative absorption and emission spectroscopy, pressure and temperature perturbations, and 4,4'-bis-anilinonaphthalene 8-sulfonate (bis-ANS) partitioning to determine the kinetics and thermodynamics of the conformational changes in AR-LBD after dihydrotestosterone (DHT) binding. In presence of DHT, the second-derivative absorption spectrum showed a red shift and a change in peak-to-peak distance. Emission intensity increased upon DHT binding, and center of spectral mass was blue shifted, denoting conformational changes resulting in more hydrophobic environment for tyrosines and tryptophans within a more compact DHT-bound receptor. In pressure perturbation calorimetry, DHT-induced energetic stabilization increased the Gibbs free energy of unfolding to 8.4 ± 1.3 kcal/mol from 3.5 ± 1.6 kcal/mol. Bis-ANS partitioning studies revealed that upon DHT binding, AR-LBD underwent biphasic rearrangement with a high activation energy (13.4 kcal/mol). An initial, molten globule-like burst phase (k 30 sec^–1) with greater solvent accessibility was followed by rearrangement (k 0.01 sec^–1), leading to a more compact conformation than apo-AR-LBD. Molecular simulations demonstrated unique sensitivity of tyrosine and tryptophan residues during pressure unfolding with rearrangement of residues in the coactivator recruitment surfaces distant from the ligand-binding pocket. In conclusion, DHT binding leads to energetic stabilization of AR-LBD domain and substantial rearrangement of residues distant from the ligand-binding pocket. DHT binding to AR-LBD involves biphasic receptor rearrangement including population of a molten globule-like intermediate state.

NURSA Molecule Pages Link:

Nuclear Receptors:   AR

Ligands:   Dihydrotestosterone