title: Kinetic and thermodynamic characterization of dihydrotestosterone-induced conformational perturbations in androgen receptor ligand-binding domain.
abstract: 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 approximately 30 sec(-1)) with greater solvent accessibility was followed by rearrangement (k approximately 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.
authors.affiliation: Section of Endocrinology, Diabetes, and Nutrition, Boston University School of Medicine, Boston, Massachusetts 02199, USA. jasuja@bu.edu
authors.forename: Ravi
authors.initials: R
authors.lastname: Jasuja
authors.forename: Jagadish
authors.initials: J
authors.lastname: Ulloor
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Yengo
authors.forename: Karen
authors.initials: K
authors.lastname: Choong
authors.forename: Andrei Y
authors.initials: AY
authors.lastname: Istomin
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Livesay
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Jacobs
authors.forename: Ronald S
authors.initials: RS
authors.lastname: Swerdloff
authors.forename: Jaroslava
authors.initials: J
authors.lastname: Miksovská
authors.forename: Randy W
authors.initials: RW
authors.lastname: Larsen
authors.forename: Shalender
authors.initials: S
authors.lastname: Bhasin
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D002151:Calorimetry / Q000379:methods; D013196:Dihydrotestosterone / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D011312:Pressure; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011944:Receptors, Androgen / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry
chemical_list: D000817:Anilino Naphthalenesulfonates; D008024:Ligands; D011944:Receptors, Androgen; D012997:Solvents; D013196:Dihydrotestosterone; D014443:Tyrosine; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate)