Kinetic and Thermodynamic Characterization of Dihydrotestosterone-Induced Conformational Perturbations in Androgen Receptor Ligand-Binding Domain
Kinetic and Thermodynamic Characterization of Dihydrotestosterone-Induced Conformational Perturbations in Androgen Receptor Ligand-Binding Domain
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DOI：
10.1210/me.2008-0304
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发表时间：
2009-08-01
影响因子：
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通讯作者：
Bhasin, Shalender
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文献类型：
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作者：
Jasuja, Ravi;Ulloor, Jagadish;Bhasin, Shalender
配体诱导的雄激素受体(AR)构象扰动在辅激活子募集和反式激活中起重要作用。然而，与激动剂结合相关的AR配体结合域(AR-LBD)中的分子重排及其动力学和热力学参数知之甚少。我们利用稳态二阶导数吸收和发射光谱、压力和温度微扰以及4，4‘-二苯基苯基-8-磺酸(bis-ANS)的分配来确定二氢睾酮(DHT)结合后AR-LBD构象变化的动力学和热力学。在DHT存在下，二阶导数吸收光谱发生红移，峰间距离发生变化。DHT结合后发射强度增加，光谱质量中心蓝移，这表明构象变化导致更紧密的DHT结合受体内酪氨酸和色氨酸的疏水环境。在压力微扰量热法中，DHT诱导的能量稳定使去折叠的Gibbs自由能从3.5+/-1.6kcal/mol增加到8.4+/-1.3kcal/mol。BIS-ANS分离研究表明，AR-LBD在DHT结合后发生两相重排，具有较高的活化能(13.4kcal/mol)。最初的熔融球状爆裂相(k类似于30秒(-1))具有更大的溶剂可及性，随后发生重排(k类似于0.01秒(-1))，导致了比apo-AR-LBD更紧密的构象。分子模拟显示，酪氨酸和色氨酸残基在压力展开过程中具有独特的敏感性，在远离配体结合口袋的辅活化子招募表面发生残基重排。总之，DHT结合导致AR-LBD结构域的能量稳定和远离配体结合口袋的残基的大量重排。DHT与AR-LBD的结合涉及两相受体重排，包括熔融的球状中间态。(分子内分泌学23：1231-1241,2009)
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 similar to 30 sec(-1)) with greater solvent accessibility was followed by rearrangement (k similar to 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. (Molecular Endocrinology 23: 1231-1241, 2009)