Allosterically Coupled Multisite Binding of Testosterone to Human Serum Albumin.
Allosterically Coupled Multisite Binding of Testosterone to Human Serum Albumin.
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DOI：
10.1210/endocr/bqaa199
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发表时间：
2021-02-01
期刊：
影响因子：
4.8
通讯作者：
Jasuja, Ravi
中科院分区：
文献类型：
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作者：
Jayaraj, Abhilash;Schwanz, Heidi A;Jasuja, Ravi
人血清白蛋白（HSA）是睾酮、其他性激素、脂肪酸和药物的载体。然而，睾酮与HSA结合的动力学及其结合位点的结构仍不完全清楚。在这里，我们使用二维核磁共振（2D NMR），荧光光谱，4，4 '-二苯胺基-1，1'-联萘-5，5 '-二磺酸二钾盐分配和平衡透析，并辅以分子建模，表征睾酮与HSA结合的动力学和结合位点的化学计量和结构位置。2D NMR研究表明，睾酮竞争性地取代了HSA上至少3个已知脂肪酸结合位点的18-[13 C]-油酸，这些位点也结合了许多药物。用荧光光谱法和平衡透析法测得的睾酮与HSA的结合等温线呈非线性，表观解离常数随睾酮和HSA浓度的不同而变化。结合等温线既不符合1：1化学计量的线性结合模型，也不符合2个独立的结合位点;结合等温线与2个或更多个变构偶联结合位点最一致。分子动力学研究表明，睾酮与HSA上脂肪酸结合位点3的结合与位点6的构象变化有关，表明这2个不同结合位点中的残基是变构偶联的。在HSA上存在多个睾酮的变构偶联结合位点。睾酮与游离脂肪酸在HSA上共享这些结合位点，游离脂肪酸可以在各种生理状态或疾病条件下从HSA中取代睾酮，影响其生物利用度。
Human serum albumin (HSA) acts as a carrier for testosterone, other sex hormones, fatty acids, and drugs. However, the dynamics of testosterone's binding to HSA and the structure of its binding sites remain incompletely understood. Here, we characterize the dynamics of testosterone's binding to HSA and the stoichiometry and structural location of the binding sites using 2-dimensional nuclear magnetic resonance (2D NMR), fluorescence spectroscopy, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt partitioning, and equilibrium dialysis, complemented by molecular modeling. 2D NMR studies showed that testosterone competitively displaced 18-[13C]-oleic acid from at least 3 known fatty acid binding sites on HSA that also bind many drugs. Binding isotherms of testosterone's binding to HSA generated using fluorescence spectroscopy and equilibrium dialysis were nonlinear and the apparent dissociation constant varied with different concentrations of testosterone and HSA. The binding isotherms neither conformed to a linear binding model with 1:1 stoichiometry nor to 2 independent binding sites; the binding isotherms were most consistent with 2 or more allosterically coupled binding sites. Molecular dynamics studies revealed that testosterone's binding to fatty acid binding site 3 on HSA was associated with conformational changes at site 6, indicating that residues in in these 2 distinct binding sites are allosterically coupled. There are multiple, allosterically coupled binding sites for testosterone on HSA. Testosterone shares these binding sites on HSA with free fatty acids, which could displace testosterone from HSA under various physiological states or disease conditions, affecting its bioavailability.