Hydrogen/deuterium-exchange (H/D-Ex) of PPARγ LBD in the presence of various modulators
Hydrogen/deuterium-exchange (H/D-Ex) of PPARγ LBD in the presence of various modulators
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DOI：
10.1110/ps.062103006
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发表时间：
2006-08-01
期刊：
影响因子：
8
通讯作者：
Griffin, Patrick R.
中科院分区：
文献类型：
--
作者：
Hamuro, Yoshitomo;Coales, Stephen J.;Griffin, Patrick R.
PPARγ是一种核受体，是一种配体依赖的转录因子，参与糖稳态和脂肪细胞的分化。PPAR伽马是各种天然和合成分子的分子靶点，包括罗格列酮等抗糖尿病药物。采用H/D-Ex结合蛋白质分解和质谱分析的方法，研究了含有或不含有调节PPARγ活性的分子的PPARγ配体结合域(LBD)的动力学。无配体PPAR-γLBD的H/D-Ex图谱表明，LBD的配体结合口袋明显比LBD的其余部分更动态。推测，为了容纳不同的配体，结合袋本质上是无序的。两种完全激动剂(罗格列酮和GW1929)、一种部分激动剂(NTZDpa)和一种共价拮抗剂(GW9662)的存在改变了PPARγLBD的动力学/构象，并减缓了蛋白质不同区域的H/D交换率。完全激动剂比部分激动剂或拮抗剂更全面地减缓H/D交换，且程度更大，这表明完全激动剂比部分激动剂或拮抗剂更能稳定PPARγLBD。一个有趣的观察是，两个完全激动剂显着稳定螺旋12，而部分激动剂和拮抗剂不干扰该区域的H/D交换。结果表明，配体结合引起的蛋白质动力学变化可能是基因激活的重要因素，H/D-Ex是分析药物先导生物活性的有效方法。
A nuclear receptor, peroxisome proliferator-activated receptor gamma (PPAR gamma), is a ligand-dependent transcription factor involved in glucose homeostasis and adipocyte differentiation. PPAR gamma is the molecular target of various natural and synthetic molecules, including anti-diabetic agents such as rosiglitazone. Amide hydrogen/deuterium-exchange (H/D-Ex), coupled with proteolysis and mass spectrometry, was applied to study the dynamics of the PPAR gamma ligand binding domain (LBD) with or without molecules that modulate PPAR gamma activity. The H/D-Ex patterns of ligand-free PPAR gamma LBD show that the ligand binding pocket of LBD is significantly more dynamic than the rest of the LBD. Presumably, the binding pocket is intrinsically disordered in order to accommodate different ligands. The presence of two full agonists (rosiglitazone and GW1929), a partial agonist (nTZDpa), an d a covalent antagonist (GW9662), changed the dynamics/conformation of PPAR gamma LBD and slowed the H/D exchange rate in various regions of the protein. The full agonists slowed the H/D exchange more globally and to a greater extent than the partial agonist or the antagonist, indicating that the full agonist stabilizes the PPAR gamma LBD more than the partial agonist or the antagonist. One interesting observation is that the two full agonists significantly stabilized helix 12 while the partial agonist and the antagonist did not perturb the H/D exchange of this region. The results showed that the change in protein dynamics induced by ligand binding may be an important factor for the activation of genes and that H/D-Ex is a useful method for analyzing the biological activity of drug leads.