Design and nuclear magnetic resonance (NMR) structure determination of the second extracellular immunoglobulin tyrosine kinase A (TrkAIg2) domain construct for binding site elucidation in drug discovery.
Design and nuclear magnetic resonance (NMR) structure determination of the second extracellular immunoglobulin tyrosine kinase A (TrkAIg2) domain construct for binding site elucidation in drug discovery.
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DOI：
10.1021/jm501307e
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发表时间：
2015-01-22
影响因子：
7.3
通讯作者：
Crump MP
中科院分区：
文献类型：
--
作者：
Shoemark DK;Williams C;Fahey MS;Watson JJ;Tyler SJ;Scoltock SJ;Ellis RZ;Wickenden E;Burton AJ;Hemmings JL;Bailey CD;Dawbarn D;Jane DE;Willis CL;Sessions RB;Allen SJ;Crump MP
酪氨酸激酶A（TrkA）受体是一种经过验证的治疗干预点，用于多种疾病。通过神经生长因子（NGF）结合第二细胞外免疫球蛋白（TrkAIg 2）结构域的TrkA活化触发细胞内信号传导级联。在外周，这促进了疼痛表型，在大脑中，促进了细胞存活或分化。可再现的结构信息和蛋白质-配体相互作用的详细验证有助于药物发现。然而，分离的TrkAlg 2结构域在不存在NGF的情况下结晶为β-链交换的二聚体，从而封闭结合表面。在这里，我们报告的设计和结构验证的第一个稳定的，具有生物活性的TrkAIg 2域的结合位点确认的核磁共振光谱。我们的结构紧密地模拟了与NGF复合的TrkAIg 2的野生型折叠（1WWW.PDB），并且1H-15 N相关光谱证实了NGF和竞争性小分子在溶液中的已知结合界面处相互作用。
The tyrosine kinase A (TrkA) receptor is a validated therapeutic intervention point for a wide range of conditions. TrkA activation by nerve growth factor (NGF) binding the second extracellular immunoglobulin (TrkAIg2) domain triggers intracellular signaling cascades. In the periphery, this promotes the pain phenotype and, in the brain, cell survival or differentiation. Reproducible structural information and detailed validation of protein–ligand interactions aid drug discovery. However, the isolated TrkAIg2 domain crystallizes as a β-strand-swapped dimer in the absence of NGF, occluding the binding surface. Here we report the design and structural validation by nuclear magnetic resonance spectroscopy of the first stable, biologically active construct of the TrkAIg2 domain for binding site confirmation. Our structure closely mimics the wild-type fold of TrkAIg2 in complex with NGF (1WWW.pdb), and the 1H–15N correlation spectra confirm that both NGF and a competing small molecule interact at the known binding interface in solution.