Structure determination of protein-ligand complexes by transferred paramagnetic shifts
Structure determination of protein-ligand complexes by transferred paramagnetic shifts
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DOI：
10.1021/ja063584z
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发表时间：
2006-10-04
影响因子：
15
通讯作者：
Otting, Gottfried
中科院分区：
文献类型：
--
作者：
John, Michael;Pintacuda, Guido;Otting, Gottfried
合理的药物设计依赖于蛋白质和低分子量先导化合物之间的复合物的三维（3D）结构的知识。一种新的核磁共振（NMR）光谱策略的基础上，从镧系离子的顺磁效应，允许快速确定的3D结构的小配体分子结合到它的蛋白质靶在溶液中，同时，它的位置和方向相对于蛋白质。该方法依赖于蛋白质靶中镧系元素离子的存在以及结合配体和游离配体之间的快速交换。配体的结合亲和力和在结合状态下经历的顺磁效应来自于天然同位素丰度下配体的浓度依赖性H-1和C-13光谱。结合蛋白质的晶体或溶液结构以及镧系元素离子的磁化率张量的先验知识，顺磁数据从简单的1D NMR光谱定义结合的配体分子相对于蛋白质的位置和取向。该方法进行了验证与镧系元素标记的N-末端结构域的Escherichia coli DNA聚合酶III，亚基θ，和胸苷之间的三元30 kDa的复合物。发现胸苷的结合模式与晶体结构中存在的胸苷一磷酸的结合模式非常相似。
Rational drug design depends on the knowledge of the three-dimensional (3D) structure of complexes between proteins and lead compounds of low molecular weight. A novel nuclear magnetic resonance (NMR) spectroscopy strategy based on the paramagnetic effects from lanthanide ions allows the rapid determination of the 3D structure of a small ligand molecule bound to its protein target in solution and, simultaneously, its location and orientation with respect to the protein. The method relies on the presence of a lanthanide ion in the protein target and on fast exchange between bound and free ligand. The binding affinity of the ligand and the paramagnetic effects experienced in the bound state are derived from concentration-dependent H-1 and C-13 spectra of the ligand at natural isotopic abundance. Combined with prior knowledge of the crystal or solution structure of the protein and of the magnetic susceptibility tensor of the lanthanide ion, the paramagnetic data define the location and orientation of the bound ligand molecule with respect to the protein from simple 1D NMR spectra. The method was verified with the ternary 30 kDa complex between the lanthanide-labeled N-terminal domain of the epsilon exonuclease subunit from the Escherichia coli DNA polymerase III, the subunit theta, and thymidine. The binding mode of thymidine was found to be very similar to that of thymidine monophosphate present in the crystal structure.