Evaluation of site-directed spin labeling for characterizing protein-ligand complexes using simulated restraints
Evaluation of site-directed spin labeling for characterizing protein-ligand complexes using simulated restraints
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DOI：
10.1016/s0006-3495(01)75785-2
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发表时间：
2001-09-01
影响因子：
3.4
通讯作者：
Constantine, KL
中科院分区：
文献类型：
--
作者：
Constantine, KL
已经进行了模拟研究，以评估在给定已知蛋白质结构的情况下，定点自旋标记用于确定蛋白质-配体复合体结构的有效性。两个蛋白质-配体复合体被用作这些研究的模型体系：二氢叶酸还原酶突变体与甲氨蝶呤络合的1.9埃分辨率的X射线结构和V-Src酪氨酸激酶SH2结构域与五个残基的络合物的1.5埃分辨率的X射线结构。氮氧化物自旋标记被模拟在五个二氢叶酸还原酶残基位置和四个SH2结构域残基位置。对于这两个体系，在能量最小化后，通过模拟退火法产生自旋标记残基的构象系综，同时保持蛋白质-配体复合体的其余部分固定。计算了配体质子和自旋标记物之间的有效距离，模拟了由H-1-核磁共振弛豫测量得到的有效距离。这些都被转换为具有几个不同精度级别的约束。然后进行了有约束的模拟退火法计算，目的是再现目标配体结合模式。还研究了加入一些补充的短距离(小于或等于5.0埃)距离约束的效果。对于二氢叶酸还原酶-甲氨蝶呤复合体，使用相对严格的自旋标记限制条件下，配体结合模式被很好地再现，但使用松散的自旋标记限制条件时，甲氨蝶呤的局部化程度较差。事实证明，短程约束和自旋标签约束是互补的。对于没有短程限制的SH2结构域-磷酸肽复合体，多肽没有定位到正确的结合槽深度，但多肽的取向和内部构象得到了较好的重现。自旋标签约束的使用与短程约束的结合导致了相对明确的结构系综。这些结果表明，由定点定向自旋标记得到的限制条件对确定结合配体的取向和构象有重要作用。准确的配体定位似乎需要几个补充的短距离限制，或者相对较紧的自旋标签限制，至少有一个自旋标记的位置，以便在后一种情况下，一些限制将配体吸引到结合口袋中。
Simulation studies have been performed to evaluate the utility of site-directed spin labeling for determining the structures of protein-ligand complexes, given a known protein structure. Two protein-ligand complexes were used as model systems for these studies: a 1.9-Angstrom -resolution x-ray structure of a dihydrofolate reductase mutant complexed with methotrexate, and a 1.5-Angstrom -resolution x-ray structure of the V-Src tyrosine kinase SH2 domain complexed with a five-residue phosphopeptide. Nitroxide spin labels were modeled at five dihydrofolate reductase residue positions and at four SH2 domain residue positions. For both systems, after energy minimization, conformational ensembles of the spin-labeled residues were generated by simulated annealing while holding the remainder of the protein-ligand complex fixed. Effective distances, simulating those that could be obtained from H-1-NMR relaxation measurements, were calculated between ligand protons and the spin labels. These were converted to restraints with several different levels of precision. Restrained simulated annealing calculations were then performed with the aim of reproducing target ligand-binding modes. The effects of incorporating a few supplementary short-range (less than or equal to5.0 Angstrom) distance restraints were also examined. For the dihydrofolate reductase-methotrexate complex, the ligand-binding mode was reproduced reasonably well using relatively tight spin-label restraints, but methotrexate was poorly localized using loose spin-label restraints. Short-range and spin-label restraints proved to be complementary. For the SH2 domain-phosphopeptide complex without the short-range restraints, the peptide did not localize to the correct depth in the binding groove; nevertheless, the orientation and internal conformation of the peptide was reproduced moderately well. Use of the spin-label restraints in conjunction with the short-range restraints resulted in relatively well defined structural ensembles. These results indicate that restraints derived from site-directed spin labeling can contribute significantly to defining the orientations and conformations of bound ligands. Accurate ligand localization appears to require either a few supplementary short-range distance restraints, or relatively tight spin-label restraints, with at least one spin label positioned so that some of the restraints draw the ligand into the binding pocket in the latter case.