采用细菌磷酸二酯酶作为实验范式来检查多种因素的影响,比如分子结构,在蛋白质表达和纯化过程中使用的配体,结晶条件和空间群,关于配体与靶酶的分子复合物的可视化。在这种情况下,使用的配体是有机磷酸盐,它们是神经毒剂和杀虫剂的片段,酶在其上充当生物清除剂。分析了通过定向进化获得的各种磷酸二酯酶构建体的12种晶体结构,分辨率高达1.38µ。apo形式和holo形式,与有机磷酸酯配体络合,被研究过。从三种不同的结晶条件获得的晶体,在四个空间群中结晶,有和没有N终端标签,用于研究这些因素对可视化酶的有机磷酸酯复合物的影响。研究表明,用于蛋白质表达的标签可以停留在活性位点并阻碍配体结合。此外,蛋白质结晶的空间群可以显着影响结合配体的可视化。还观察到结晶沉淀剂可以与,甚至排除,配体结合,导致误报或导致候选药物的不正确识别。共结晶条件之一能够定义在离去基团分离后容纳与有机磷酸酯底物的几种产物的P原子相连的取代基的空间。磷酸二酯酶与有机磷酸酯产物的配合物的晶体结构显示,P-O键的两个部分带电的O原子与暴露的β-Zn2离子和掩埋的α-Zn2离子的相互作用距离相似。这表明两种Zn2离子都在稳定底物水解的过渡态中起作用。总的来说,这项研究为研究配体-蛋白质复合物的晶体结构所涉及的挑战和考虑因素提供了有价值的见解,强调仔细的实验设计和严格的数据分析的重要性,以确保所得的磷酸二酯酶-有机磷酸酯结构的准确性和可靠性。
A bacterial phosphotriesterase was employed as an experimental paradigm to examine the effects of multiple factors, such as the molecular constructs, the ligands used during protein expression and purification, the crystallization conditions and the space group, on the visualization of molecular complexes of ligands with a target enzyme. In this case, the ligands used were organophosphates that are fragments of the nerve agents and insecticides on which the enzyme acts as a bioscavenger. 12 crystal structures of various phosphotriesterase constructs obtained by directed evolution were analyzed, with resolutions of up to 1.38 Å. Both apo forms and holo forms, complexed with the organophosphate ligands, were studied. Crystals obtained from three different crystallization conditions, crystallized in four space groups, with and without N-terminal tags, were utilized to investigate the impact of these factors on visualizing the organophosphate complexes of the enzyme. The study revealed that the tags used for protein expression can lodge in the active site and hinder ligand binding. Furthermore, the space group in which the protein crystallizes can significantly impact the visualization of bound ligands. It was also observed that the crystallization precipitants can compete with, and even preclude, ligand binding, leading to false positives or to the incorrect identification of lead drug candidates. One of the co-crystallization conditions enabled the definition of the spaces that accommodate the substituents attached to the P atom of several products of organophosphate substrates after detachment of the leaving group. The crystal structures of the complexes of phosphotriesterase with the organophosphate products reveal similar short interaction distances of the two partially charged O atoms of the P-O bonds with the exposed β-Zn2+ ion and the buried α-Zn2+ ion. This suggests that both Zn2+ ions have a role in stabilizing the transition state for substrate hydrolysis. Overall, this study provides valuable insights into the challenges and considerations involved in studying the crystal structures of ligand-protein complexes, highlighting the importance of careful experimental design and rigorous data analysis in ensuring the accuracy and reliability of the resulting phosphotriesterase-organophosphate structures.