Structural Control of Nonnative Ligand Binding in Engineered Mutants of Phosphoenolpyruvate Carboxykinase.
Structural Control of Nonnative Ligand Binding in Engineered Mutants of Phosphoenolpyruvate Carboxykinase.
复制标题
磷酸烯醇丙酮酸羧激酶工程突变体中非天然配体结合的结构控制。
DOI：
10.1021/acs.biochem.8b00963
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发表时间：
2018
期刊：
影响因子：
2.9
通讯作者：
Tainer,JohnA
中科院分区：
文献类型：
--
作者：
Tang,HenryYH;Shin,DavidS;Hura,GregL;Yang,Yue;Hu,Xiaoyu;Lightstone,FeliceC;McGee,MatthewD;Padgett,HalS;Yannone,StevenM;Tainer,JohnA
改变配体结合和活性的识别的蛋白质工程具有巨大的潜力。在这里，大肠杆菌磷酸烯醇丙酮酸羧激酶 (PEPCK) 的配体结合，将草酰乙酸转化为 CO2 和磷酸烯醇丙酮酸，作为糖异生的第一个关键步骤，被设计为适应替代配体作为具有结构信息的示例性系统。根据我们在假定的 CO2 结合位点处对 PEPCK 活性位点中碳酸氢盐结合的鉴定，我们探测了非天然配体与排列成类似于碳酸氢盐几何形状的三个氧原子的结合。测定并分析了与非天然配体硫代硫酸盐和甲磺酸盐结合的 PEPCK 和点突变体以及应变 ATP 和重新定向的草酰乙酸中间体以及意外的碳酸氢盐的晶体结构。这些突变成功地改变了结合的配体位置和方向及其特异性：突变的 PEPCK 结合硫代硫酸盐或甲磺酸盐，但不会同时结合两者。计算预测了甲磺酸盐结合突变体，并揭示了活性位点有序溶剂的释放对配体结合产生强烈影响。除了非天然配体结合之外，一种突变体还改变了 Mn2+ 配位球：该突变体不是典型的八面体配体排列，而是只有五配位排列。通过这项工作，所有下游事件所需的配体结合、位置和金属离子辅因子几何形状的关键特征可以通过少量突变进行设计，以深入了解蛋白质-配体识别的基本基础。通过结构和计算知识，设计突变和随机突变的组合有助于对配体结合和活性的预定变化进行稳健设计，以工程化蛋白质功能。
Protein engineering to alter recognition underlying ligand binding and activity has enormous potential. Here, ligand binding forEscherichia coliphosphoenolpyruvate carboxykinase (PEPCK), which converts oxaloacetate into CO2and phosphoenolpyruvate as the first committed step in gluconeogenesis, was engineered to accommodate alternative ligands as an exemplary system with structural information. From our identification of bicarbonate binding in the PEPCK active site at the supposed CO2binding site, we probed binding of nonnative ligands with three oxygen atoms arranged to resemble the bicarbonate geometry. Crystal structures of PEPCK and point mutants with bound nonnative ligands thiosulfate and methanesulfonate along with strained ATP and reoriented oxaloacetate intermediates and unexpected bicarbonate were determined and analyzed. The mutations successfully altered the bound ligand position and orientation and its specificity: mutated PEPCKs bound either thiosulfate or methanesulfonate but never both. Computational calculations predicted a methanesulfonate binding mutant and revealed that release of the active site ordered solvent exerts a strong influence on ligand binding. Besides nonnative ligand binding, one mutant altered the Mn2+coordination sphere: instead of the canonical octahedral ligand arrangement, the mutant in question had an only five-coordinate arrangement. From this work, critical features of ligand binding, position, and metal ion cofactor geometry required for all downstream events can be engineered with small numbers of mutations to provide insights into fundamental underpinnings of protein–ligand recognition. Through structural and computational knowledge, the combination of designed and random mutations aids in the robust design of predetermined changes to ligand binding and activity to engineer protein function.