From P_II Signaling to Metabolite Sensing: A Novel 2-Oxoglutarate Sensor That Details P_II - NAGK Complex Formation

The widespread PII signal transduction proteins are known for integrating signals of nitrogen and energy supply and regulating cellular behavior by interacting with a multitude of target proteins. The PII protein of the cyanobacterium Synechococcus elongatus forms complexes with the controlling enzyme of arginine synthesis, N-acetyl-L-glutamate kinase (NAGK) in a 2-oxoglutarate- and ATP/ADP-dependent manner. Fusing NAGK and PII proteins to either CFP or YFP yielded a FRET sensor that specifically responded to 2-oxoglutarate. The impact of the fluorescent tags on PII and NAGK was evaluated by enzyme assays, surface plasmon resonance spectroscopy and isothermal calorimetric experiments. The developed FRET sensor provides real-time data on PII - NAGK interaction and its modulation by the effector molecules ATP, ADP and 2-oxoglutarate in vitro. Additionally to its utility to monitor 2-oxoglutarate levels, the FRET assay provided novel insights into PII - NAGK complex formation: (i) It revealed the formation of an encounter-complex between PII and NAGK, which holds the proteins in proximity even in the presence of inhibitors of complex formation; (ii) It revealed that the PII T-loop residue Ser49 is neither essential for complex formation with NAGK nor for activation of the enzyme but necessary to form a stable complex and efficiently relieve NAGK from arginine inhibition; (iii) It showed that arginine stabilizes the NAGK hexamer and stimulates PII - NAGK interaction.广泛分布的PII信号转导蛋白（PII signal transduction proteins）以整合氮与能量供应信号、通过与众多靶蛋白相互作用调控细胞行为而为人熟知。蓝细菌长聚球藻（Synechococcus elongatus）的PII蛋白可与精氨酸合成的关键调控酶——N-乙酰-L-谷氨酸激酶（NAGK）形成复合物，该过程依赖2-氧代戊二酸（2-oxoglutarate）以及ATP/ADP的存在。将NAGK与PII蛋白分别融合至青色荧光蛋白（cyan fluorescent protein, CFP）或黄色荧光蛋白（yellow fluorescent protein, YFP）后，可得到一种可特异性响应2-氧代戊二酸的荧光共振能量转移（Fluorescence Resonance Energy Transfer, FRET）传感器。研究人员通过酶活测定、表面等离子体共振光谱法以及等温滴定量热实验，评估了荧光标签对PII与NAGK蛋白的影响。所构建的FRET传感器可在体外实时获取PII与NAGK的相互作用数据，以及效应分子ATP、ADP与2-氧代戊二酸对该相互作用的调控信息。除可用于监测2-氧代戊二酸水平外，该FRET检测方法还为PII-NAGK复合物的形成机制提供了全新认知：(i) 揭示了PII与NAGK之间存在遭遇复合物（encounter complex），即便在复合物形成抑制剂存在的情况下，该复合物仍可使两种蛋白保持邻近状态；(ii) 证实PII蛋白的T环残基丝氨酸49（Ser49）既不是与NAGK形成复合物以及激活该酶的必需位点，却是形成稳定复合物并有效解除NAGK受精氨酸抑制所必需的位点；(iii) 表明精氨酸可稳定NAGK六聚体，并促进PII与NAGK的相互作用。