支架蛋白INAD的PDZ5结构域点突变对其氧化还原电势的影响

Redox Potential Influence on PDZ5 Domain of Scaffold Protein INAD by Its Point Mutation

INAD(inactivation,no after-potential D,INAD)蛋白是一种典型的支架蛋白,其核心结构由5个PDZ结构域串联组成,定位于果蝇光感受器的微绒毛上,负责介导G蛋白偶联级联视觉信号通路,调控果蝇视觉信号的传导。其第五个PDZ结构域通过在氧化态和还原态构象之间进行转换的方式对光信号做出反应。单独的PDZ5具有较低的氧化还原电势,以氧化态构象稳定存在;当其与相邻的结构域PDZ4相互作用后,会使自身的一对二硫键(Cys606/Cys645)打开,氧化还原电势被大幅度提高约330 m V,从而稳定在还原态构象。但是,PDZ4调控PDZ5氧化电势的机制尚不清楚。作者主要探讨了INAD-PDZ5变构效应对其自身氧化还原电势的调控,对PDZ5上参与PDZ4相互作用的位点设计了点突变,借助生物化学手段发现仅单突变即可引起PDZ5的氧化还原电势改变。该发现进一步从分子层面阐述了PDZ4对PDZ5氧化还原电势的调控机制。这种通过外围通路调控蛋白质内部二硫键的断开与形成,从而调控自身氧化还原电势的机制,也是一种前所未有的蛋白质氧化方式。

INAD(inactivation, no after-potential D, INAD) is a typical scaffold protein mainly made up of five PDZ domains. It localizes within the microvilli of fly photoreceptor cells and actively participates in the G protein-coupled visual signaling pathway. The fifth PDZ domain cycles between oxidized and reduced forms in response to light. Isolated PDZ5 has relatively low redox potential, which is stable in oxidized state. But the formation of PDZ4/5 complex keeps PDZ5 in the reduced state by disrupting the Cys606/Cys645 disulfide bond and raising the redox potential by around 330 m V, and its molecular mechanism, however, is not clear yet. Here the authors studied the redox potential of PDZ5 controled by its allosteric effect. They constructed mutations of residues on PDZ5 which take part in binding with PDZ4 and observed raise in PDZ5 redox potential. This work partially explains the control mechanism of redox potential in a molecular level. The external network located at the surface of PDZ5 could allosterically regulate its internal disulfide-mediated oxidation, and this represents an unprecedented protein oxidation mechanism.