大肠杆菌YacG锌指结构的金属结合及功能特性

Characterization of Metal Binding and Function of Zinc-finger Motif in Escherichia coli YacG

Yac G蛋白是一种能够抑制大肠杆菌促旋酶(E.coli gyrase)活性的内源性小分子蛋白质,仅由65个氨基酸残基组成。核磁共振(NMR)研究发现,Yac G结构中含有1个Cys-X2-Cys-X15-CysX3-Cys序列的锌指结构域,然而其作用并不清楚。本研究发现,在添加外源锌或者铁的M9基础培养基中,表达并纯化得到分别含有锌和铁的Yac G蛋白,而在同时添加铁和L-半胱氨酸的M9基础培养基中可以纯化得到含有铁硫簇的蛋白质。这表明,Yac G不仅是一个锌指蛋白,也是铁结合或铁硫簇结合蛋白。定点突变实验发现,Yac G锌指结构中的4个半胱氨酸残基突变后,其结合的锌、铁、铁硫簇的含量都显著下降。这提示,锌结合、铁结合以及铁硫簇结合的位点均位于锌指结构域中的4个半胱氨酸残基。体内Yac G过表达实验显示,用IPTG在大肠杆菌体内诱导表达野生型Yac G蛋白会导致其生长明显受到抑制,而过表达突变体蛋白(Yac G-C12/28S)对其生长的抑制作用将会减弱。体外实验进一步发现,锌结合、铁结合以及铁硫簇结合形式的Yac G蛋白对E.coli gyrase促DNA螺旋活性的抑制作用没有明显差别,但是锌指结构突变体蛋白(Yac G-C12/28S)对gyrase活性的抑制作用显著减弱。这说明,完整的锌指结构对Yac G抑制gyrase活性的功能具有重要作用。此研究有可能为gyrase抑制剂类抗生素药物的研发提供有用的线索。

Yac G is an endogenous protein that inhibits the activity of E. coli gyrase. It only contains 65 amino acids among which a novel zinc-finger motif with sequences of Cys-X2-Cys-X15-Cys-X3-Cys was found by NMR. However,the role of zinc-finger motif in Yac G is still unknown. Here,we found that Yac G could bind certain amount of zinc or iron if purified from E. coli cells grown in M9 minimal medium supplemented with exogenous zinc or iron respectively. Interestingly,iron-sulfur cluster was detected in Yac G when it was purified from M9 minimal medium supplemented with both iron and L-cysteine,indicating that Yac G is not only a zinc binding protein,but also an iron binding or iron-sulfur protein.The site-directed mutagenesis studies showed that mutations of the cysteine residues in zinc-finger motif would significantly weaken zinc,iron or iron-sulfur cluster binding in Yac G,suggesting that they may share the same binding sites in Yac G. The in vivo overexpression experiment showed that induction of wild-type Yac G by IPTG largely inhibited the cell growth,while inhibition of Yac G-C12/28 S mutant overexpression on cell growth was weaker than that of wild-type. In vitro DNA supercoiling assay further showed that the binding of zinc,iron,or iron-sulfur cluster to wild-type Yac G would not influence the inhibitory effect on gyrase activity discriminatingly,while double site mutations of Yac G（ C12/28S） was less capable of inhibiting supercoiling activity of gyrase. These results suggest that the intact zinc-finger motif in Yac G may play an important role for the protein function. This work may provide helpful insight for developing therapeutic drugs targeting gyrase.