【摘要】：Yac G蛋白是一種能夠抑制大腸桿菌促旋酶(E.coli gyrase)活性的內(nèi)源性小分子蛋白質(zhì),僅由65個(gè)氨基酸殘基組成。核磁共振(NMR)研究發(fā)現(xiàn),Yac G結(jié)構(gòu)中含有1個(gè)Cys-X2-Cys-X15-CysX3-Cys序列的鋅指結(jié)構(gòu)域,然而其作用并不清楚。本研究發(fā)現(xiàn),在添加外源鋅或者鐵的M9基礎(chǔ)培養(yǎng)基中,表達(dá)并純化得到分別含有鋅和鐵的Yac G蛋白,而在同時(shí)添加鐵和L-半胱氨酸的M9基礎(chǔ)培養(yǎng)基中可以純化得到含有鐵硫簇的蛋白質(zhì)。這表明,Yac G不僅是一個(gè)鋅指蛋白,也是鐵結(jié)合或鐵硫簇結(jié)合蛋白。定點(diǎn)突變實(shí)驗(yàn)發(fā)現(xiàn),Yac G鋅指結(jié)構(gòu)中的4個(gè)半胱氨酸殘基突變后,其結(jié)合的鋅、鐵、鐵硫簇的含量都顯著下降。這提示,鋅結(jié)合、鐵結(jié)合以及鐵硫簇結(jié)合的位點(diǎn)均位于鋅指結(jié)構(gòu)域中的4個(gè)半胱氨酸殘基。體內(nèi)Yac G過表達(dá)實(shí)驗(yàn)顯示,用IPTG在大腸桿菌體內(nèi)誘導(dǎo)表達(dá)野生型Yac G蛋白會導(dǎo)致其生長明顯受到抑制,而過表達(dá)突變體蛋白(Yac G-C12/28S)對其生長的抑制作用將會減弱。體外實(shí)驗(yàn)進(jìn)一步發(fā)現(xiàn),鋅結(jié)合、鐵結(jié)合以及鐵硫簇結(jié)合形式的Yac G蛋白對E.coli gyrase促DNA螺旋活性的抑制作用沒有明顯差別,但是鋅指結(jié)構(gòu)突變體蛋白(Yac G-C12/28S)對gyrase活性的抑制作用顯著減弱。這說明,完整的鋅指結(jié)構(gòu)對Yac G抑制gyrase活性的功能具有重要作用。此研究有可能為gyrase抑制劑類抗生素藥物的研發(fā)提供有用的線索。
[Abstract]:Yac G protein is an endogenous small molecule protein which can inhibit the activity of Escherichia coli (E.coli gyrase) and consists of only 65 amino acid residues. A zinc finger domain with Cys-X2-Cys-X15-CysX3-Cys sequence was found in the structure of Yac G by nuclear magnetic resonance (NMR), but its role is not clear. In this study, we found that Yac G protein containing zinc and iron was expressed and purified in M9 basal medium supplemented with exogenous zinc or iron, respectively. The protein containing iron and sulfur clusters could be purified from M9 basal medium supplemented with iron and L-cysteine. This indicates that Yac G is not only a zinc finger protein, but also an iron-bound or iron-sulfur cluster binding protein. The results of site-directed mutation showed that the contents of bound zinc and iron-sulfur clusters decreased significantly after four cysteine residues of Yac G zinc finger structure were mutated. The results suggested that the sites of zinc binding, iron binding and iron-sulfur cluster binding were all located at four cysteine residues in the zinc-finger domain. Overexpression of Yac G in vivo showed that the expression of wild-type Yac G protein induced by IPTG in Escherichia coli resulted in obvious inhibition of its growth, but the inhibitory effect of overexpression mutant protein (Yac G-C12/28S) on its growth was weakened. It was further found that there was no significant difference in the inhibitory effects of zinc binding, iron binding and iron-sulfur cluster binding Yac G protein on the DNA helical activity induced by E.coli gyrase in vitro. However, zinc finger structural mutant protein (Yac G-C12/28S) inhibited the activity of gyrase significantly. This suggests that the intact zinc finger structure plays an important role in the inhibition of gyrase activity by Yac G. This study may provide useful clues for the development of gyrase inhibitor antibiotics.
【作者單位】： 溫州醫(yī)科大學(xué)檢驗(yàn)醫(yī)學(xué)院生命科學(xué)學(xué)院檢驗(yàn)醫(yī)學(xué)教育部重點(diǎn)實(shí)驗(yàn)室;溫州市中心醫(yī)院風(fēng)濕免疫科;
【基金】：國家自然科學(xué)基金項(xiàng)目(No.31100576) 浙江省公益技術(shù)研究項(xiàng)目(No.2016C33027)資助~~
【分類號】：R378

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