Rapid Identification of Secondary Structure and Binding Site Residues in an Intrinsically Disordered Protein Segment.
Rapid Identification of Secondary Structure and Binding Site Residues in an Intrinsically Disordered Protein Segment.
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DOI：
10.3389/fgene.2021.755292
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发表时间：
2021
影响因子：
3.7
通讯作者：
Varadarajan R
中科院分区：
文献类型：
--
作者：
Chandra S;Chattopadhyay G;Varadarajan R
结核分枝杆菌含有MazEF家族的9个毒素-抗毒素（TA）系统。由于MazF毒素在结核分枝杆菌持续性和疾病中的作用，MazEF TA模块非常重要。MazE抗毒素具有一个无序的c端结构域，可以结合毒素MazF并中和其核糖核酸内酶活性。然而，迄今为止，大多数MazEF TA复合物的结构仍未得到解决，使抗毒素的结构和功能信息模糊不清。我们提出了一种简便的方法来鉴定无序抗毒素上的毒素结合残基。利用带电荷残基扫描诱变技术筛选酵母表面显示的MazE6抗毒素文库，以对抗纯化的同源伙伴MazF6毒素。结合残基是通过流式细胞术检测与配体结合的相对减少来破译的。我们已经用这种方法鉴定了假定的抗毒素界面残基和抗毒素在MazEF6 TA系统中相互作用时获得的局部结构，同样的方法很容易适用于其他内在无序的蛋白质区域。
Mycobacterium tuberculosis harbours nine toxin-antitoxin (TA) systems of the MazEF family. MazEF TA modules are of immense importance due to the perceived role of the MazF toxin in M. tuberculosis persistence and disease. The MazE antitoxin has a disordered C-terminal domain that binds the toxin, MazF and neutralizes its endoribonuclease activity. However, the structure of most MazEF TA complexes remains unsolved till date, obscuring structural and functional information about the antitoxins. We present a facile method to identify toxin binding residues on the disordered antitoxin. Charged residue scanning mutagenesis was used to screen a yeast surface displayed MazE6 antitoxin library against its purified cognate partner, the MazF6 toxin. Binding residues were deciphered by probing the relative reduction in binding to the ligand by flow cytometry. We have used this to identify putative antitoxin interface residues and local structure attained by the antitoxin upon interaction in the MazEF6 TA system and the same methodology is readily applicable to other intrinsically disordered protein regions.
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影响因子：
5
作者：
Bhasin M;Varadarajan R
通讯作者：
Varadarajan R
影响因子：
3.2
作者：
Jorgensen, Mikkel G.;Pandey, Deo P.;Gerdes, Kenn
通讯作者：
Gerdes, Kenn
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
DOI：
10.1073/pnas.0505089102
发表时间：
2005-11-08
影响因子：
11.1
作者：
Bajaj, K;Chakrabarti, P;Varadarajan, R
通讯作者：
Varadarajan, R
影响因子：
5.7
作者：
Aghera, Nilesh K.;Prabha, Jyothi;Varadarajan, Raghavan
通讯作者：
Varadarajan, Raghavan