Chandra, S and Chattopadhyay, G and Varadarajan, R (2021) Rapid Identification of Secondary Structure and Binding Site Residues in an Intrinsically Disordered Protein Segment. In: Frontiers in Genetics, 12 .
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Abstract
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. © Copyright © 2021 Chandra, Chattopadhyay and Varadarajan.
| Item Type: | Journal Article |
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| Publication: | Frontiers in Genetics |
| Publisher: | Frontiers Media S.A. |
| Additional Information: | The copyright for this article belongs to Authors |
| Keywords: | antitoxin; aspartic acid; bacterial vector; intrinsically disordered protein; lymphocyte antibody; membrane protein, amplification refractory mutation system polymerase chain reaction; Article; autofluorescence; Bacillus subtilis; bacterial strain; binding affinity; binding assay; binding site; bioinformatics; cloning; controlled study; diagnostic test accuracy study; EBY100 strain; Escherichia coli; flow cytometry; fluorescence activated cell sorting; gene; genetic conservation; histogram; mazE6 antitoxin gene; mazF6 antitoxin gene; mutagenesis; mutant; Mycobacterium tuberculosis; nonhuman; plasmid; polymerase chain reaction; prediction; protein expression; protein interaction; protein protein interaction; protein purification; protein secondary structure; Saccharomyces cerevisiae; Sanger sequencing; sequence homology; site directed mutagenesis; structure activity relation; toxin-antitoxin system; X ray crystallography; yeast |
| Department/Centre: | Division of Biological Sciences > Molecular Biophysics Unit |
| Date Deposited: | 21 Dec 2021 05:54 |
| Last Modified: | 21 Dec 2021 05:54 |
| URI: | http://eprints.iisc.ac.in/id/eprint/70703 |
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