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Combining cysteine scanning with chemical labeling to map protein-protein interactions and infer bound structure in an intrinsically disordered region
The Mycobacterium tuberculosis genome harbours nine toxin-antitoxin (TA) systems of the mazEF family. These consist of two proteins, a toxin and an antitoxin, encoded in an operon. While the toxin has a conserved fold, the antitoxins are structurally diverse and the toxin binding region is typically...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9585320/ https://www.ncbi.nlm.nih.gov/pubmed/36275627 http://dx.doi.org/10.3389/fmolb.2022.997653 |
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author | Ahmed, Shahbaz Chattopadhyay, Gopinath Manjunath, Kavyashree Bhasin, Munmun Singh, Neelam Rasool, Mubashir Das, Sayan Rana, Varsha Khan, Neha Mitra, Debarghya Asok, Aparna Singh, Ramandeep Varadarajan, Raghavan |
author_facet | Ahmed, Shahbaz Chattopadhyay, Gopinath Manjunath, Kavyashree Bhasin, Munmun Singh, Neelam Rasool, Mubashir Das, Sayan Rana, Varsha Khan, Neha Mitra, Debarghya Asok, Aparna Singh, Ramandeep Varadarajan, Raghavan |
author_sort | Ahmed, Shahbaz |
collection | PubMed |
description | The Mycobacterium tuberculosis genome harbours nine toxin-antitoxin (TA) systems of the mazEF family. These consist of two proteins, a toxin and an antitoxin, encoded in an operon. While the toxin has a conserved fold, the antitoxins are structurally diverse and the toxin binding region is typically intrinsically disordered before binding. We describe high throughput methodology for accurate mapping of interfacial residues and apply it to three MazEF complexes. The method involves screening one partner protein against a panel of chemically masked single cysteine mutants of its interacting partner, displayed on the surface of yeast cells. Such libraries have much lower diversity than those generated by saturation mutagenesis, simplifying library generation and data analysis. Further, because of the steric bulk of the masking reagent, labeling of virtually all exposed epitope residues should result in loss of binding, and buried residues are inaccessible to the labeling reagent. The binding residues are deciphered by probing the loss of binding to the labeled cognate partner by flow cytometry. Using this methodology, we have identified the interfacial residues for MazEF3, MazEF6 and MazEF9 TA systems of M. tuberculosis. In the case of MazEF9, where a crystal structure was available, there was excellent agreement between our predictions and the crystal structure, superior to those with AlphaFold2. We also report detailed biophysical characterization of the MazEF3 and MazEF9 TA systems and measured the relative affinities between cognate and non-cognate toxin–antitoxin partners in order to probe possible cross-talk between these systems. |
format | Online Article Text |
id | pubmed-9585320 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-95853202022-10-22 Combining cysteine scanning with chemical labeling to map protein-protein interactions and infer bound structure in an intrinsically disordered region Ahmed, Shahbaz Chattopadhyay, Gopinath Manjunath, Kavyashree Bhasin, Munmun Singh, Neelam Rasool, Mubashir Das, Sayan Rana, Varsha Khan, Neha Mitra, Debarghya Asok, Aparna Singh, Ramandeep Varadarajan, Raghavan Front Mol Biosci Molecular Biosciences The Mycobacterium tuberculosis genome harbours nine toxin-antitoxin (TA) systems of the mazEF family. These consist of two proteins, a toxin and an antitoxin, encoded in an operon. While the toxin has a conserved fold, the antitoxins are structurally diverse and the toxin binding region is typically intrinsically disordered before binding. We describe high throughput methodology for accurate mapping of interfacial residues and apply it to three MazEF complexes. The method involves screening one partner protein against a panel of chemically masked single cysteine mutants of its interacting partner, displayed on the surface of yeast cells. Such libraries have much lower diversity than those generated by saturation mutagenesis, simplifying library generation and data analysis. Further, because of the steric bulk of the masking reagent, labeling of virtually all exposed epitope residues should result in loss of binding, and buried residues are inaccessible to the labeling reagent. The binding residues are deciphered by probing the loss of binding to the labeled cognate partner by flow cytometry. Using this methodology, we have identified the interfacial residues for MazEF3, MazEF6 and MazEF9 TA systems of M. tuberculosis. In the case of MazEF9, where a crystal structure was available, there was excellent agreement between our predictions and the crystal structure, superior to those with AlphaFold2. We also report detailed biophysical characterization of the MazEF3 and MazEF9 TA systems and measured the relative affinities between cognate and non-cognate toxin–antitoxin partners in order to probe possible cross-talk between these systems. Frontiers Media S.A. 2022-10-07 /pmc/articles/PMC9585320/ /pubmed/36275627 http://dx.doi.org/10.3389/fmolb.2022.997653 Text en Copyright © 2022 Ahmed, Chattopadhyay, Manjunath, Bhasin, Singh, Rasool, Das, Rana, Khan, Mitra, Asok, Singh and Varadarajan. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Molecular Biosciences Ahmed, Shahbaz Chattopadhyay, Gopinath Manjunath, Kavyashree Bhasin, Munmun Singh, Neelam Rasool, Mubashir Das, Sayan Rana, Varsha Khan, Neha Mitra, Debarghya Asok, Aparna Singh, Ramandeep Varadarajan, Raghavan Combining cysteine scanning with chemical labeling to map protein-protein interactions and infer bound structure in an intrinsically disordered region |
title | Combining cysteine scanning with chemical labeling to map protein-protein interactions and infer bound structure in an intrinsically disordered region |
title_full | Combining cysteine scanning with chemical labeling to map protein-protein interactions and infer bound structure in an intrinsically disordered region |
title_fullStr | Combining cysteine scanning with chemical labeling to map protein-protein interactions and infer bound structure in an intrinsically disordered region |
title_full_unstemmed | Combining cysteine scanning with chemical labeling to map protein-protein interactions and infer bound structure in an intrinsically disordered region |
title_short | Combining cysteine scanning with chemical labeling to map protein-protein interactions and infer bound structure in an intrinsically disordered region |
title_sort | combining cysteine scanning with chemical labeling to map protein-protein interactions and infer bound structure in an intrinsically disordered region |
topic | Molecular Biosciences |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9585320/ https://www.ncbi.nlm.nih.gov/pubmed/36275627 http://dx.doi.org/10.3389/fmolb.2022.997653 |
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