Bajaj, P and Manjunath, K and Varadarajan, R (2022) Structural and functional determinants inferred from deep mutational scans. In: Protein Science, 31 (7).
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Abstract
Mutations that affect protein binding to a cognate partner primarily occur either at buried residues or at exposed residues directly involved in partner binding. Distinguishing between these two categories based solely on mutational phenotypes is challenging. The bacterial toxin CcdB kills cells by binding to DNA Gyrase. Cell death is prevented by binding to its cognate antitoxin CcdA, at an extended interface that partially overlaps with the GyrA binding site. Using the CcdAB toxin–antitoxin (TA) system as a model, a comprehensive site-saturation mutagenesis library of CcdB was generated in its native operonic context. The mutational sensitivity of each mutant was estimated by evaluating the relative abundance of each mutant in two strains, one resistant and the other sensitive to the toxic activity of the CcdB toxin, through deep sequencing. The ability to bind CcdA was inferred through a RelE reporter gene assay, since the CcdAB complex binds to its own promoter, repressing transcription. By analyzing mutant phenotypes in the CcdB-sensitive, CcdB-resistant, and RelE reporter strains, it was possible to assign residues to buried, CcdA interacting or GyrA interacting sites. A few mutants were individually constructed, expressed, and biophysically characterized to validate molecular mechanisms responsible for the observed phenotypes. Residues inferred to be important for antitoxin binding, are also likely to be important for rejuvenating CcdB from the CcdB–Gyrase complex. Therefore, even in the absence of structural information, when coupled to appropriate genetic screens, such high-throughput strategies can be deployed for predicting structural and functional determinants of proteins.
| Item Type: | Journal Article |
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| Publication: | Protein Science |
| Publisher: | John Wiley and Sons Inc |
| Additional Information: | The copyright for this article belongs to the Author(s). |
| Keywords: | antitoxin; bacterial toxin; ccda antitoxin; ccdb toxin; DNA topoisomerase (ATP hydrolysing) A; unclassified drug; antitoxin; bacterial protein; DNA topoisomerase (ATP hydrolysing), Article; bacterial gene; bacterial strain; binding site; cell death; controlled study; crystal structure; enzyme active site; Escherichia coli; gene repression; in vivo study; molecular library; multiplexed deep sequencing; nonhuman; operon; phenotype; promoter region; protein expression; protein function; protein structure; rele gene; reporter gene; sequence analysis; site-saturation mutagenesis; chemistry; genetics; metabolism; mutation, Antitoxins; Bacterial Proteins; DNA Gyrase; Escherichia coli; Mutation |
| Department/Centre: | Division of Biological Sciences > Molecular Biophysics Unit |
| Date Deposited: | 19 Sep 2022 09:32 |
| Last Modified: | 19 Sep 2022 09:32 |
| URI: | https://eprints.iisc.ac.in/id/eprint/76606 |
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