Structural and functional determinants inferred from deep mutational scans.

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  • Additional Information
    • Source:
      Publisher: Cold Spring Harbor Laboratory Press Country of Publication: United States NLM ID: 9211750 Publication Model: Print Cited Medium: Internet ISSN: 1469-896X (Electronic) Linking ISSN: 09618368 NLM ISO Abbreviation: Protein Sci Subsets: MEDLINE
    • Publication Information:
      Publication: 2001- : Woodbury, NY : Cold Spring Harbor Laboratory Press
      Original Publication: New York, N.Y. : Cambridge University Press, c1992-
    • Subject Terms:
      Antitoxins*/genetics ; Bacterial Proteins*/chemistry; DNA Gyrase/chemistry ; DNA Gyrase/genetics ; DNA Gyrase/metabolism ; Escherichia coli/genetics ; Mutation
    • 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.
      (© 2022 The Protein Society.)
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    • Contributed Indexing:
      Keywords: active-site; fitness; gene regulation; protein structure prediction; residue burial
    • Accession Number:
      0 (Antitoxins)
      0 (Bacterial Proteins)
      EC 5.99.1.3 (DNA Gyrase)
    • Publication Date:
      Date Created: 20220628 Date Completed: 20220630 Latest Revision: 20230703
    • Publication Date:
      20240105
    • Accession Number:
      PMC9202547
    • Accession Number:
      10.1002/pro.4357
    • Accession Number:
      35762712