Scientific Reports (Jun 2022)

Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

  • Rebekah P. Dyer,
  • Hariny M. Isoda,
  • Gabriela S. Salcedo,
  • Gaetano Speciale,
  • Madison H. Fletcher,
  • Linh Q. Le,
  • Yi Liu,
  • Karen Brami-Cherrier,
  • Shiazah Z. Malik,
  • Edwin J. Vazquez-Cintron,
  • Andrew C. Chu,
  • David C. Rupp,
  • Birgitte P. S. Jacky,
  • Thu T. M. Nguyen,
  • Benjamin B. Katz,
  • Lance E. Steward,
  • Sudipta Majumdar,
  • Amy D. Brideau-Andersen,
  • Gregory A. Weiss

DOI
https://doi.org/10.1038/s41598-022-13617-z
Journal volume & issue
Vol. 12, no. 1
pp. 1 – 11

Abstract

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Abstract The botulinum neurotoxin serotype A (BoNT/A) cuts a single peptide bond in SNAP25, an activity used to treat a wide range of diseases. Reengineering the substrate specificity of BoNT/A’s protease domain (LC/A) could expand its therapeutic applications; however, LC/A’s extended substrate recognition (≈ 60 residues) challenges conventional approaches. We report a directed evolution method for retargeting LC/A and retaining its exquisite specificity. The resultant eight-mutation LC/A (omLC/A) has improved cleavage specificity and catalytic efficiency (1300- and 120-fold, respectively) for SNAP23 versus SNAP25 compared to a previously reported LC/A variant. Importantly, the BoNT/A holotoxin equipped with omLC/A retains its ability to form full-length holotoxin, infiltrate neurons, and cleave SNAP23. The identification of substrate control loops outside BoNT/A’s active site could guide the design of improved BoNT proteases and inhibitors.