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 Malik, Edwin Vazquez‐Cintron, Andrew C. Chu, D. Rupp, Birgitte P. S. Jacky, Thu Trang Nguyễn, Benjamin B. Katz, Lance E. Steward, Sudipta Majumdar, Amy Brideau-Andersen, Gregory A. Weiss
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.