Comparative protein engineering redirects the specificity of Clostridium botulinum proteases.
Botulinum neurotoxin serotypes A and E (BoNT/A and BoNT/E) cleave SNAP25 and are widely used in therapeutic applications. Redirecting the substrate specificity of their protease domains, LC/A and LC/E, could expand their utility to new therapeutic targets. Here, we report a comparative protein engineering strategy that integrates prior mutagenesis, bioinformatics, and structural insights to reprogram LC protease specificity. Directed evolution yielded a 14-mutation LC/A variant with 273-fold greater specificity for SNAP23 than a previously reported engineered protease. Insights from LC/A engineering then guided six rounds of directed evolution to generate an 8-mutation LC/E variant with a 26,000-fold increase in SNAP29 cleavage and no detectable activity toward SNAP25. Importantly, both engineered proteases retain their altered substrate preferences under physiologically relevant substrate and salt concentrations. Together, these findings establish comparative protein engineering as an effective framework for retargeting botulinum neurotoxin proteases.