Screening and Engineering of Ancestral Sortase Es That Enable High-Fidelity Enzyme Design with Activity Trade-Off

Abstract

Protein grafting techniques that enable conjugation of diverse compounds to target proteins endow them with new functions. Sortase-mediated ligation (SML) is one such method in which sortases catalyze transpeptidation between a substrate protein or peptide bearing a cell wall sorting signal (CWSS) and a nucleophile containing an N-terminal triglycine motif. In this study, we identified novel sortase Es suitable for SML by combining in silico enzyme screening with ancestral sequence reconstruction (ASR). Eight ancestral sortases Es (1AcSE–8AcSE) were designed, of which four (1AcSE, 2AcSE, 3AcSE, and 8AcSE) were expressed in soluble form and exhibited moderate thermal stability. HPLC analysis using Ac-YNL(A/P)ETGA and GGGKY peptides revealed that 2AcSE displayed no activity toward Ac-YNLPETGA, suggesting its unique substrate specificity. Structural analysis of 2AcSE indicated that differences in Loop A, which corresponds with the β3−β4 loop, may contribute to its specificity. We designed ΔAcSE5, a variant of a previously characterized high-activity AcSE5, by replacing its Loop A (residues 56′–61′, GEAPLK) with the shorter TG motif from 2AcSE. ΔAcSE5 exhibited improved specificity for Ac-YNLAETGA over Ac-YNLPETGA, with a trade-off in activity and reduced byproduct formation during conjugation of a shark antibody to GGG-Venus. These findings demonstrate that database-driven screening and structure–function analysis of AcSEs can guide the design of novel sortase E variants optimized for SML applications.