Abstract Staphylococcus aureus sortase A (SrtA) is a membrane‐anchored cysteine transpeptidase that recognizes the Leu–Pro–X–Thr–Gly (LPXTG) motif in surface proteins and covalently incorporates them into the bacterial cell wall. Although wild‐type SrtA enables site‐specific protein ligation in protein engineering, its practical application is restricted by its inherently low catalytic efficiency. A previously engineered pentamutant (P94R/D160N/D165A/K190E/K196T) exhibits roughly a hundredfold increase in catalytic efficiency compared with the wild‐type SrtA. Here, we report the crystal structure of this pentamutant variant in its ligand‐free form, lacking any bound LPXTG peptide, at a resolution of 1.93 Å. Remarkably, the high‐resolution structure reveals that key active‐site loops adopt conformations nearly identical to those of the covalent SrtA–LPXTG analog complex previously determined by Nuclear Magnetic Resonance (NMR). The results demonstrate that the pentamutant stabilizes a substrate‐ready state without requiring the loop rearrangements induced by the binding of the LPXTG substrate, providing the molecular basis of its improved substrate recognition for enhanced catalytic efficiency. These insights provide a structural foundation for the future design of more efficient SrtA‐based ligases for bioconjugation application.
Park et al. (Tue,) studied this question.