Structural basis for proenzyme maturation, substrate recognition, and ligation by a hyperactive peptide asparaginyl ligase.

Abstract

Peptide ligases are versatile enzymes that can be utilized for precise protein conjugation for bioengineering applications. Hyperactive peptide asparaginyl ligases (PALs), such as butelase-1, belong to a small class of enzymes from cyclotide-producing plants that can perform site-specific, rapid ligation reactions after a target peptide asparagine/aspartic acid (Asx) residue binds to the active site of the ligase. How PALs specifically recognize their polypeptide substrates has remained elusive, especially at the prime binding side of the enzyme. Here we report crystal structures that capture VyPAL2, a catalytically efficient PAL from Viola yedoensis, in an activated state, with and without a bound substrate. The bound structure shows one ligase with the N-terminal polypeptide tail from another ligase molecule trapped at its active site, revealing how Asx inserts in the enzyme's S1 pocket and why a hydrophobic residue is required at the P2' position. Besides illustrating the anchoring role played by P1 and P2' residues, these results uncover a role for the Gatekeeper residue at the surface of the S2 pocket in shifting the nonprime portion of the substrate and, as a result, the activity toward ligation or hydrolysis. These results suggest a picture for proenzyme maturation in the vacuole and will inform the rational design of peptide ligases with tailored specificities.