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

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Peptide ligases, particularly hyperactive peptide asparaginyl ligases (PALs), are crucial for precise protein conjugation in bioengineering.
• PALs, found in cyclotide-producing plants, facilitate rapid, site-specific ligation reactions.
• The precise mechanism of polypeptide substrate recognition by PALs, especially at the prime binding site, remains poorly understood.

Purpose of the Study:

• To elucidate the substrate recognition mechanism of hyperactive peptide asparaginyl ligases (PALs).
• To determine the structural basis for the specificity of PALs, using VyPAL2 from Viola yedoensis as a model.
• To inform the rational design of peptide ligases with enhanced and tailored specificities.

Main Methods:

• X-ray crystallography to capture crystal structures of VyPAL2.
• Structural analysis of VyPAL2 in both apo (unbound) and holo (substrate-bound) states.
• Analysis of substrate-enzyme interactions within the active site, including the S1 and S2 pockets.

Main Results:

• Crystal structures revealed VyPAL2 in an activated state, with and without a bound substrate.
• The bound structure showed an N-terminal polypeptide tail from another ligase molecule occupying the active site, elucidating Asx insertion into the S1 pocket.
• Identification of a requirement for a hydrophobic residue at the P2' position and the anchoring roles of P1 and P2' residues.
• Uncovered a role for the Gatekeeper residue in the S2 pocket in modulating substrate binding and influencing ligation versus hydrolysis activity.

Conclusions:

• The study provides atomic-level insights into how PALs recognize their polypeptide substrates, particularly the insertion of Asx into the S1 pocket and the role of the P2' residue.
• The Gatekeeper residue's function in the S2 pocket is critical for determining the enzyme's activity (ligation vs. hydrolysis).
• These findings offer a mechanistic understanding that will guide the engineering of novel peptide ligases with specificities tailored for diverse bioengineering applications and suggest a model for proenzyme maturation.