Structural basis of ribosomal peptide macrocyclization in plants

Area of Science:

• Biochemistry
• Structural Biology
• Drug Discovery

Background:

• Constrained, cyclic peptides encoded by plant genes are emerging as promising drug leads.
• Asparaginyl endopeptidases (AEPs) are plant enzymes evolved for head-to-tail peptide ligation (macrocyclization).
• The mechanism of macrocyclization by solvent-exposed plant AEPs remains poorly understood.

Purpose of the Study:

• To elucidate the structural basis of macrocyclization by plant asparaginyl endopeptidases (AEPs).
• To provide a framework for engineering AEPs for enhanced production of cyclic peptide drug leads.

Main Methods:

• X-ray crystallography of an active sunflower (Helianthus annuus) AEP.
• Active site analysis and identification of a tetrahedral intermediate.
• Site-directed mutagenesis of catalytic residues.

Main Results:

• The crystal structure of a plant AEP revealed a binding mode for peptide macrocyclization via a tetrahedral intermediate.
• Mutagenesis studies demonstrated the ability to alter the ratio of cyclic to acyclic products.
• AEPs from species lacking natural cyclic peptides can perform macrocyclization under specific pH conditions.

Conclusions:

• Structural characterization of plant AEP provides mechanistic insights into peptide macrocyclization.
• This work lays the foundation for engineering AEPs to generate novel cyclic peptide drug candidates.
• The findings suggest broader applicability of AEPs in peptide engineering beyond their natural roles.