Repurposing a plant peptide cyclase for targeted lysine acylation

Area of Science:

• Biochemistry
• Protein Engineering
• Synthetic Biology

Background:

• Transpeptidases are valuable for protein engineering but typically function only at protein termini.
• Existing methods for internal protein labeling often require large recognition tags or non-standard components, limiting their applicability.
• There is a need for enzymatic tools that enable versatile internal protein modification.

Purpose of the Study:

• To develop a novel enzymatic strategy for labeling lysine side chains within proteins.
• To repurpose an engineered asparaginyl ligase for versatile isopeptide ligations.
• To demonstrate the utility of this method for chemoenzymatic synthesis and protein conjugation.

Main Methods:

• Engineered an asparaginyl ligase to accept internal lysine residues as substrates.
• Identified a specific dipeptide motif (lysine with adjacent leucine) that facilitates ligation.
• Utilized the ligase for intra- and intermolecular isopeptide bond formation at internal lysine side chains.
• Applied the method for synthesizing peptides with non-native topology and conjugating modified peptides to proteins.

Main Results:

• Successfully repurposed an engineered asparaginyl ligase for internal protein labeling at lysine side chains.
• Demonstrated efficient isopeptide ligation mediated by an internal lysine-leucine motif.
• Achieved chemoenzymatic synthesis of peptides with novel C-terminus-to-side chain linkages.
• Showcased the conjugation of chemically modified peptides to recombinant proteins.

Conclusions:

• The engineered asparaginyl ligase provides a versatile platform for internal protein labeling via lysine side chains.
• This approach overcomes limitations of existing methods, enabling new possibilities in protein engineering and bioconjugation.
• The developed method facilitates the creation of proteins with unique structural and functional properties.