Repurposing a plant peptide cyclase for targeted lysine acylation.

Transpeptidases are powerful tools for protein engineering but are largely restricted to acting at protein backbone termini. Alternative enzymatic approaches for internal protein labelling require bulky recognition motifs or non-proteinogenic reaction partners, potentially restricting which proteins can be modified or the types of modification that can be installed. Here we report a strategy for labelling lysine side chain ε-amines by repurposing an engineered asparaginyl ligase, which naturally catalyses peptide head-to-tail cyclization, for versatile isopeptide ligations that are compatible with peptidic substrates. We find that internal lysines with an adjacent leucine residue mimic the conventional N-terminal glycine-leucine substrate. This dipeptide motif enables efficient intra- or intermolecular ligation through internal lysine side chains, minimally leaving an asparagine C-terminally linked to the lysine side chain via an isopeptide bond. The versatility of this approach is demonstrated by the chemoenzymatic synthesis of peptides with non-native C terminus-to-side chain topology and the conjugation of chemically modified peptides to recombinant proteins.

Native and Engineered Cyclic Disulfide-Rich Peptides as Drug Leads.

Bioactive peptides are a highly abundant and diverse group of molecules that exhibit a wide range of structural and functional variation. Despite their immense therapeutic potential, bioactive peptides have been traditionally perceived as poor drug candidates, largely due to intrinsic shortcomings that reflect their endogenous heritage, i.e., short biological half-lives and poor cell permeability. In this review, we examine the utility of molecular engineering to insert bioactive sequences into constrained scaffolds with desired pharmaceutical properties. Applying lessons learnt from nature, we focus on molecular grafting of cyclic disulfide-rich scaffolds (naturally derived or engineered), shown to be intrinsically stable and amenable to sequence modifications, and their utility as privileged frameworks in drug design.

Enzymatic C-to-C Protein Ligation.

Transpeptidase-catalyzed protein and peptide modifications have been widely utilized for generating conjugates of interest for biological investigation or therapeutic applications. However, all known transpeptidases are constrained to ligating in the N-to-C orientation, limiting the scope of attainable products. Here, we report that an engineered asparaginyl ligase accepts diverse incoming nucleophile substrate mimetics, particularly when a means of selectively quenching the reactivity of byproducts released from the recognition sequence is employed. In addition to directly catalyzing formation of l-/d- or α-/ß-amino acid junctions, we find C-terminal Leu-ethylenediamine (Leu-Eda) motifs to be bona fide mimetics of native N-terminal Gly-Leu sequences. Appending a C-terminal Leu-Eda to synthetic peptides or, via an intein-splicing approach, to recombinant proteins enables direct transpeptidase-catalyzed C-to-C ligations. This work significantly expands the synthetic scope of enzyme-catalyzed protein transpeptidation reactions.

Enzymatic C-Terminal Protein Engineering with Amines.

Chemoenzymatic protein and peptide modification is a powerful means of generating defined, homogeneous conjugates for a range of applications. However, the use of transpeptidases is limited by the need to prepare synthetic peptide conjugates to be ligated, bulky recognition tags remaining in the product, and inefficient substrate turnover. Here, we report a peptide/protein labeling strategy that utilizes a promiscuous, engineered transpeptidase to irreversibly incorporate diverse, commercially available amines at a C-terminal asparagine. To demonstrate the utility of this approach, we prepare a protein-drug conjugate, generate a genetically inaccessible C-to-C protein fusion, and site specifically label both termini of a single protein in sequential steps.

Asparaginyl Ligases: New Enzymes for the Protein Engineer's Toolbox.

Enzyme-catalysed site-specific protein modifications enable the precision manufacture of conjugates for the study of protein function and/or for therapeutic or diagnostic applications. Asparaginyl ligases are a class of highly efficient transpeptidases with the capacity to modify proteins bearing only a tripeptide recognition motif. Herein, we review the types of protein modification that are accessible using these enzymes, including N- and C-terminal protein labelling, head-to-tail cyclisation, and protein-protein conjugation. We describe the progress that has been made to engineer highly efficient ligases as well as efforts to chemically manipulate the enzyme reaction to favour product formation. These enzymes are powerful additions to the protein engineer's toolbox.

Improved Asparaginyl-Ligase-Catalyzed Transpeptidation via Selective Nucleophile Quenching.

The use of enzymes for the site-specific modification of proteins/peptides has become a highly accessible, widespread approach to study protein/peptide functions or to generate therapeutic conjugates. Asparaginyl endopeptidases (AEPs) that preferentially catalyze transpeptidation reactions (AEP ligases) have emerged as enticing alternatives to established approaches, such as bacterial sortases, due to their catalytic efficiency and short tripeptide recognition motifs. However, under standard conditions, a substantial excess of the nucleophile to be conjugated is needed to reach desirable yields. Herein we report a versatile approach to shift the AEP-catalyzed transpeptidation equilibrium toward product formation via selectively quenching the nucleophilicity of the competing leaving-group peptide. Our metal-complexation-based strategy enables efficient peptide/protein labeling at the N- or C-terminus with near-equimolar concentrations of nucleophile label. Furthermore, we show that this approach can enhance protein-protein ligation and facilitate the formation of transpeptidation products that are otherwise unattainable.