Asparaginyl ligases are powerful tools for peptide and protein engineering due to their ability to efficiently catalyze a variety of site-specific transpeptidation reactions. Although engineering efforts have enhanced the transpeptidation efficiency of several enzymes, attempts to modify their substrate specificity have been more limited. In a recent study, we produced the first asparaginyl ligase with engineered P2' substrate specificity by mutating Tyr188 to Ala in OaAEP1. Here, we report the engineering of two additional asparaginyl ligases from different plant families, VyPAL2 and butelase 1. We show that mutating the corresponding Tyr residue located in the S2' pocket of these enzymes also expands their substrate scope, enabling the mutant enzymes to process substrates for peptide cyclization, protein-protein ligation, and N-terminal protein labeling that their parent enzymes process poorly. These findings further establish the role of the conserved S2' Tyr residue as a general determinant of substrate specificity for asparaginyl ligases and provide a path toward more extensive engineering efforts.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a well-established target for lowering cholesterol and is abundantly present in the extracellular space. Inhibitors of PCSK9 have achieved marked success in the clinic, but an alternative strategy for therapeutic modulation is emerging through the degradation of PCSK9. This novel strategy has been enabled by the identification of cell surface receptors such as the asialoglycoprotein receptor (ASGPR), which mediates the lysosomal degradation of extracellular ligands. Given the importance of this therapeutic mechanism, we investigated the synthesis of bifunctional molecules comprising Tri-GalNAc (an ASGPR binder) with a peptide inhibitor we previously reported. In addition to chemical synthesis, we report a novel method for the production of Tri-GalNAc-conjugated peptides, involving the use of enzymatically mediated ligation postsynthesis. We demonstrate that both the synthetic constructs and chemoenzymatic constructs have the intended structures and in vitro activities. While these molecules did not show cellular activities, the chemical and biochemical methods reported here could be broadly applied to the construction of LYTACs in general. One significant challenge that this work overcomes is the C-terminal attachment of Tri-GalNAc, which remains hitherto a difficult experimental task for not only peptides but also larger biologics in particular.

The transmembrane protein (TMEM) 233 mediates Excelsatoxin A (ExTxA)-induced pain by removing fast inactivation of the sodium channel Nav1.7. In contrast, TMEM233 itself seems to stabilize the inactivated state of Nav1.7. ExTxA-induced activation of dorsal root ganglion (DRG) neurons is only partly inhibited by tetrodotoxin (TTX), possibly indicating that the TTX-resistant sodium channel Nav1.8 is also modulated by ExTxA. To address this possibility, we performed patch clamp and calcium imaging experiments on mouse DRG neurons and on neuroblastoma ND7/23 and CHO cells expressing Nav1.8 and TMEM233. ExTxA-induced calcium influx in DRG neurons was almost completely inhibited by TTX applied in combination with the selective Nav1.8-inhibitor suzetrigine (VX-548). ExTxA removed fast inactivation of TTX-resistant sodium currents in DRG neurons, as well as of recombinant human or rat Nav1.8 channels co-expressed with TMEM233 in ND7/23 or CHO cells. The co-expression of Nav1.8 and TMEM233 was associated with an ExTxA-independent hyperpolarizing shift of the voltage-dependencies of fast and slow inactivation, an impeded recovery from fast inactivation and an increased use-dependent inhibition by the local anesthetic lidocaine. These effects were constant across physiological temperatures (~10°C, 21°C and 37°C), and TMEM233 also modulated temperature-dependent properties of Nav1.8. Our data suggest that ExTxA-induced pain is likely to involve a TMEM233-mediated regulation of Nav1.8. Furthermore, TMEM233 seems to be a relevant interacting protein of Nav1.8 that modulates the channel's distinct functional and pharmacological properties. These data warrant further investigations into how TMEM233 regulates nociceptor excitability.

The ability to precisely modify proteins and peptides is fundamental to studying their function and creating new variants or topologies with improved properties. Recent studies have transformed the scope of transpeptidases as versatile tools for site-specific modification of proteins and peptides. The engineered asparaginyl ligase OaAEP1 is an ultrafast transpeptidase that stands out owing to its ability to efficiently catalyze a diverse range of modifications that extend well beyond its natural function to generate backbone cyclic peptides in plants. In this Protocol Extension, we describe a framework for the design and application of noncanonical reactions catalyzed by OaAEP1 that provide access to engineered products with customized terminal or side-chain modifications. The reactions proceed cleanly under mild, nondenaturing conditions and can be applied to a broad array of substrates produced by chemical synthesis or recombinant expression, including folded proteins and peptides. After preparing the required substrates and reagents (~5 d) and expressing the recombinant enzyme in Escherichia coli (~3 d), OaAEP1-catalyzed reactions can be carried out in a matter of minutes to hours. We describe methods for installing non-native C-terminal modifications, including by conjugating commercially available nonpeptidic amines (reactive handles, carbohydrates and so on) or ligating a reversed (retro) substrate mimetic that enables production of genetically inaccessible C-to-C fusions. We also describe procedures for OaAEP1-catalyzed side-chain modification of proteins and peptides, which can be applied to generate side-chain-to-tail macrocyclic products, to label a specific side-chain amine with a dye or other reporter tag, or to produce defined protein-cyclic peptide fusions.

The majority of ant species are venomous, and in most lineages, peptide toxins dominate venom composition. Here we investigated the venom of the ant Tetraponera rufonigra (subfamily Pseudomyrmecinae). The major active component, Tr1a, is a homodimeric peptide that is cytolytic, activates mammalian sensory neurons and is insecticidal. Structural analyses show that Tr1a consists of two identical peptide chains arranged as antiparallel α-helices and covalently linked by three interchain disulfide bonds, forming a symmetrical amphipathic dimer. We demonstrate that dimeric venom peptides with similar biological activities have evolved independently multiple times across ants. The repeated evolution of dimeric venom peptides in ants highlights the evolutionary plasticity of ant venom and the role of convergence in shaping ant venom composition.

Cyclotides are a unique class of head-to-tail cyclic peptides with exceptional stability, making them promising scaffolds for therapeutic and agrochemical applications. Their biosynthesis in plants involves asparaginyl endopeptidases (AEPs), which catalyze backbone cyclization through transpeptidation. This chapter presents a detailed chemoenzymatic method for producing cyclotides using AEP-mediated cyclization, focusing on the model cyclotide kalata B1. The method leverages the high efficiency and specificity of AEPs, enabling cyclization of folded substrates without the need for protecting groups or harsh chemical reagents. This approach is scalable and adaptable to other cyclotides and bioactive peptides, offering a robust platform for generating stable, cyclic peptides with enhanced therapeutic potential.

Transpeptidases are valuable enzymes for peptide and protein engineering due to their ease of use and highly defined substrate specificities. Asparaginyl ligases are a class of highly efficient transpeptidases. Engineered asparaginyl ligases with orthogonal substrate specificities would provide access to new types of sequential transpeptidation regimes, but no such engineered variants have been reported thus far. Here, we engineer the widely used asparaginyl ligase OaAEP1 for altered substrate specificity. We find that a single amino acid substitution, Y188A, facilitates the recognition of a substrate sequence that is essentially unmodified by the parent enzyme or an alternative Y188W mutant. This orthogonality enables controlled sequential reactions for the generation of a dual N- and C-terminally labeled protein and the one-pot synthesis of two distinct cyclic peptides from a single linear synthetic peptide precursor. Introducing the equivalent mutation in a consensus-designed asparaginyl ligase facilitates similarly altered substrate specificity, suggesting that the Tyr188 residue is a general determinant of the asparaginyl ligase substrate specificity.

Disulfide-rich peptides (DRPs) have evolved intricate topologies to carry out a wide range of bioactivities throughout nature, e.g., in fungi, insects, plants and animals, and have proven applications in medicine and agriculture. To discover novel DRPs, it is now routine to screen DRP libraries for target affinity, but target binding does not necessarily correlate with function. This study reports an innovative platform for screening of DRP libraries based on the functional endpoint of biochemical reactions within picoliter-sized water-in-oil droplets. We leveraged yeast secretory expression to ensure proper assembly of disulfide connectivity, and thus peptide shape, and engineered customizable strains for facile detection of function (i.e., protease inhibitory activity) for libraries of DRPs. Rapid enrichment of a potent trypsin inhibitor (MCoTI-II) from a >100 000 pool of randomized variants across four rounds of selection was achieved, far exceeding the library sizes explored previously for peptide systems in droplet microfluidics. This developed platform provides a foundation to explore the functional engineering of DRPs.

Cyclic disulfide-rich peptides have become increasingly popular in drug development because their structures enhance molecular stability and allow for mutagenesis to introduce non-native functions. This review focuses on yeast-based platform technologies and their utility in advancing cyclic disulfide-rich peptides as drug modalities and for large-scale biomanufacturing. These technologies include yeast surface display which facilitates the screening of large libraries to develop peptide binders with strong affinity and selectivity for protein targets, while maintaining the innate high stability of the peptide scaffold via protease-based selection pressure. We also describe a recently developed platform that leverages yeast’s ability to secrete correctly folded disulfide-rich peptides while simultaneously displaying peptide or protein tags on their surfaces. In combination with microfluidics technology, the platform creates single-cell yeast-in-droplets reactors, enabling the screening of large libraries based on functional output rather than solely on binding affinity. After identifying cyclic peptide candidates through library-based discovery, these candidates can be produced using a versatile yeast-based bioproduction platform. Traditionally, cyclic disulfide-rich peptides are produced through solid-phase synthesis, a method that generates significant amounts of toxic waste. In contrast, yeast-based bioproduction offers an environmentally sustainable alternative. It has the capability to produce structurally distinct peptides with minimal adjustments and is easily scalable using microbial fermenters, making it an ideal choice for large-scale production.

α-Conotoxin Vc1.1 is a disulfide-rich peptide and a promising drug candidate for treating neuropathic and chronic pain. Backbone cyclization was applied to enhance its drug-like properties, resulting in improved serum stability and oral bioavailability. However, this modification also adversely affected its stability and activity in simulated intestinal fluid (SIF). To address these adverse effects, we explored the use of polyethylene glycol (PEG) linkers as substitutes for peptide backbone cyclization linkers. PEG linkers are smaller, more flexible, and more stable than peptide linkers. Furthermore, previous studies have demonstrated that PEG backbone linkers can enhance the activity of conotoxins. In this study, we synthesized four PEG-backboned cyclic Vc1.1 (cVc1.1) analogues with varying lengths of PEG linkers and used a chemo-enzymatic method to cyclize these analogues. Their structure, stability, and activity were subsequently evaluated. Although the results revealed that PEG linkers preserved the SIF stability and activity of cVc1.1, they highlighted the crucial role of the peptide's helical structure in maintaining its stability and activity. Additionally, this work introduces a novel approach for synthesizing cyclic conotoxins.

Cyclotides are plant-derived peptides characterized by a head-to-tail cyclic backbone and a cystine knot motif comprised of three disulfide bonds. Formation of this motif via in vitro oxidative folding can be challenging and can result in misfolded isomers with nonnative disulfide connectivities. Here, we investigated the effect of beta-turn nucleation on cyclotide oxidative folding. Two types of beta-turn mimics were grafted into kalata B1, individually replacing each of the four beta- turns in the folded cyclotide. Insertion of D-Pro-Gly into loop 5 was beneficial to the folding of both cyclic kB1 and a linear form of the peptide. The linear grafted analog folded four- times faster in aqueous conditions than cyclic kB1 in optimized conditions. Additionally, the cyclic analogue folded without the need for redox agents by transitioning through a native-like intermediate that was on-pathway to product formation. Kalata B1 is from the M & ouml;bius subfamily of cyclotides. Grafting D-Pro-Gly into loop 5 of cyclotides from two other subfamilies also had a beneficial effect on folding. Our findings demonstrate the importance of a beta-turn nucleation site for cyclotide oxidative folding, which could be adopted as a chemical strategy to improve the in vitro folding of diverse cystine-rich peptides.

Cyclotides are a diverse class of plant-derived cyclic, disulfide-rich peptides with a unique cyclic cystine knot topology. Their remarkable structural stability and resistance to proteolytic degradation can lead to improved pharmacokinetics and oral activity as well as selectivity and high enzymatic stability. Thus, cyclotides have emerged as powerful scaffold molecules for designing peptide-based therapeutics. The chemical engineering of cyclotides has generated novel peptide ligands of G protein-coupled receptors (GPCRs), today's most exploited drug targets. However key challenges potentially limit the widespread use of cyclotides in molecular grafting applications. Folding of cyclotides containing bioactive epitopes remains a major bottleneck in cyclotide synthesis. Here we present a modular 'plug and play' approach that effectively bypasses problems associated with the oxidative folding of cyclotides. By grafting onto a pre-formed acyclic cyclotide-like scaffold we show that difficult-to-graft sequences can be easily obtained and can target GPCRs with nanomolar affinities and potencies. We further show the suitability of this new method to graft other complex epitopes including structures with additional disulfide bonds that are not readily available via currently employed chemical methods, thus fully unlocking cyclotides to be used in drug design applications.

Cyclotides are cysteine-rich plant-derived peptides composed of 28-37 amino acids with a head-to-tail cyclic backbone and a knotted arrangement of three conserved disulfide bonds. Their beneficial biophysical properties make them promising molecules for pharmaceutical and agricultural applications. The Violaceae plant family is the major cyclotide-producing family, and to date, every examined plant from this family has been found to contain cyclotides. The presence of cyclotides in Viola communis was inferred by mass spectroscopy previously, but their sequences and properties had yet to be explored. In this study, the occurrence of cyclotides in this plant was investigated using proteomics and transcriptomics. Twenty cyclotides were identified at the peptide level, including two new members from the bracelet (Vcom1) and Möbius (Vcom2) subfamilies. Structural analysis of these newly identified peptides demonstrated a similar fold compared with cyclotides from the same respective subfamilies. Biological assays of Vcom1 and Vcom2 revealed them to be cytotoxic to Sf9 insect cell lines, with Vcom1 demonstrating higher potency than Vcom2. The results suggest that they could be further explored as insecticidal agents and confirm earlier general findings that bracelet cyclotides have more potent insecticidal activity than their Möbius relatives. Seven new cyclotide-like sequences were observed in the transcriptome of V. communis, highlighting the Violaceae as a rich source for new cyclotides with potential insecticidal activity. An analysis of sequences flanking the cyclotide domain in the various precursors from V. communis and other Violaceae plants revealed new insights into cyclotide processing and suggested the possibility of two alternative classes of N-terminal processing enzymes for cyclotide biosynthesis.

Topological transformations and permutations of proteins have attracted significant interest as strategies to generate new protein functionalities or stability. These efforts have mainly been inspired by naturally occurring post-translational modifications, such as head-to-tail cyclization, circular permutation, or lasso-like entanglement. Such approaches can be realized experimentally via genetic encoding, in the case of circular permutation, or via enzymatic processing, in the case of cyclization. Notably, these previously described strategies leave the polypeptide backbone orientation unaltered. Here we describe an unnatural protein permutation, the protein domain inversion, whereby a C-terminal portion of a protein is enzymatically inverted from the canonical N-to-C to a C-to-C configuration with respect to the N-terminal part of the protein. The closest conceptually analogous biological process is perhaps the inversion of DNA segments as catalyzed by recombinases. We achieve these inversions using an engineered sortase A, a widely used transpeptidase. Our reactions proceed efficiently under mild conditions at 4–25 °C and are compatible with entirely heterologously-produced protein substrates.

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels that play a role in neurotransmission and pain sensation. The snake venom-derived peptides, mambalgins, exhibit potent analgesic effects in rodents by inhibiting central ASIC1a and peripheral ASIC1b. Despite their distinct species- and subtype-dependent pharmacology, previous structure-function studies have focussed on the mambalgin interaction with ASIC1a. Currently, the specific channel residues responsible for this pharmacological profile, and the mambalgin pharmacophore at ASIC1b remain unknown. Here we identify non-conserved residues at the ASIC1 subunit interface that drive differences in the mambalgin pharmacology from rat ASIC1a to ASIC1b, some of which likely do not make peptide binding interactions. Additionally, an amino acid variation below the core binding site explains potency differences between rat and human ASIC1. Two regions within the palm domain, which contribute to subtype-dependent effects for mambalgins, play key roles in ASIC gating, consistent with subtype-specific differences in the peptides mechanism. Lastly, there is a shared primary mambalgin pharmacophore for ASIC1a and ASIC1b activity, with certain peripheral peptide residues showing variant-specific significance for potency. Through our broad mutagenesis studies across various species and subtype variants, we gain a more comprehensive understanding of the pharmacophore and the intricate molecular interactions that underlie ligand specificity. These insights pave the way for the development of more potent and targeted peptide analogues required to advance our understating of human ASIC1 function and its role in disease.

The asparaginyl ligase [C247A]OaAEP1 is shown to ligate secondary amine nucleophiles to peptide and protein C-termini. These reactions can be extended to protein dual labelling, including one-pot dual labelling directly at the ligation junction.

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 epsilon-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. Plant asparaginyl endopeptidases that function preferentially as transpeptidases naturally catalyse the head-to-tail cyclization of plant peptides. Using substrate mimicry and reaction optimization, their function has now been repurposed to catalyse intermolecular isopeptide bond formation on diverse peptide and protein substrates.

G protein-coupled receptors are among the most widely studied classes of drug targets. A major challenge in this field is to develop ligands that will selectively modulate a single receptor subtype to overcome the disadvantages of undesired "off target" effects caused by lack of target and thus signaling specificity. In the current study, we explored ligand design for the melanocortin 4 receptor (MC4R) since it is an attractive target for developing antiobesity drugs. Endogenously, the receptor is activated by peptide ligands, i.e., three melanocyte-stimulating hormones (α-MSH, β-MSH, and γ-MSH) and by adrenocorticotropic hormone. Therefore, we utilized a peptide drug design approach, utilizing "molecular grafting" of pharmacophore peptide sequence motifs onto a stable nature-derived peptide scaffold. Specifically, protegrin-4-like-peptide-1 (Pr4LP1) and arenicin-1-like-peptide-1 (Ar3LP1) fully activated MC4R in a functional cAMP assay with potencies of 3.7 and 1.0 nM, respectively. In a nanoluciferase complementation assay with less signal amplification, the designed peptides fully recruited mini-Gs with subnanomolar and nanomolar potencies. Interestingly, these novel peptide MC4R ligands recruited β-arrestin-2 with ∼2-fold greater efficacies and ∼20-fold increased potencies as compared to the endogenous α-MSH. The peptides were inactive at related MC1R and MC3R in a cAMP accumulation assay. These findings highlight the applicability of animal-derived disulfide-rich scaffolds to design pathway and subtype selective MC4R pharmacological probes. In the future, this approach could be exploited to develop functionally selective ligands that could offer safer and more effective obesity drugs.

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.