Thioether Cross-links Research Articles

Structure and Function of a Class III Metal-Independent Lanthipeptide Synthetase.

In bacteria, Ser/Thr protein kinase-like sequences are found as part of large multidomain polypeptides that biosynthesize lanthipeptides, a class of natural products distinguished by the presence of thioether cross-links. The kinase domain phosphorylates Ser or Thr residues in the peptide substrates. Subsequent β-elimination by a lyase domain yields electrophilic dehydroamino acids, which can undergo cyclase domain-catalyzed cyclization to yield conformationally restricted, bioactive compounds. Here, we reconstitute the biosynthetic pathway for a class III lanthipeptide from Bacillus thuringiensis NRRL B-23139, including characterization of a two-component protease for leader peptide excision. We also describe the first crystal structures of a class III lanthipeptide synthetase, consisting of the lyase, kinase, and cyclase domains, in various states including complexes with its leader peptide and nucleotide. The structure shows interactions between all three domains that result in an active conformation of the kinase domain. Biochemical analysis demonstrates that the three domains undergo movement upon binding of the leader peptide to establish interdomain allosteric interactions that stabilize this active form. These studies inform on the regulatory mechanism of substrate recognition and provide a framework for engineering of variants of biotechnological interest.

Intermolecular electron transfer in radical SAM enzymes as a new paradigm for reductive activation

Radical S-adenosyl-L-methionine (rSAM) enzymes bind one or more Fe-S clusters and catalyze transformations that produce complex and structurally diverse natural products. One of the clusters, a 4Fe-4S cluster, binds and reductively cleaves SAM to generate the 5'-deoxyadenosyl radical, which initiates the catalytic cycle by H-atom transfer from the substrate. The role(s) of the additional auxiliary Fe-S clusters (ACs) remains largely enigmatic. The rSAM enzyme PapB catalyzes the formation of thioether cross-links between the β-carbon of an Asp and a Cys thiolate found in the PapA peptide. One of the twoACs in the protein binds to the substrate thiol where, upon formation of a thioether bond, one reducing equivalent is returned to the protein. However, for the next catalytic cycle to occur, the protein must undergo an electronic state isomerization, returning the electron to the SAM-binding cluster. Using a series of iron-sulfur cluster deletion mutants, our data support a model whereby the isomerization is an obligatorily intermolecular electron transfer event that can be mediated by redox active proteins or small molecules, likely via the second AC in PapB. Surprisingly, a mixture of FMN and NADPH is sufficient to support both the reductive and the isomerization steps. These findings lead to a new paradigm involving intermolecular electron transfer steps in the activation of rSAM enzymes that require multiple iron-sulfur clusters for turnover. The implications of these results for the biological activation of rSAM enzymes are discussed.

Peptide Selenocysteine Substitutions Reveal Direct Substrate-Enzyme Interactions at Auxiliary Clusters in Radical S-Adenosyl-l-methionine Maturases.

Radical S-adenosyl-l-methionine (SAM) enzymes leverage the properties of one or more iron- and sulfide-containing metallocenters to catalyze complex and radical-mediated transformations. By far the most populous superfamily of radical SAM enzymes are those that, in addition to a 4Fe-4S cluster that binds and activates the SAM cofactor, also bind one or more additional auxiliary clusters (ACs) of largely unknown catalytic significance. In this report we examine the role of ACs in two RS enzymes, PapB and Tte1186, that catalyze formation of thioether cross-links in ribosomally synthesized and post-translationally modified peptides (RiPPs). Both enzymes catalyze a sulfur-to-carbon cross-link in a reaction that entails H atom transfer from an unactivated C-H to initiate catalysis, followed by formation of a C-S bond to yield the thioether. We show that both enzymes tolerate substitution of SeCys instead of Cys at the cross-linking site, allowing the systems to be subjected to Se K-edge X-ray spectroscopy. The EXAFS data show a direct interaction with the Fe of one of the ACs in the Michaelis complex, which is replaced with a Se-C interaction under reducing conditions that lead to the product complex. Site-directed deletion of the clusters in Tte1186 provide evidence for the identity of the AC. The implications of these observations in the context of the mechanism of these thioether cross-linking enzymes are discussed.

Photoinduced Dithiolane Crosslinking for Multiresponsive Dynamic Hydrogels.

While many hydrogels are elastic networks crosslinked by covalent bonds, viscoelastic hydrogels with adaptable crosslinks are increasingly being developed to better recapitulate time and position-dependent processes found in many tissues. In this work, 1,2-dithiolanes are presented as dynamic covalent photocrosslinkers of hydrogels, resulting in disulfide bonds throughout the hydrogel that respond to multiple stimuli. Using lipoic acid as a model dithiolane, disulfide crosslinks are formed under physiological conditions, enabling cell encapsulation via an initiator-free light-induced dithiolane ring-opening photopolymerization. The resulting hydrogels allow for multiple photoinduced dynamic responses including stress relaxation, stiffening, softening, and network functionalization using a single chemistry, which can be supplemented by permanent reaction with alkenes to further control network properties and connectivity using irreversible thioether crosslinks. Moreover, complementary photochemical approaches are used to achieve rapid and complete sample degradation via radical scission and post-gelation network stiffening when irradiated in the presence of reactive gel precursor. The results herein demonstrate the versatility of this material chemistry to study and direct 2D and 3D cell-material interactions. This work highlights dithiolane-based hydrogel photocrosslinking as a robust method for generating adaptable hydrogels with a range of biologically relevant mechanical and chemical properties that are varied on demand.

Leveraging Substrate Promiscuity of a Radical S-Adenosyl-L-methionine RiPP Maturase toward Intramolecular Peptide Cross-Linking Applications.

Radical S-adenosyl-l-methionine (RS) enzymes operate on a variety of substrates and catalyze a wide range of complex radical-mediated transformations. Radical non-α-carbon thioether peptides (ranthipeptides) are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs). The RS enzyme PapB catalyzes the formation of thioether cross-links between Cys/Asp (or Cys/Glu) residues located in six Cys-X3-Asp/Glu motifs. In this report, using a minimal substrate that contains a single cross-link motif, we explore the substrate scope of the PapB and show that the enzyme is highly promiscuous and will accept a variety of Cys-Xn-Asp sequences where n = 0–6. Moreover, we show that the enzyme will introduce in-line and nested thioether cross-links independently in peptide sequences that contain two motifs derived from the wild-type sequence. Additionally, the enzyme accepts peptides that contain d-amino acids at either the Cys or the Asp position. These observations are leveraged to produce a thioether cyclized analogue of the FDA-approved therapeutic agent octreotide, with a Cys-Glu cross-link replacing the disulfide that is found in the drug. These findings highlight the remarkable substrate tolerance of PapB and show the utility of RS RiPP maturases in biotechnological applications.

Rational Design of a Dual-Targeting Natural Toxin-Like Bicyclic Peptide for Selective Bioenergetic Blockage in Cancer Cells.

Stapled α-helical peptides emerge as one of the attractive peptidomimetics which can efficiently penetrate the cell membrane to access intracellular targets. However, the incorporation of a highly lipophilic cross-link may lead to nonspecific membrane toxicity in certain cases. Here, we report a new class of thioether-tethered bicyclic α-helical peptide to mimic the highly constrained loop-helix structure of natural toxins with the dual-targeting ability for both cell-surface receptors and intracellular targets. The thioether cross-links are introduced to replace the redox-sensitive disulfide bonds in natural toxins via a photoinduced thiol-yne reaction followed by macrolactamization. As a proof of concept, αVβ3 integrin targeting ligand was grafted into one of the macrocycles in the bicyclic scaffold, while a mitochondria-targeting proapoptotic motif was introduced into the other macrocycle stabilized by an i, i + 7 alkyl thioether cross-link to recapitulate its α-helical conformation. The obtained dual-targeting bicyclic α-helical BIRK peptides showed highly stable α-helical conformation in the presence of denaturants or under high temperature. Notably, BIRK peptides could induce selective cell death in αVβ3 integrin-positive B16F10 cells by interfering with the bioenergetic functions of mitochondria. This work provides a new avenue to design and stabilize α-helical peptides in a highly constrained bicyclic loop-helix scaffold with dual functionality.

Exploring structural signatures of the lanthipeptide prochlorosin 2.8 using tandem mass spectrometry and trapped ion mobility-mass spectrometry.

Lanthipeptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by intramolecular thioether cross-links formed between a dehydrated serine/threonine (dSer/dThr) and a cysteine residue. Prochlorosin 2.8 (Pcn2.8) is a class II lanthipeptide that exhibits a non-overlapping thioether ring pattern, for which no biological activity has been reported yet. The variant Pcn2.8[16RGD] has been shown to bind tightly to the αvβ3 integrin receptor. In the present work, tandem mass spectrometry, using collision-induced dissociation (CID) and electron capture dissociation (ECD), and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) were used to investigate structural signatures for the non-overlapping thioether ring pattern of Pcn2.8. CID experiments on Pcn2.8 yielded bi and yj fragments between the thioether cross-links, evidencing the presence of a non-overlapping thioether ring pattern. ECD experiments of Pcn2.8 showed a significant increase of hydrogen migration events near the residues involved in the thioether rings with a more pronounced effect at the dehydrated residues as compared to the cysteine residues. The high-resolution mobility analysis, aided by site-directed mutagenesis ([P8A], [P11A], [P12A], [P8A/P11A], [P8A/P12A], [P11A/P12A], and [P8A/P11A/P12A] variants), demonstrated that Pcn2.8 adopts cis/trans-conformations at Pro8, Pro11, and Pro12 residues. These observations were complementary to recent NMR findings, for which only the Pro8 residue was evidenced to adopt cis/trans-orientations. This study highlights the analytical power of the TIMS-MS/MS workflow for the structural characterization of lanthipeptides and could be a useful tool in our understanding of the biologically important structural elements that drive the thioether cyclization process.

Late-Stage Hydrocarbon Conjugation and Cyclisation in Synthetic Peptides and Proteins.

The conventional S-alkylation of cysteine relies upon using activated electrophiles. Here we demonstrate high-yielding and selective S-alkylation and S-lipidation of cysteines in unprotected synthetic peptides and proteins by using weak electrophiles and a Zn2+ promoter. Linear or branched iodoalkanes can S-alkylate cysteine in an unprotected 38-residue Myc peptide fragment and in a 91-residue miniprotein Omomyc, thus highlighting selective late-stage synthetic modifications. Metal-assisted cysteine alkylation is also effective for incorporating dehydroalanine into unprotected peptides and for peptide cyclisation via aliphatic thioether crosslinks, including customising macrocycles to stabilise helical peptides for enhanced uptake and delivery to proteins inside cells. Chemoselective and efficient late-stage Zn2+ -promoted cysteine alkylation in unprotected peptides and proteins promises many useful applications.

Precursor peptide-targeted mining of more than one hundred thousand genomes expands the lanthipeptide natural product family

BackgroundLanthipeptides belong to the ribosomally synthesized and post-translationally modified peptide group of natural products and have a variety of biological activities ranging from antibiotics to antinociceptives. These peptides are cyclized through thioether crosslinks and can bear other secondary post-translational modifications. While lanthipeptide biosynthetic gene clusters can be identified by the presence of genes encoding characteristic enzymes involved in the post-translational modification process, locating the precursor peptides encoded within these clusters is challenging due to their short length and high sequence variability, which limits the high-throughput exploration of lanthipeptide biosynthesis. To address this challenge, we enhanced the predictive capabilities of Rapid ORF Description & Evaluation Online (RODEO) to identify members of all four known classes of lanthipeptides.ResultsUsing RODEO, we mined over 100,000 bacterial and archaeal genomes in the RefSeq database. We identified nearly 8500 lanthipeptide precursor peptides. These precursor peptides were identified in a broad range of bacterial phyla as well as the Euryarchaeota phylum of archaea. Bacteroidetes were found to encode a large number of these biosynthetic gene clusters, despite making up a relatively small portion of the genomes in this dataset. A number of these precursor peptides are similar to those of previously characterized lanthipeptides, but even more were not, including potential antibiotics. One such new antimicrobial lanthipeptide was purified and characterized. Additionally, examination of the biosynthetic gene clusters revealed that enzymes installing secondary post-translational modifications are more widespread than initially thought.ConclusionLanthipeptide biosynthetic gene clusters are more widely distributed and the precursor peptides encoded within these clusters are more diverse than previously appreciated, demonstrating that the lanthipeptide sequence-function space remains largely underexplored.

Photopatternable, Branched Polymer Hydrogels Based on Linear Macromonomers for 3D Cell Culture Applications.

Photochemical ligation strategies in hydrogel materials are crucial to model spatiotemporal phenomena that occur in the natural extracellular matrix. We here describe the use of cyclic 1,2-dithiolanes to cross-link with norbornene on linear poly(ethylene glycol) polymers through UV irradiation in a rapid and byproduct-free manner, resulting in branched macromolecular architectures and hydrogel materials from low-viscosity precursor solutions. Oscillatory rheology and NMR data indicate the one-pot formation of thioether and disulfide cross-links. Spatial and temporal control of the hydrogel mechanical properties and functionality was demonstrated by oscillatory rheology and confocal microscopy. A cytocompatible response of NIH 3T3 fibroblasts was observed within these materials, providing a foothold for further exploration of this photoactive cross-linking moiety in the biomedical field.

Bioprocess Development for Lantibiotic Ruminococcin-A Production in Escherichia coli and Kinetic Insights Into LanM Enzymes Catalysis.

Ruminococcin-A (RumA) is a peptide antibiotic with post-translational modifications including thioether cross-links formed from non-canonical amino acids, called lanthionines, synthesized by a dedicated lanthionine-generating enzyme RumM. RumA is naturally produced by Ruminococcus gnavus, which is part of the normal bacterial flora in the human gut. High activity of RumA against pathogenic Clostridia has been reported, thus allowing potential exploitation of RumA for clinical applications. However, purifying RumA from R. gnavus is challenging due to low production yields (<1 μg L–1) and difficulties to cultivate the obligately anaerobic organism. We recently reported the reconstruction of the RumA biosynthesis machinery in Escherichia coli where the fully modified and active peptide was expressed as a fusion protein together with GFP. In the current study we developed a scale-up strategy for the biotechnologically relevant heterologous production of RumA, aimed at overproducing the peptide under conditions comparable with those in industrial production settings. To this end, glucose-limited fed-batch cultivation was used. Firstly, parallel cultivations were performed in 24-microwell plates using the enzyme-based automated glucose-delivery cultivation system EnPresso® B to determine optimal conditions for IPTG induction. We combined the bioprocess development with ESI-MS and tandem ESI-MS to monitor modification of the precursor peptide (preRumA) during bioreactor cultivation. Dehydration of threonine and serine residues in the core peptide, catalyzed by RumM, occurs within 1 h after IPTG induction while formation of thioether cross-bridges occur around 2.5 h after induction. Our data also supplies important information on modification kinetics especially with respect to the fluctuations observed in the various dehydrated precursor peptide versions or intermediates produced at different time points during bioreactor cultivation. Overall, protein yields obtained from the bioreactor cultivations were >120 mg L–1 for the chimeric construct and >150 mg L–1 for RumM. The correlation observed between microscale and lab-scale bioreactor cultivations suggests that the process is robust and realistically applicable to industrial-scale conditions.

Assessing the Flexibility of the Prochlorosin 2.8 Scaffold for Bioengineering Applications.

Cyclization is a common strategy to confer proteolytic resistance to peptide scaffolds. Thus, cyclic peptides have been the focus of extensive bioengineering efforts. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a superfamily of peptidic natural products that often contain macrocycles. In the RiPP family of lanthipeptides, macrocyclization is accomplished through formation of thioether cross-links between cysteines and dehydrated serines/threonines. The recent production of lanthipeptide libraries and development of methods to display lanthipeptides on yeast or phage highlights their potential for bioengineering and synthetic biology. In this regard, the prochlorosins are especially promising as the corresponding class II lanthipeptide synthetase ProcM matures numerous precursor peptides with diverse core peptide sequences. To facilitate future bioengineering projects, one of its native substrates, ProcA2.8, was subjected in this study to in-depth mutational analysis to test the limitations of ProcM-mediated cyclization. Alanine scan mutagenesis was performed on all residues within the two rings, and multiple prolines were introduced at various positions. Moreover, mutation, deletion, and insertion of residues in the region linking the two lanthionine rings was tested. Additional residues were also introduced or deleted from either ring, and inversion of ring forming residues was attempted to generate diastereomers. The findings were used for epitope grafting of the RGD integrin binding epitope within prochlorosin 2.8, resulting in a low nanomolar affinity binder of the αvβ3 integrin that was more stable toward proteolysis and displayed higher affinity than the linear counterpart.

Influence of Sulfur-Containing Diamino Acid Structure on Covalently Crosslinked Copolypeptide Hydrogels.

Biologically occurring non-canonical di-α-amino acids were converted into new di-N-carboxyanhydride (di-NCA) monomers in reasonable yields with high purity. Five different di-NCAs were separately copolymerized with tert-butyl-l-glutamate NCA to obtain covalently crosslinked copolypeptides capable of forming hydrogels with varying crosslinker density. Comparison of hydrogel properties with residue structure revealed that different di-α-amino acids were not equivalent in crosslink formation. Notably, l-cystine was found to produce significantly weaker hydrogels compared to l-homocystine, l-cystathionine, and l-lanthionine, suggesting that l-cystine may be a sub-optimal choice of di-α-amino acid for preparation of copolypeptide networks. The di-α-amino acid crosslinkers also provided different chemical stability, where disulfide crosslinks were readily degraded by reduction, and thioether crosslinks were stable against reduction. This difference in response may provide a means to fine tune the reduction sensitivity of polypeptide biomaterial networks.

Development and Application of Yeast and Phage Display of Diverse Lanthipeptides.

Peptide display has enabled identification and optimization of ligands to many targets. These ligands are usually linear or disulfide-containing peptides that are vulnerable to proteolysis or reduction. We report yeast surface and phage display of lanthipeptides, macrocyclic ribosomally synthesized and post-translationally modified peptides (RiPPs). Lanthipeptides contain multiple thioether cross-links that bestow their biological activities. We developed C-terminal yeast display of the class II lanthipeptides lacticin 481 and haloduracin β, and randomization of the C-ring of the former was used to select tight binders to αvβ3 integrin. This represents the first examples of bacterial RiPP production in Saccharomyces cerevisiae for identification of variants with new biological activities. We also report N-terminal phage display of the class I lanthipeptide nisin and randomization of its A- and B-rings to enrich binders to a small molecule, lipid II. The successful display and randomization of both class I and II lanthipeptides demonstrates the versatility and potential of RiPP display.

Incorporation of Nonproteinogenic Amino Acids in Class I and II Lantibiotics.

Lantibiotics are ribosomally synthesized and post-translationally modified peptide natural products that contain thioether cross-links formed by lanthionine and methyllanthionine residues. They exert potent antimicrobial activity against Gram-positive bacteria. We herein report production of analogues of two lantibiotics, lacticin 481 and nisin, that contain nonproteinogenic amino acids using two different strategies involving amber stop codon suppression technology. These methods complement recent alternative approaches to incorporate nonproteinogenic amino acids into lantibiotics.

Intramolecular Thioether Crosslinking to Increase the Proteolytic Stability of Affibody Molecules.

Protein therapeutics suffer from low oral bioavailability, mainly due to poor membrane permeability and digestion by gastrointestinal proteases. To improve proteolytic stability, intramolecular thioether crosslinks were introduced into a three-helix affibody molecule binding the human epidermal growth factor receptor (EGFR). Solid-phase peptide synthesis was used to produce an unmodified control protein domain and three different crosslinked protein domain variants: one with a thioether crosslink between the N-terminal lysine residue and a cysteine residue in the second loop region (denoted K4), a second with a crosslink between the C-terminal lysine residue and a cysteine residue in the first loop region (denoted K58), and a third with crosslinks in both positions (denoted K4K58). Circular dichroism (CD) and surface-plasmon-resonance-based (SPR-based) biosensor studies of the protein domains showed that the three-helix structure and high-affinity binding to EGFR were preserved in the crosslinked protein domains. In vitro digestion by gastrointestinal proteases demonstrated that the crosslinked protein domains showed increased stability towards pepsin and towards a combination of trypsin and chymotrypsin.

Mechanistic Understanding of Lanthipeptide Biosynthetic Enzymes.

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) that display a wide variety of biological activities, from antimicrobial to antiallodynic. Lanthipeptides that display antimicrobial activity are called lantibiotics. The post-translational modification reactions of lanthipeptides include dehydration of Ser and Thr residues to dehydroalanine and dehydrobutyrine, a transformation that is carried out in three unique ways in different classes of lanthipeptides. In a cyclization process, Cys residues then attack the dehydrated residues to generate the lanthionine and methyllanthionine thioether cross-linked amino acids from which lanthipeptides derive their name. The resulting polycyclic peptides have constrained conformations that confer their biological activities. After installation of the characteristic thioether cross-links, tailoring enzymes introduce additional post-translational modifications that are unique to each lanthipeptide and that fine-tune their activities and/or stability. This review focuses on studies published over the past decade that have provided much insight into the mechanisms of the enzymes that carry out the post-translational modifications.

Synthesis and Bioactivity of Diastereomers of the Virulence Lanthipeptide Cytolysin.

Cytolysin, a two-component lanthipeptide comprising cytolysin S (CylLS″) and cytolysin L (CylLL″), is the only family member to exhibit lytic activity against mammalian cells in addition to synergistic antimicrobial activity. A subset of the thioether cross-links of CylLS″ and CylLL″ have ll stereochemistry instead of the canonical dl stereochemistry in all previously characterized lanthipeptides. The synthesis of a CylLS″ variant with dl stereochemistry is reported. Its antimicrobial activity was found to be decreased, but not its lytic activity against red blood cells. Hence, the unusual ll stereochemistry is not responsible for the lytic activity.

Tuning Hydrogel Properties to Promote the Assembly of Salivary Gland Spheroids in 3D

Current treatments for chronic xerostomia, or "dry mouth", do not offer long-term therapeutic benefits for head and neck cancer survivors previously treated with curative radiation. Towards the goal of creating tissue-engineered constructs for the restoration of salivary gland functions, we developed new hyaluronic acid (HA)-based hydrogels using thiolated HA (HA-SH) and acrylated HA (HA-AES) with a significant molecular weight mismatch. Four hydrogel formulations with varying HA concentration, (1-2.4 wt%) and thiol/acrylate ratios (2/1 to 36/1) and elastic moduli (G': 35 to 1897 Pa, 2 h post-mixing) were investigated. In our system, thiol/acrylate reaction was initiated rapidly upon mixing of HA-SH/HA-AES to establish thioether crosslinks with neighboring ester groups, and spontaneous sulfhydryl oxidation occurred slowly over several days to install a secondary network. The concurrent reactions cooperatively create a cell-permissive network to allow for cell expansion and aggregation. Multicellular spheroids formed readily from a robust ductal epithelial cell line (Madin-Darby Canine Kidney, MDCK cells) in all hydrogel formulations investigated. Primary salivary human stem/progenitor cells (hS/PCs), on the other hand, are sensitive to the synthetic extracellular environment, and organized acini-like structures with an average diameter of 50 µm were obtained only in gels with G' ≤ 216 Pa and a thiol/acrylate ratio ≥18. The spheroid size and size distribution were dependent on the HA content in the hydrogel. Cells in hS/PC spheroids formed tight junctions (occludin), remained viable and proliferative, secreted structural proteins (collagen IV and laminin) found in the basement membrane and maintained key stem/progenitor markers. We conclude that incorporation of time-dependent, dynamic features into a covalently crosslinked HA network produces an adaptable hydrogel framework that promotes hS/PC assembly and supports early aspects of salivary morphogenesis, key to reconstitution of a fully functional implantable salivary gland.

Radical SAM Enzyme‐Catalyzed Formation of Thioether Crosslinks in Ribosomal‐Produced Peptides: Formation of the RiPP Natural Product Thuricin CD by the Radical SAM Enzymes TrnC and TrnD

Radical SAM Enzyme‐Catalyzed Formation of Thioether Crosslinks in Ribosomal‐Produced Peptides: Formation of the RiPP Natural Product Thuricin CD by the Radical SAM Enzymes TrnC and TrnD