Thomas A. Knappe

Isolation and Structural Characterization of Capistruin, a Lasso Peptide Predicted from the Genome Sequence of Burkholderia thailandensis E264

Lasso peptides are a structurally unique class of bioactive peptides characterized by a knotted arrangement, where the C-terminus threads through an N-terminal macrolactam ring. Although ribosomally synthesized, only the gene cluster for the best studied lasso peptide MccJ25 from Escherichia coli consisting of the precursor protein McjA and the processing and immunity proteins McjB, McjC, and McjD is known. Through genome mining studies, we have identified homologues of all four proteins in Burkholderia thailandensis E264 and predicted this strain to produce a lasso peptide. Here we report the successful isolation of the predicted peptide, named capistruin. Upon optimization of the fermentation conditions, mass spectrometric and NMR structural studies proved capistruin to adopt a novel lasso fold. Heterologous production of the lasso peptide in Escherichia coli showed that the identified genes are sufficient for the biosynthesis of capistruin, which exhibits antimicrobial activity against closely related Burkholderia and Pseudomonas strains. In general, our rational approach should be widely applicable for the isolation of new lasso peptides to explore their high structural stability and diverse biological activity.

The radical SAM enzyme AlbA catalyzes thioether bond formation in subtilosin A

Subtilosin A is a 35-residue, ribosomally synthesized bacteriocin encoded by the sbo-alb operon of Bacillus subtilis. It is composed of a head-to-tail circular peptide backbone that is additionally restrained by three unusual thioether bonds between three cysteines and the α-carbon of one threonine and two phenylalanines, respectively. In this study, we demonstrate that these bonds are synthesized by the radical S-adenosylmethionine enzyme AlbA, which is encoded by the sbo-alb operon and comprises two [4Fe-4S] clusters. One [4Fe-4S] cluster is coordinated by the prototypical CXXXCXXC motif and is responsible for the observed S-adenosylmethionine cleavage reaction, whereas the second [4Fe-4S] cluster is required for the generation of all three thioether linkages. On the basis of the obtained results, we propose a new radical mechanism for thioether bond formation. In addition, we show that AlbA-directed substrate transformation is leader-peptide dependent, suggesting that thioether bond formation is the first step during subtilosin A maturation.

Insights into the Biosynthesis and Stability of the Lasso Peptide Capistruin

Capistruin is a 19-residue ribosomally synthesized lasso peptide encoded by the capABCD gene cluster in Burkholderia thailandensis. It is composed of an N-terminal 9-residue macrolactam ring, through which the 10-residue C-terminal tail is threaded. Using a heterologous capistruin production system in Escherichia coli, we have generated 48 mutants of the precursor protein CapA to gain insights into capistruin biosynthesis. Only 4 residues (Gly1, Arg11, Val12, and Ile13) of the lasso sequence were found to be critical for maturation. Tandem mass spectrometric fragmentation studies of capistruin F16A/F18A proved Arg15 to be responsible for the trapping of the C-terminal tail. Substituting Arg15 and Phe16 by alanine revealed a temperature-sensitive capistruin derivative, which unfolds into a branched cyclic peptide upon heating. In conclusion, our global mutagenic approach revealed a low overall specificity of the biosynthetic machinery and important structure-stability correlations.

Introducing Lasso Peptides as Molecular Scaffolds for Drug Design: Engineering of an Integrin Antagonist

Thumbnail image of graphical abstract Tightening the noose: Lasso peptides are a class of stable bacterial peptides with unique characteristics that encourage their application in drug design. Epitope grafting of the integrin binding motif RGD onto the lasso structure of microcin J25 converts the knotted peptide into a nanomolar integrin antagonist (see picture). Engineered lasso peptides can therefore be used for pharmacophore presentation.

Dissecting the Maturation Steps of the Lasso Peptide Microcin J25 in vitro

Microcin J25 is the archetype of a growing class of bacterial ribosomal peptides possessing a knotted topology (lasso peptides). It consists of an eight‐residue macrolactam ring through which the C‐terminal tail is threaded. It is biosynthesized as a precursor that is processed by two maturation enzymes (McjB/McjC). Insights into the mechanism of microcin J25 biosynthesis have been provided previously by mutagenesis of the precursor peptide in vivo. In this study we have demonstrated distinct functions of McjB and McjC in vitro for the first time, based on the detection of reaction intermediates. McjB was characterized as a new ATP‐dependent cysteine protease, whereas McjC was confirmed to be a lactam synthetase. The two enzymes were functionally interdependent, likely forming a structural complex. Their substrate preference was directly investigated with the aid of mutated precursor peptides. Depending on the substitutions, microcin J25 variants with either a lasso or branched‐cyclic topology could be generated in vitro.

The glucagon receptor antagonist BI‐32169 constitutes a new class of lasso peptides

The glucagon receptor antagonist BI‐32169, recently isolated from Streptomyces sp., was described as a bicyclic peptide, although its primary structure comprises conserved elements of class I and class II lasso peptides. Tandem mass spectrometric and nuclear magnetic resonance spectroscopic studies revealed that BI‐32169 is a lasso‐structured peptide constituting the new class III of lasso peptides. The determined lasso fold opens new avenues to improve the promising biological activity of BI‐32169.

The Antibacterial Threaded-lasso Peptide Capistruin Inhibits Bacterial RNA Polymerase

Capistruin, a ribosomally synthesized, post-translationally modified peptide produced by Burkholderia thailandensis E264, efficiently inhibits growth of Burkholderia and closely related Pseudomonas strains. The functional target of capistruin is not known. Capistruin is a threaded-lasso peptide (lariat peptide) consisting of an N-terminal ring of nine amino acids and a C-terminal tail of 10 amino acids threaded through the ring. The structure of capistruin is similar to that of microcin J25 (MccJ25), a threaded-lasso antibacterial peptide that is produced by some strains of Escherichia coli and targets DNA-dependent RNA polymerase (RNAP). Here, we show that capistruin, like MccJ25, inhibits wild type E. coli RNAP but not mutant, MccJ25-resistant, E. coli RNAP. We show further that an E. coli strain resistant to MccJ25, as a result of a mutation in an RNAP subunit gene, exhibits resistance to capistruin. The results indicate that the structural similarity of capistruin and MccJ25 reflects functional similarity and suggest that the functional target of capistruin, and possibly other threaded-lasso peptides, is bacterial RNAP.

Erythrochelin - a hydroxamate-type siderophore predicted from the genome of Saccharopolyspora erythraea

The class of nonribosomally assembled siderophores encompasses a multitude of structurally diverse natural products. The genome of the erythromycin‐producing strain Saccharopolyspora erythraea contains 25 secondary metabolite gene clusters that are mostly considered to be orphan, including two that are responsible for siderophore assembly. In the present study, we report the isolation and structural elucidation of the hydroxamate‐type tetrapeptide siderophore erythrochelin, the first nonribosomal peptide synthetase‐derived natural product of S. erythraea. In an attempt to substitute the traditional activity assay‐guided isolation of novel secondary metabolites, we have employed a dedicated radio‐LC‐MS methodology to identify nonribosomal peptides of cryptic gene clusters in the industrially relevant strain. This methodology was based on transcriptome data and adenylation domain specificity prediction and resulted in the detection of a radiolabeled ornithine‐inheriting hydroxamate‐type siderophore. The improvement of siderophore production enabled the elucidation of the overall structure via NMR and MSn analysis and hydrolysate‐derivatization for the determination of the amino acid configuration. The sequence of the tetrapeptide siderophore erythrochelin was determined to be d‐α‐N‐acetyl‐δ‐N‐acetyl‐δ‐N‐hydroxyornithine‐d‐serine‐cyclo(l‐δ‐N‐hydroxyornithine‐l‐δ‐N‐acetyl‐δ‐N‐hydroxyornithine). The results derived from the structural and functional characterization of erythrochelin enabled the proposal of a biosynthetic pathway. In this model, the tetrapeptide is assembled by the nonribosomal peptide synthetase EtcD, involving unusual initiation‐ and cyclorelease‐mechanisms.

Rational improvement of the affinity and selectivity of integrin binding of grafted lasso peptides.

Integrins moderate diverse important functions in the human body and are promising targets in cancer therapy. Hence, the selective inhibition of specific integrins is of great medicinal interest. Here, we report the optimization of a grafted lasso peptide, yielding MccJ25(RGDF), which is a highly potent and selective αvβ3 integrin inhibitor. Furthermore, its NMR structure was elucidated and employed in a molecular dynamics approach, revealing information about the integrin binding mode and selectivity profile of MccJ25(RGDF).

Topoisomer Differentiation of Molecular Knots by FTICR MS: Lessons from Class II Lasso Peptides

Lasso peptides constitute a class of bioactive peptides sharing a knotted structure where the C-terminal tail of the peptide is threaded through and trapped within an N-terminal macrolactam ring. The structural characterization of lasso structures and differentiation from their unthreaded topoisomers is not trivial and generally requires the use of complementary biochemical and spectroscopic methods. Here we investigated two antimicrobial peptides belonging to the class II lasso peptide family and their corresponding unthreaded topoisomers: microcin J25 (MccJ25), which is known to yield two-peptide product ions specific of the lasso structure under collision-induced dissociation (CID), and capistruin, for which CID does not permit to unambiguously assign the lasso structure. The two pairs of topoisomers were analyzed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) upon CID, infrared multiple photon dissociation (IRMPD), and electron capture dissociation (ECD). CID and ECD spectra clearly permitted to differentiate MccJ25 from its non-lasso topoisomer MccJ25-Icm, while for capistruin, only ECD was informative and showed different extent of hydrogen migration (formation of c•/z from c/z•) for the threaded and unthreaded topoisomers. The ECD spectra of the triply-charged MccJ25 and MccJ25-lcm showed a series of radical b-type product ions