Generation of Lasso Peptide-Based ClpP Binders

The Clp protease system fulfills a plethora of important functions in bacteria. It consists of a tetradecameric ClpP barrel holding the proteolytic centers and two hexameric Clp-ATPase rings, which recognize, unfold, and then feed substrate proteins into the ClpP barrel for proteolytic degradation. Flexible loops carrying conserved tripeptide motifs protrude from the Clp-ATPases and bind into hydrophobic pockets (H-pockets) on ClpP. Here, we set out to engineer microcin J25 (MccJ25), a ribosomally synthesized and post-translationally modified peptide (RiPP) of the lasso peptide subfamily, by introducing the conserved tripeptide motifs into the lasso peptide loop region to mimic the Clp-ATPase loops. We studied the capacity of the resulting lasso peptide variants to bind to ClpP and affect its activity. From the nine variants generated, one in particular (12IGF) was able to activate ClpP from Staphylococcus aureus and Bacillus subtilis. While 12IGF conferred stability to ClpP tetradecamers and stimulated peptide degradation, it did not trigger unregulated protein degradation, in contrast to the H-pocket-binding acyldepsipeptide antibiotics (ADEPs). Interestingly, synergistic interactions between 12IGF and ADEP were observed

Discovery of a Unique Structural Motif in Lanthipeptide Synthetases for Substrate Binding and Interdomain Interactions

Class III lanthipeptide synthetases catalyze the formation of lanthionine/methyllanthionine and labionin crosslinks. We present here the 2.40 Å resolution structure of the kinase domain of a class III lanthipeptide synthetase CurKC from the biosynthesis of curvopeptin. A unique structural subunit for leader binding, named leader recognition domain (LRD), was identified. The LRD of CurKC is responsible for the recognition of the leader peptide and for mediating interactions between the lyase and kinase domains. LRDs are highly conserved among the kinase domains of class III and class IV lanthipeptide synthetases. The discovery of LRDs provides insight into the substrate recognition and domain organization in multidomain lanthipeptide synthetases

Das Potenzial von RiPPs in medizinisch-biotechnologischen Anwendungen

Ribosomally synthesized and post-translationally modified proteins (RiPPs) are an interesting natural product superfamily. Their underlying biosynthetic principles, where recognition sites are strictly separated from the regions that are modified in the substrate, make them interesting for utilization in medicinal and biotechnological applications. Here, the promiscuity of RiPP biosynthetic enzymes will be described for examples from the lasso and lanthipeptide subfamilies and current developments as well as future directions in this field will be discussed.TU Berlin, Open-Access-Mittel – 202

Generation of Lasso Peptide-Based ClpP Binders

The Clp protease system fulfills a plethora of important functions in bacteria. It consists of a tetradecameric ClpP barrel holding the proteolytic centers and two hexameric Clp-ATPase rings, which recognize, unfold, and then feed substrate proteins into the ClpP barrel for proteolytic degradation. Flexible loops carrying conserved tripeptide motifs protrude from the Clp-ATPases and bind into hydrophobic pockets (H-pockets) on ClpP. Here, we set out to engineer microcin J25 (MccJ25), a ribosomally synthesized and post-translationally modified peptide (RiPP) of the lasso peptide subfamily, by introducing the conserved tripeptide motifs into the lasso peptide loop region to mimic the Clp-ATPase loops. We studied the capacity of the resulting lasso peptide variants to bind to ClpP and affect its activity. From the nine variants generated, one in particular (12IGF) was able to activate ClpP from Staphylococcus aureus and Bacillus subtilis. While 12IGF conferred stability to ClpP tetradecamers and stimulated peptide degradation, it did not trigger unregulated protein degradation, in contrast to the H-pocket-binding acyldepsipeptide antibiotics (ADEPs). Interestingly, synergistic interactions between 12IGF and ADEP were observed

Investigation of Substrate Recognition and Biosynthesis in Class IV Lanthipeptide Systems

Lanthipeptides belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs) and are subdivided into four classes. The first two classes have been heavily studied, but less is known about classes III and IV. The lanthipeptide synthetases of classes III and IV share a similar organization of protein domains: A lyase domain at the N-terminus, a central kinase domain, and a C-terminal cyclase domain. Here, we provide deeper insight into class IV enzymes (LanLs). A series of putative producer strains was screened to identify production conditions of four new venezuelin-like lanthipeptides, and an <i>Escherichia coli</i> based heterologous production system was established for a fifth. The latter not only allowed production of fully modified core peptide but was also employed as the basis for mutational analysis of the precursor peptide to identify regions important for enzyme recognition. These experiments were complemented by <i>in vitro</i> binding studies aimed at identifying the region of the leader peptide recognized by the LanL enzymes as well as determining which domain of the enzyme is recognizing the substrate peptide. Combined, these studies revealed that the kinase domain is mediating the interaction with the precursor peptide and that a putatively α-helical stretch of residues at the center to N-terminal region of the leader peptide is important for enzyme recognition. In addition, a combination of <i>in vitro</i> assays and tandem mass spectrometry was used to elucidate the order of dehydration events in these systems

Lasso Peptides: An Intriguing Class of Bacterial Natural Products

ConspectusNatural products of peptidic origin often represent a rich source of medically relevant compounds. The synthesis of such polypeptides in nature is either initiated by deciphering the genetic code on the ribosome during the translation process or driven by ribosome-independent processes. In the latter case, highly modified bioactive peptides are assembled by multimodular enzymes designated as nonribosomal peptide synthetases (NRPS) that act as a protein-template to generate chemically diverse peptides. On the other hand, the ribosome-dependent strategy, although relying strictly on the 20–22 proteinogenic amino acids, generates structural diversity by extensive post-translational-modification. This strategy seems to be highly distributed in all kingdoms of life. One example for this is the lasso peptides, which are an emerging class of ribosomally assembled and post-translationally modified peptides (RiPPs) from bacteria that were first described in 1991.A wide range of interesting biological activities are known for these compounds, including antimicrobial, enzyme inhibitory, and receptor antagonistic activities. Since 2008, genome mining approaches allowed the targeted isolation and characterization of such molecules and helped to better understand this compound class and their biosynthesis. Their defining structural feature is a macrolactam ring that is threaded by the C-terminal tail and held in position by sterically demanding residues above and below the ring, resulting in a unique topology that is reminiscent of a lariat knot. The ring closure is achieved by an isopeptide bond formed between the N-terminal α-amino group of a glycine, alanine, serine, or cysteine and the carboxylic acid side chain of an aspartate or glutamate, which can be located at positions 7, 8, or 9 of the amino acid sequence.In this Account, we discuss the newest findings about these compounds, their biosynthesis, and their physicochemical properties. This includes the suggested mechanism through which the precursor peptide is enzymatically processed into a mature lasso peptide and crucial residues for enzymatic recognition. Furthermore, we highlight new insights considering the protease and thermal stability of lasso peptides and discuss why seven amino acid residue rings are likely to be the lower limit feasible for this compound class. To elucidate their fascinating three-dimensional structures, NMR spectroscopy is commonly employed. Therefore, the general methodology to elucidate these structures by NMR will be discussed and pitfalls for these approaches are highlighted. In addition, new tools provided by recent investigations to assess and prove the lasso topology without a complete structure elucidation will be summarized. These include techniques like ion mobility–mass spectrometry and a combined approach of thermal and carboxypeptidase treatment with subsequent LC-MS analysis. Nevertheless, even though much was learned about these compounds in recent years, their true native function and the exact enzymatic mechanism of their maturation remain elusive

Caulosegnins I–III: A Highly Diverse Group of Lasso Peptides Derived from a Single Biosynthetic Gene Cluster

Lasso peptides are natural products of ribosomal origin with a unique knotted structural fold. Even though only a few of them are known, recent reports of newly isolated lasso peptides were scarce. In this work, we report the identification of a novel lasso peptide gene cluster from <i>Caulobacter segnis</i>, that produces three new lasso peptides (caulosegnins I, II, and III) using a single biosynthetic machinery. These lasso peptides possess different ring sizes and amino acid sequences. In this study, we have developed a system for enhanced lasso peptide production to allow isolation of these compounds through heterologous expression in <i>Escherichia coli</i>. We were able to elucidate the structure of the most abundant lasso peptide caulosegnin I via NMR spectroscopic analysis and performed a thorough mutational analysis that gave insight into their biosynthesis and revealed important factors affecting the stabilization of the lasso fold in general. The caulosegnins also show a diverse behavior when subjected to thermal denaturation, which is exceptional as all lasso peptides were believed to have an intrinsic high thermal stability

Specific fragmentation of mechanically interlocked peptides

National audienc

General rules of fragmentation evidencing lasso structures in CID and ETD

International audienceLasso peptides constitute a structurally unique class of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by a mechanically interlocked structure in which the C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. Tandem mass spectrometry using collision induced dissociation (CID) and electron capture dissociation (ECD) have shown previously different fragmentation patterns for capistruin, microcin J25 and their corresponding branched-cyclic forms in which the C-terminal tail is unthreaded. In order to develop general rules that unambiguously discriminate the lasso and branched-cyclic topologies, this report presents experimental evidence for a set of twenty-one lasso peptides analyzed by CID and electron transfer dissociation (ETD). CID experiments on lasso peptides specifically yielded mechanically interlocked species with associated bi and yj fragments. For class II lasso peptides, these lasso-specific fragments were observed only for peptides in which the loop, located above the macrolactam ring, was strictly longer than four amino acid residues. For class I and III lasso peptides, part of the C-terminal tail remains covalently linked to the macrolactam ring by disulfide bonds; associated bi and yj fragments therefore do not clearly constitute a signature of the lasso topology. ETD experiments of lasso peptides showed a significant increase of hydrogen migration events in the loop region when compared to their branched-cyclic topoisomers, leading to the formation of specific ci˙/z′j fragments for all lasso peptides, regardless of their class and loop size. Our experiments enabled us to establish general rules for obtaining structural details from CID and ETD fragmentation patterns, obviating the need for structure determination by NMR or X-ray crystallography