Julian D. Hegemann

Lasso Peptides: An Intriguing Class of Bacterial Natural Products

Natural 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.

Lasso peptides from proteobacteria: Genome mining employing heterologous expression and mass spectrometry

Lasso peptides are natural products with a unique three dimensional structure resembling a lariat knot. They are from ribosomal origin and are post-translationally modified by two enzymes (B and C), one of which shares little similarity to enzymes outside of lasso peptide biosynthetic gene clusters and as such is a useful target for genome mining. In this study, we demonstrate a B protein-centric genome mining approach through which we were able to identify 102 putative lasso peptide biosynthetic gene clusters from a total of 87 different proteobacterial strains. Ten of these clusters were cloned into the pET41a expression vector, optimized through incorporation of a ribosomal binding site and heterologously expressed in Escherichia coli BL21(DE3). All 12 predicted lasso peptides (namely burhizin, caulonodin I, caulonodin II, caulonodin III, rhodanodin, rubrivinodin, sphingonodin I, sphingonodin II, syanodin I, sphingopyxin I, sphingopyxin II, and zucinodin) were detected by high-resolution Fourier transform mass spectrometry and their proposed primary structure was confirmed through tandem mass spectrometry. High yields (ranging from 0.4 to 5.2 mg/L) were observable for eight of these compounds, while thermostability assays revealed five new representatives of heat labile lasso peptides.

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 Caulobacter segnis, 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 Escherichia coli. 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.

The Astexin-1 Lasso Peptides: Biosynthesis, Stability, and Structural Studies

Lasso peptides are a large family of natural products that owe their name to a unique structure formed by a side chain to backbone macrocyclization, resembling a knotted lasso. The unique structure has significant impact on their biological and physical properties, as lasso peptides are usually more stable than linear ones. Current work examines stability, structure, and biosynthesis of recently discovered lasso peptide astexin-1, a heat-sensitive lasso peptide. The obtained results revealed a new lasso structure with a tight loop and long tail as well as narrow specificity of the maturation machinery for some essential residues associated with the protease processing site, involved in macrolactam ring formation and entrapment of the tail. Using the astexin-1 structure, it was possible to rationally construct a thermostable variant of this lasso peptide.

Xanthomonins I–III: A New Class of Lasso Peptides with a Seven‐Residue Macrolactam Ring

Lasso peptides belong to the class of ribosomally synthesized and post-translationally modified peptides. Their common distinguishing feature is an N-terminal macrolactam ring that is threaded by the C-terminal tail. This lasso fold is maintained through steric interactions. The isolation and characterization of xanthomonins I-III, the first lasso peptides featuring macrolactam rings consisting of only seven amino acids, is now presented. The crystal structure of xanthomonin I and the NMR structure of xanthomonin II were also determined. A total of 25 variants of xanthomonin II were generated to probe different aspects of the biosynthesis, stability, and fold maintenance. These mutational studies reveal the limits such a small ring imposes on the threading and show that every plug amino acid larger than serine is able to maintain a heat-stable lasso fold in the xanthomonin II scaffold.

The PqqD homologous domain of the radical SAM enzyme ThnB is required for thioether bond formation during thurincin H maturation

Thurincin H is a 31‐residue, ribosomally synthesized bacteriocin originating from the thn operon of Bacillus thuringiensis SF361. It is the only known sactipeptide carrying four thioether bridges between four cysteines and the α‐carbons of a serine, an asparagine and two threonine residues. By analysis of the thn operon and use of in vitro studies we now reveal that ThnB is a radical S‐adenosylmethionine (SAM) enzyme containing two [4Fe–4S] clusters. Furthermore, we confirm the involvement of ThnB in the formation of the thioether bonds present within the structure of thurincin H. Finally, we show that the PqqD homologous N‐terminal domain of ThnB is essential for maturation of the thurincin H precursor peptide, but not for the SAM cleavage activity of ThnB.

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).

Ion Mobility–Mass Spectrometry of Lasso Peptides: Signature of a Rotaxane Topology

Ion mobility mass spectrometry data were collected on a set of five class II lasso peptides and their branched-cyclic topoisomers prepared in denaturing solvent conditions with and without sulfolane as a supercharging agent. Sulfolane was shown not to affect ion mobility results and to allow the formation of highly charged multiply protonated molecules. Drift time values of low charged multiply protonated molecules were found to be similar for the two peptide topologies, indicating the branched-cyclic peptide to be folded in the gas phase into a conformation as compact as the lasso peptide. Conversely, high charge states enabled a discrimination between lasso and branched-cyclic topoisomers, as the former remained compact in the gas phase while the branched-cyclic topoisomer unfolded. Comparison of the ion mobility mass spectrometry data of the lasso and branched-cyclic peptides for all charge states, including the higher charge states obtained with sulfolane, yielded three trends that allowed differentiation of the lasso form from the branched-cyclic topology: low intensity of highly charged protonated molecules, even with the supercharging agent, low change in collision cross sections with increasing charge state of all multiply protonated molecules, and narrow ion mobility peak widths associated with the coexistence of fewer conformations and possible conformational changes.

Characterization of caulonodin lasso peptides revealed unprecedented N-terminal residues and a precursor motif essential for peptide maturation

Lasso peptides, a peculiar family of ribosomally assembled and post-translationally modified peptides (RiPPs), possess a fascinating 3D structure, which can confer rigidity and stability against chemical and thermal denaturation. Their distinctive “lariat knot” structure is accountable for their antibacterial, enzyme inhibitory and receptor antagonist activities. While the biosynthetic machinery was recently characterized, the rules concerning the formation of this unique lasso structure on the basis of their peptide sequences remain elusive. Restrictions such as the length of the peptide, the size of the ring, or the nature of the amino acids associated with the lasso fold stabilization were recently overhauled by the identification of new members of this RiPP family. In this work we demonstrate the isolation of four genome-mining-predicted lasso peptides featuring the unprecedented amino acids serine or alanine at position 1 of the core peptide. By a mutational approach we were able to predict the lasso fold for four peptides (caulonodins IV to VII). This prediction was confirmed for caulonodin V by the full elucidation of its 3D-structure via NMR and for caulonodin VI by the determination of long range NOE-contacts. Furthermore, the substrate specificity of the biosynthetic machinery for the atypical position 1 was probed. Additionally, utilizing the recent growth of functional lasso peptide precursor sequences we were able to identify a conserved motif in the C-terminal part of the leader peptide through bioinformatics analysis. Employing an extensive in vivo analysis for substitution tolerance of the biosynthetic machinery in this conserved region confirmed the significance of several residues, indicating that the predicted motif is very likely a general leader peptide recognition sequence specific for lasso peptide maturation.

Insights into the Unique Phosphorylation of the Lasso Peptide Paeninodin

Lasso peptides are a new class of ribosomally synthesized and post-translationally modified peptides and thus far are only isolated from proteo- and actinobacterial sources. Typically, lasso peptide biosynthetic gene clusters encode enzymes for biosynthesis and export but not for tailoring. Here, we describe the isolation of the novel lasso peptide paeninodin from the firmicute Paenibacillus dendritiformis C454 and reveal within its biosynthetic cluster a gene encoding a kinase, which we have characterized as a member of a new class of lasso peptide-tailoring kinases. By employing a wide variety of peptide substrates, it was shown that this novel type of kinase specifically phosphorylates the C-terminal serine residue while ignoring those located elsewhere. These experiments also reveal that no other recognition motif is needed for efficient enzymatic phosphorylation of the C-terminal serine. Furthermore, through comparison with homologous HPr kinases and subsequent mutational analysis, we confirmed the essential catalytic residues. Our study reveals how lasso peptides are chemically diversified and sets the foundation for rational engineering of these intriguing natural products.