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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. [ABSTRACT FROM AUTHOR]

Lasso peptides are a class of bioactive ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by a mechanically interlocked topology, where the C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. BI-32169 is a class III lasso peptide containing one disulfide bond that further stabilizes the lasso structure. In contrast to its branched-cyclic analog, BI-32169 has higher stability and is known to exert a potent inhibitory activity against the human glucagon 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) experiments were carried out to evidence specific structural signatures of the two topologies. CID experiments showed similar fragmentation patterns for the two topoisomers, where a part of the C-terminal tail remains covalently linked to the macrolactam ring by the disulfide bond, which cannot clearly constitute a signature of the lasso topology. ECD experiments of BI-32169 showed an increase of hydrogen migration events in the loop region when compared with those of its branched-cyclic topoisomer evidencing specific structural signatures for the lasso topology. The high mobility resolving power of TIMS resulted in the identification of multiple conformations for the protonated species but did not allow the clear differentiation of the two topologies in mixture. When in complex with cesium metal ions, a reduced number of conformations led to a clear identification of the two structures. Experiments reducing and alkylating the disulfide bond of BI-32169 showed that the lasso structure is preserved and heat stable and the associated conformational changes provide new insights about the role of the disulfide bond in the inhibitory activity against the human glucagon receptor. [ABSTRACT FROM AUTHOR]