Exploring Structural Signatures of the Lanthipeptide Prochlorosin 2.8 Using Tandem Mass Spectrometry and Trapped Ion Mobility-mass Spectrometry

Overview

Journal Anal Bioanal Chem

Specialty Chemistry

Date 2021 Jun 9

PMID 34105020

Citations 7

Authors

Kevin Jeanne Dit Fouque

Julian D Hegemann

Miguel Santos-Fernandez

Tung T Le

Mario Gomez-Hernandez

Wilfred A van der Donk

Francisco Fernandez-Lima

Affiliations

Soon will be listed here.

Abstract

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 b and y 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.

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References

Lohans C, Li J, Vederas J . Structure and biosynthesis of carnolysin, a homologue of enterococcal cytolysin with D-amino acids. J Am Chem Soc. 2014; 136(38):13150-3. DOI: 10.1021/ja5070813. View

Mohimani H, Kersten R, Liu W, Wang M, Purvine S, Wu S . Automated genome mining of ribosomal peptide natural products. ACS Chem Biol. 2014; 9(7):1545-51. PMC: 4215869. DOI: 10.1021/cb500199h. View

Hegemann J, van der Donk W . Investigation of Substrate Recognition and Biosynthesis in Class IV Lanthipeptide Systems. J Am Chem Soc. 2018; 140(17):5743-5754. PMC: 5932250. DOI: 10.1021/jacs.8b01323. View

Chiu J, Tillett D, Dawes I, March P . Site-directed, Ligase-Independent Mutagenesis (SLIM) for highly efficient mutagenesis of plasmids greater than 8kb. J Microbiol Methods. 2008; 73(2):195-8. DOI: 10.1016/j.mimet.2008.02.013. View

Zirah S, Afonso C, Linne U, Knappe T, Marahiel M, Rebuffat S . Topoisomer differentiation of molecular knots by FTICR MS: lessons from class II lasso peptides. J Am Soc Mass Spectrom. 2011; 22(3):467-79. DOI: 10.1007/s13361-010-0028-1. View

Kersten R, Yang Y, Xu Y, Cimermancic P, Nam S, Fenical W . A mass spectrometry-guided genome mining approach for natural product peptidogenomics. Nat Chem Biol. 2011; 7(11):794-802. PMC: 3258187. DOI: 10.1038/nchembio.684. View

Sit C, Yoganathan S, Vederas J . Biosynthesis of aminovinyl-cysteine-containing peptides and its application in the production of potential drug candidates. Acc Chem Res. 2011; 44(4):261-8. DOI: 10.1021/ar1001395. View

Fujinami D, -Mahin A, Elsayed K, Islam M, Nagao J, Roy U . The lantibiotic nukacin ISK-1 exists in an equilibrium between active and inactive lipid-II binding states. Commun Biol. 2018; 1:150. PMC: 6156582. DOI: 10.1038/s42003-018-0150-3. View

Hsu S, Breukink E, Tischenko E, Lutters M, de Kruijff B, Kaptein R . The nisin-lipid II complex reveals a pyrophosphate cage that provides a blueprint for novel antibiotics. Nat Struct Mol Biol. 2004; 11(10):963-7. DOI: 10.1038/nsmb830. View

10.

Urban J, Moosmeier M, Aumuller T, Thein M, Bosma T, Rink R . Phage display and selection of lanthipeptides on the carboxy-terminus of the gene-3 minor coat protein. Nat Commun. 2017; 8(1):1500. PMC: 5686179. DOI: 10.1038/s41467-017-01413-7. View

11.

Li B, Sher D, Kelly L, Shi Y, Huang K, Knerr P . Catalytic promiscuity in the biosynthesis of cyclic peptide secondary metabolites in planktonic marine cyanobacteria. Proc Natl Acad Sci U S A. 2010; 107(23):10430-5. PMC: 2890784. DOI: 10.1073/pnas.0913677107. View

12.

Montalban-Lopez M, Zhou L, Buivydas A, van Heel A, Kuipers O . Increasing the success rate of lantibiotic drug discovery by Synthetic Biology. Expert Opin Drug Discov. 2012; 7(8):695-709. DOI: 10.1517/17460441.2012.693476. View

13.

Repka L, Chekan J, Nair S, van der Donk W . Mechanistic Understanding of Lanthipeptide Biosynthetic Enzymes. Chem Rev. 2017; 117(8):5457-5520. PMC: 5408752. DOI: 10.1021/acs.chemrev.6b00591. View

14.

Silveira J, Michelmann K, Ridgeway M, Park M . Fundamentals of Trapped Ion Mobility Spectrometry Part II: Fluid Dynamics. J Am Soc Mass Spectrom. 2016; 27(4):585-95. DOI: 10.1007/s13361-015-1310-z. View

15.

Dit Fouque K, Lavanant H, Zirah S, Hegemann J, Fage C, Marahiel M . General rules of fragmentation evidencing lasso structures in CID and ETD. Analyst. 2018; 143(5):1157-1170. DOI: 10.1039/c7an02052j. View

16.

Pierson N, Chen L, Russell D, Clemmer D . Cis-trans isomerizations of proline residues are key to bradykinin conformations. J Am Chem Soc. 2013; 135(8):3186-92. PMC: 3624761. DOI: 10.1021/ja3114505. View

17.

Escano J, Smith L . Multipronged approach for engineering novel peptide analogues of existing lantibiotics. Expert Opin Drug Discov. 2015; 10(8):857-70. DOI: 10.1517/17460441.2015.1049527. View

18.

Bobeica S, Dong S, Huo L, Mazo N, McLaughlin M, Jimenez-Oses G . Insights into AMS/PCAT transporters from biochemical and structural characterization of a double Glycine motif protease. Elife. 2019; 8. PMC: 6363468. DOI: 10.7554/eLife.42305. View

19.

Fernandez-Lima F, Kaplan D, Suetering J, Park M . Gas-phase separation using a trapped ion mobility spectrometer. Int J Ion Mobil Spectrom. 2013; 14(2-3). PMC: 3807855. DOI: 10.1007/s12127-011-0067-8. View

20.

Fernandez-Lima F, Kaplan D, Park M . Note: Integration of trapped ion mobility spectrometry with mass spectrometry. Rev Sci Instrum. 2012; 82(12):126106. PMC: 3248236. DOI: 10.1063/1.3665933. View