Assessing the Flexibility of the Prochlorosin 2.8 Scaffold for Bioengineering Applications

Julian D. Hegemann, Silvia C. Bobeica, Mark C. Walker, Ian R. Bothwell, Wilfred Adrianus van der Donk

Research output: Contribution to journalArticle

Abstract

Cyclization is a common strategy to confer proteolytic resistance to peptide scaffolds. Thus, cyclic peptides have been the focus of extensive bioengineering efforts. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a superfamily of peptidic natural products that often contain macrocycles. In the RiPP family of lanthipeptides, macrocyclization is accomplished through formation of thioether cross-links between cysteines and dehydrated serines/threonines. The recent production of lanthipeptide libraries and development of methods to display lanthipeptides on yeast or phage highlights their potential for bioengineering and synthetic biology. In this regard, the prochlorosins are especially promising as the corresponding class II lanthipeptide synthetase ProcM matures numerous precursor peptides with diverse core peptide sequences. To facilitate future bioengineering projects, one of its native substrates, ProcA2.8, was subjected in this study to in-depth mutational analysis to test the limitations of ProcM-mediated cyclization. Alanine scan mutagenesis was performed on all residues within the two rings, and multiple prolines were introduced at various positions. Moreover, mutation, deletion, and insertion of residues in the region linking the two lanthionine rings was tested. Additional residues were also introduced or deleted from either ring, and inversion of ring forming residues was attempted to generate diastereomers. The findings were used for epitope grafting of the RGD integrin binding epitope within prochlorosin 2.8, resulting in a low nanomolar affinity binder of the αvβ3 integrin that was more stable toward proteolysis and displayed higher affinity than the linear counterpart.

Original languageEnglish (US)
Pages (from-to)1204-1214
Number of pages11
JournalACS synthetic biology
Volume8
Issue number5
DOIs
StatePublished - May 17 2019

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Keywords

  • RiPP
  • epitope grafting
  • integrin
  • lanthipeptide
  • prochlorosin
  • substrate scope

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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