Assessing the Flexibility of the Prochlorosin 2.8 Scaffold for Bioengineering Applications

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 posttranslationally-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 crosslinks 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 towards proteolysis and displayed higher affinity than the linear counterpart.

Mechanisms and Evolution of Diversity-Generating RiPP Biosynthesis

“…Intriguingly, although their core peptide sequences are highly diverse, there are some hints that they are not random. For instance, engineering studies have shown that the substrate tolerance of ProcM is not limitless and that unlike the natural substrates that have been investigated, which generally make single ring patterns, non-natural substrates in which the positions of Ser/Cys are altered often lead to incomplete cyclization [109]. These observations suggest that despite their diversity, the core peptides in the genomes may have undergone selection to be good substrates for ProcM.…”

Minimal lactazole scaffold for in vitro production of pseudo-natural thiopeptides

“…These studies resulted in the discovery of lanthipeptide inhibitors of HIV budding process, 15 urokinase plasminogen activator 16 and α v β 3 integrin binders. 13 Similarly, we anticipate that the integration of the FIT-Laz system with powerful in vitro screening techniques such as mRNA display 66 will provide access to artificial thiopeptides with desirable pharmacological profiles for drug discovery purposes.…”

Das Potenzial von RiPPs in medizinisch-biotechnologischen Anwendungen

“…Eine dieser Varianten zeigte ebenfalls eine hohe Bindungsaffi nität zu dem αvβ3-Integrin (Abb. 2E, [9]).…”

“…Durch die Nutzung degenerierter Oligonukleotid-Primer konnte eine Bibliothek aus etwa 10 6 verschiedenen Prochlorosin-2.8-Varianten erzeugt werden [10]. Durch Screening dieser Bibliothek konnte ein Inhibitor der Protein-Protein-Interaktion zwischen dem HIV-p6-Protein und der UEV-Domäne des humanen Proteins TSG101 identifi ziert werden [9]. Diese Wechselwirkung ist essenziell für die Knospung von HIV-Partikeln in infi zierten Zellen.…”