Peptidase Activation by a Leader Peptide-Bound RiPP Recognition Element

Area of Science:

• Biochemistry
• Molecular Biology
• Bioinformatics

Background:

• Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a diverse class of natural products.
• Lasso peptide biosynthesis relies on RiPP precursor recognition elements (RREs) and leader peptidases.
• The precise interaction mechanism between RREs and leader peptidases remains incompletely understood.

Purpose of the Study:

• To elucidate the RRE:leader peptidase interaction mechanism in lasso peptide biosynthesis.
• To define the structural and functional roles of RRE domains in protease activity regulation.
• To experimentally validate bioinformatics-guided predictions of the RRE-leader peptidase interface.

Main Methods:

• Evolutionary covariance analysis of thousands of predicted RiPP biosynthetic gene clusters (BGCs).
• Biochemical and biophysical experiments including chemical shift perturbation and paramagnetic relaxation enhancement.
• Cell-free biosynthesis and purified protein assays, including a novel p-nitroanilide-based leader peptidase assay.

Main Results:

• Identified a conserved hydrophobic β-sandwich-like interface formed by interacting RRE and leader peptidase domains.
• Demonstrated a dual role for RRE domains: delivering precursor peptides and forming part of the S2 proteolytic pocket.
• Showed that RREs are essential for leader peptidase activity, with diminished activity at substoichiometric levels.

Conclusions:

• The RRE domain is integral to the leader peptidase active site, regulating lasso peptide processing.
• RRE binding to the leader peptide recognition sequence is required for full proteolytic activation.
• This study reveals a novel mechanism for protease activity regulation mediated by the RRE domain.