Peptidase Activation by a Leader Peptide-Bound RiPP Recognition Element

Abstract

The RiPP precursor recognition element (RRE) is a conserved domain found in many prokaryotic ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic gene clusters (BGCs). RREs bind with high specificity and affinity to a recognition sequence within the N-terminal leader region of RiPP precursor peptides. Lasso peptide biosynthesis involves an RRE-dependent leader peptidase, which is discretely encoded or fused to the RRE as a di-domain protein. Here we leveraged thousands of predicted BGCs to define the RRE:leader peptidase interaction through evolutionary covariance analysis. Each interacting domain contributes a three-stranded β-sheet to form a hydrophobic β-sandwich-like interface. The bioinformaticsguided predictions were experimentally confirmed using proteins from discrete and fused lasso peptide BGC architectures. Support for the domain−domain interface derived from chemical shift perturbation, paramagnetic relaxation enhancement experiments, and rapid variant activity screening using cell-free biosynthesis. Further validation of selected variants was performed with purified proteins. We developed a p-nitroanilide-based leader peptidase assay to illuminate the role of RRE domains. Our data show that RRE domains play a dual function. RRE domains deliver the precursor peptide to the leader peptidase, and the rate is saturable as expected for a substrate. RRE domains also partially compose the elusive S2 proteolytic pocket that binds the penultimate threonine of lasso leader peptides. Because the RRE domain is required to form the active site, leader peptidase activity is greatly diminished when the RRE domain is supplied at substoichiometric levels. Full proteolytic activation requires RRE engagement with the recognition sequence-containing portion of the leader peptide. Together, our observations define a new mechanism for protease activity regulation.

Lasso Peptides: Exploring the Folding Landscape of Nature’s Smallest Interlocked Motifs

“…Lasso peptides are an expanding class of compact bioactive ribosomally synthesized and post-translationally modified peptide natural products (RiPPs) characterized by an interlocked lasso fold. − These cyclic entangled structures (Figure ) can range from 13 to 33 residues in length , and result from an isopeptide bond between the N-terminal amine and a side chain carboxylate, which forms a seven to nine residue macrolactam ring that is threaded by the C-terminal peptide tail. ,− Once folded and cyclized, lasso peptides are extremely stable with their threaded tails held below the ring by steric locks, called plugs, formed by bulky side chains . In biological systems, lasso peptide maturation begins with the interaction of the lasso precursor leader sequence with a RiPP precursor recognition element (RRE) that guides its cleavage by a leader peptidase, which enables a lasso cyclase to fold the lasso motif and catalyze the isopeptide bond formation. ,,, Once formed, the value of lasso peptides in nature is demonstrated by their diverse biological activities, including enzyme inhibition, receptor antagonism, and antibiotic effects, ,− which are likely facilitated by their thermal stability, resistance to degradation, and rigid globular shapes. ,, Accordingly, the lasso scaffold could be invaluable in the design of novel therapeutics that are sized between small molecules and biologics, ,,− which can lead to reduced immunogenicity and the capacity for bioavailability. , Collectively, lasso peptides warrant further investigation.…”

“…8 In biological systems, lasso peptide maturation begins with the interaction of the lasso precursor leader sequence with a RiPP precursor recognition element (RRE) that guides its cleavage by a leader peptidase, which enables a lasso cyclase to fold the lasso motif and catalyze the isopeptide bond formation. 1,3,9,10 Once formed, the value of lasso peptides in nature is demonstrated by their diverse biological activities, including enzyme inhibition, receptor antagonism, and antibiotic effects, 4,11−14 which are likely facilitated by their thermal stability, resistance to degradation, and rigid globular shapes. 4,15,16 Accordingly, the lasso scaffold could be invaluable in the design of novel therapeutics that are sized between small molecules and biologics, 3,13,17−19 which can lead to reduced immunogenicity and the capacity for bioavailability.…”

“…The production of lasso peptides has been considerably expanded through the development of heterologous expression and cell-free biosynthesis approaches. 3,7,16,21,22 However, an adaptable chemical synthesis platform would provide further access to this scaffold and unnatural variants. 23,24 A central limitation in the development of a chemical synthesis platform is the requirement that the lasso precursor be pre-folded prior to isopeptide bond formation; otherwise, the nonthreaded conformation (herein referred to as a tadpole) would be obtained.…”

Micrococcin cysteine-to-thiazole conversion through transient interactions between a scaffolding protein and two modification enzymes

“…Modifications in TOMM precursor peptides are orchestrated by an E1-like scaffold protein, a YcaO cyclodehydratase, and a dehydrogenase; in some cases, a highly divergent Ocin-ThiF-like protein is also present (16,21,26,41,42). The E1-like and Ocin-ThiF-like proteins are involved with leader peptide binding through an RRE domain that features a winged helix-turn-helix (wHTH) structure comprised of three -helices and a three-stranded -sheet (15). The purpose of the leader peptide is to recruit the biosynthetic enzymes, but they have shown variations in their roles in the biosynthesis of diverse TOMMs (20,(43)(44)(45).…”

Bioinformatics-guided discovery of biaryl-linked lasso peptides

“…The leader portion harbors the recognition sequence, which directs the enzymatic processing events via interaction with the RiPP precursor recognition element (RRE). 4 Meanwhile, the core region receives all of the post-translational modifications (PTMs). 5 Upon RRE binding, leader peptidolysis is initiated by the co-occurring (B) protein, releasing the core peptide as a substrate for the ATP-dependent lasso cyclase (C).…”