Steric complementarity directs sequence promiscuous leader binding in RiPP biosynthesis

Abstract

Enzymes that generate ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products have garnered significant interest, given their ability to produce large libraries of chemically diverse scaffolds. Such RiPP biosynthetic enzymes are predicted to bind their corresponding peptide substrates through sequence-specific recognition of the leader sequence, which is removed after the installation of posttranslational modifications on the core sequence. The conservation of the leader sequence within a given RiPP class, in otherwise disparate precursor peptides, further supports the notion that strict sequence specificity is necessary for leader peptide engagement. Here, we demonstrate that leader binding by a biosynthetic enzyme in the lasso peptide class of RiPPs is directed by a minimal number of hydrophobic interactions. Biochemical and structural data illustrate how a single leader-binding domain can engage sequence-divergent leader peptides using a conserved motif that facilitates hydrophobic packing. The presence of this simple motif in noncognate peptides results in low micromolar affinity binding by binding domains from several different lasso biosynthetic systems. We also demonstrate that these observations likely extend to other RiPP biosynthetic classes. The portability of the binding motif opens avenues for the engineering of semisynthetic hybrid RiPP products.

An Amphipathic Alpha‐Helix Guides Maturation of the Ribosomally‐Synthesized Lipolanthines

“…At horough understanding of lipolanthine/lanthipeptide biosynthesis and its regulation is therefore essential to increase production yields and to engineer variants with improved pharmacological properties.T he fundamental step in any RiPP maturation is the specific recruitment of the precursor peptide substrate by the modifying enzyme(s) through molecular recognition of the cognate LP. [19,20] Recent studies have already addressed some aspects of LP recognition in various classes of prokaryotic RiPPs. [20,21] This led to the proposal of designated RiPP precursor peptide recognition elements (RREs) in multiple modifying enzymes (e.g., lasso peptides,l inear-azoline containing peptides and thiopeptides).…”

“…[19,20] Recent studies have already addressed some aspects of LP recognition in various classes of prokaryotic RiPPs. [20,21] This led to the proposal of designated RiPP precursor peptide recognition elements (RREs) in multiple modifying enzymes (e.g., lasso peptides,l inear-azoline containing peptides and thiopeptides). [21] Forl anthipeptides however, only the dehydratase NisB (Nisin) has been experimentally shown to contain an RRE, which recognizes ac onserved F-D/N-L-N/D motif found in various class-I lanthipeptide LPs,w hereas no RRE was identified in class-II, -III or -IV lanthipeptide systems yet.…”

Exploring the conformational landscape of a lanthipeptide synthetase using native mass spectrometry

“…rapidly expanding with the discovery and characterization of many new biosynthetic pathways, elucidation of the structural features that endow these enzymes with their relaxed substrate specificities and interesting catalytic properties have lagged behind. Nevertheless, high-resolution structures have been solved for a handful of enzymes involved in the biosynthesis of several types of RiPP natural products including the class I and II lanthipeptides, [6][7][8][9][10] cyanobactins, [11][12] orbitides, 13 sactipeptides, 14 lasso peptides, 15 thiopeptides, 16 and others. [17][18][19] In several systems, co-crystal structures of the RiPP biosynthetic enzyme bound to its cognate precursor peptide have revealed the presence of a winged helix-turn-helix motif (termed the RiPP recognition element 20 Recently, we reported the use hydrogen-deuterium exchange mass spectrometry (HDX-MS) as a versatile biophysical tool for studying the structural dynamics of RiPP biosynthetic enzymes.…”

Eine amphipathische alpha‐Helix lenkt die Modifizierung ribosomal‐synthetisierter Lipolanthine

“…Ein umfassendes Verständnis der Lipolanthin‐/Lanthipeptid‐Biosynthese und ihrer Regulation ist essenziell, um Produktionsausbeuten zu erhöhen und um Varianten mit verbesserten pharmakologischen Eigenschaften zu konstruieren. Der fundamentale Schritt in jeder RiPP‐Reifung ist die spezifische Rekrutierung des Vorläuferpeptidsubstrats durch modifizierende Enzyme mittels molekularer Erkennung des zugehörigen LP . Neuere Studien haben sich bereits mit Aspekten der LP‐Erkennung in verschiedenen Klassen prokaryotischer RiPPs beschäftigt .…”

“…Der fundamentale Schritt in jeder RiPP‐Reifung ist die spezifische Rekrutierung des Vorläuferpeptidsubstrats durch modifizierende Enzyme mittels molekularer Erkennung des zugehörigen LP . Neuere Studien haben sich bereits mit Aspekten der LP‐Erkennung in verschiedenen Klassen prokaryotischer RiPPs beschäftigt . So wurden bestimmte RiPP‐Vorläuferpeptid‐Erkennungselemente (precursor peptide recognition elements, RREs) in verschiedenen modifizierenden Enzymen (z.…”