Structural Basis for a Dual Function ATP Grasp Ligase That Installs Single and Bicyclic ω-Ester Macrocycles in a New Multicore RiPP Natural Product

Abstract

Among the ribosomally synthesized and post-translationally modified peptide (RiPP) natural products, “graspetides” (formerly known as microviridins) contain macrocyclic esters and amides that are formed by ATP-grasp ligase tailoring enzymes using the side chains of Asp/Glu as acceptors and Thr/Ser/Lys as donors. Graspetides exhibit diverse patterns of macrocylization and connectivities exemplified by microviridins, that have a caged tricyclic core, and thuringin and plesiocin that feature a “hairpin topology” with cross-strand ω-ester bonds. Here, we characterize chryseoviridin, a new type of multicore RiPP encoded by Chryseobacterium gregarium DS19109 (Phylum Bacteroidetes) and solve a 2.44 Å resolution crystal structure of a quaternary complex consisting of the ATP-grasp ligase CdnC bound to ADP, a conserved leader peptide and a peptide substrate. HRMS/MS analyses show that chryseoviridin contains four consecutive five- or six-residue macrocycles ending with a microviridin-like core. The crystal structure captures respective subunits of the CdnC homodimer in the apo or substrate-bound state revealing a large conformational change in the B-domain upon substrate binding. A docked model of ATP places the γ-phosphate group within 2.8 Å of the Asp acceptor residue. The orientation of the bound substrate is consistent with a model in which macrocyclization occurs in the N- to C-terminal direction for core peptides containing multiple Thr/Ser-to-Asp macrocycles. Using systematically varied sequences, we validate this model and identify two- or three-amino acid templating elements that flank the macrolactone and are required for enzyme activity in vitro. This work reveals the structural basis for ω-ester bond formation in RiPP biosynthesis.

Phylogenomic analysis of the diversity of graspetides and proteins involved in their biosynthesis

“…Leader peptides are known to play an important role in the processing of many RiPPs [ 25 ]. Microviridin-like precursors contain the highly conserved “PFFARFL” sequence in the leader, which interacts with the middle subdomain of ATP-grasp, and this interaction is necessary to initiate catalysis [ 15 , 20 , 26 ]. We analyzed consensus sequences of leader peptides of all largest clusters of identified precursors and found that, in all these clusters, the leader region contained several conserved residues, typically, often within the 30 N-terminal residues (Fig.…”

“…Genes are shown by block arrows, roughly to scale, color code is provided in the inset on the right upper corner. Chryseobacterium gregarium system in which CdnA3 peptides was experimentally characterized is shown separately (16S rRNA gene in this genome is incomplete and cannot be included in the tree reconstruction) [ 20 ]. Numbers below the precursor genes correspond to 4 clades in the respective tree (Additional file 5 : Figure S5).…”

“…Consensus for multiple alignment of each cluster was determined as described in “Material and Methods” section. Conserved positions that can be involved in formation of ester or amide bonds are highlighted by red and correspond to the experimentally determined linkages [ 20 ], GG-motifs are highlighted by blue and characteristic microviridin group leader motif is highlighted by green …”

“…The formation of these linkages is catalyzed by two paralogous ATP-grasp enzymes encoded in the respective loci [ 18 ]. Subsequently, graspetides were characterized including marinostatin [ 19 ] and chryseoviridin [ 20 ], distant homologs of microviridin, and plesiocin and thuringinin that lack sequence similarity to the former two peptides or to each other [ 21 , 22 ]. Plesiocin and thuringinin require a single ATP-grasp enzyme to make all the linkages [ 21 , 22 ].…”

Structure-Based Engineering of Peptide Macrocyclases for the Chemoenzymatic Synthesis of Microviridins

“…Additionally, modeling full-length precursor peptide into MdnC shows a short distance for the precursor peptide to bind in one monomer, while the distance for in trans binding across the homodimer is better accommodated (Figure S8). This model is consistent with the recently published crystal structure of the CdnC ATP-grasp ligase, which was crystallized as an asymmetric homodimer with ADP and leader peptide bound in both monomers, whereas the core peptide was bound in only one monomer …”