posted on 2021-05-24, 14:04authored byGengxiang Zhao, Dalibor Kosek, Hong-Bing Liu, Shannon I. Ohlemacher, Brittney Blackburne, Anastasia Nikolskaya, Kira S. Makarova, Jiadong Sun, Clifton E. Barry III, Eugene V. Koonin, Fred Dyda, Carole A. Bewley
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.