Synthetic cycle of the initiation module of a formylating nonribosomal peptide synthetase

Abstract

Nonribosomal peptide synthetases (NRPSs) are very large proteins that produce small peptide molecules with wide-ranging biological activities, including environmentally friendly chemicals and many widely used therapeutics. NRPSs are macromolecular machines, with modular assembly-line logic, a complex catalytic cycle, moving parts and many active sites. In addition to the core domains required to link the substrates, they often include specialized tailoring domains, which introduce chemical modifications and allow the product to access a large expanse of chemical space. It is still unknown how the NRPS tailoring domains are structurally accommodated into megaenzymes or how they have adapted to function in nonribosomal peptide synthesis. Here we present a series of crystal structures of the initiation module of an antibiotic-producing NRPS, linear gramicidin synthetase. This module includes the specialized tailoring formylation domain, and states are captured that represent every major step of the assembly-line synthesis in the initiation module. The transitions between conformations are large in scale, with both the peptidyl carrier protein domain and the adenylation subdomain undergoing huge movements to transport substrate between distal active sites. The structures highlight the great versatility of NRPSs, as small domains repurpose and recycle their limited interfaces to interact with their various binding partners. Understanding tailoring domains is important if NRPSs are to be utilized in the production of novel therapeutics.

New Structural Data Reveal the Motion of Carrier Proteins in Nonribosomal Peptide Synthesis

“…The secondary and tertiary structures of PCP domains are highly conserved, with only minor deviations from the prototypical four‐helix bundle being documented to date 19, 20, 21, 22, 23, 24, 25, 26, 27. At the level of primary structure, PCPs are more variable,21 which gives rise to variations in local shape and charge distributions of the exposed and buried surfaces.…”

Interdomain and Intermodule Organization in Epimerization Domain Containing Nonribosomal Peptide Synthetases

“…[31][32][33][34][35] Recently, structures of intact modules of 'holo'-NRPS fragments, with the fundamental prosthetic group Ppant were solved. 36,37 The conformational changes of individual domains in the intact modules allows the carrier domain to transfer intermediates between domains while inducing partner domains to adopt their catalytically competent conformations. Additionally, the role of inter-domain linker regions in the assembly line mechanism is under-explored.…”

“…38,39,40 To address issues of structure and function, research strategies using functional probes utilizing structurally restricted carrier domains suitable for X-ray crystallographic analysis has provided useful structural insights. 32,34,37,39,40 In this study, we describe the structure of both the holo and apo forms of the PCP-E didomain of the GrsA, the first module of the cyclic peptide antibiotic (gramicidin S) synthetase. The biosynthesis of gramicidin S in Bacillus brevis is catalyzed by the NRPS GrsA and GrsB; in which, five modules incorporate ten amino acids cyclized into the final cyclic peptide (Figure 1).…”

Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture

“…Negative stain electron microscopy corroborated the dynamic NRPS module observation and showed multiple locations for the thioesterase domain. A second recent study presented multiple structures of LgrA, the initiation module of the linear gramicidin NRPS (25), which contains a formyltransferase domain upstream of the adenylation and PCP domains. The LgrA structures showed an additional subdomain movement within the adenylation domain that delivers the PCP to the formyltransferase domain.…”