Photo-crosslink analysis in nonribosomal peptide synthetases reveals aberrant gel migration of branched crosslink isomers and spatial proximity between non-neighboring domains

Abstract

Nonribosomal peptide synthetases (NRPSs) are large, multi-modular enzyme templates for the biosynthesis of important peptide natural products. Modules are composed of a set of semi-autonomous domains that facilitate the individual...

Intermediary conformations linked to the directionality of the aminoacylation pathway of nonribosomal peptide synthetases

“…In fact, when mapping these regions on the structure of holo-PCP-E, 41 the reciprocal changes in HD exchange of seq10–seq12 relative to those of seq1–seq9 would be consistent with a loosening of the PCP-E interaction upon increased PCP binding to the A domain, as expected. 42 Further investigation will be required in the future to address the PCP-E interaction in detail.…”

Exploring modular reengineering strategies to redesign the teicoplanin non-ribosomal peptide synthetase

“…The lack of structural data for large NRPS biosynthetic protein constructs limits our understanding the structural role of different NRPS inter-modular linkers that connect different modules. Recent dimodular NRPS protein X-ray structures 41 as well as photocrosslinking studies 42 provides us with an understanding of the exibility of the NRPS biosynthetic machinery, which suggests that linker exchange in multi-modular NRPS proteins could well alter domain-domain motion and potentially prevent productive substrate delivery to downstream peptide processing domains. However, further structural investigations of large multi-modular NRPS constructs are needed in order to deliver the molecular insights into the roles that such linkers play in NRPS-mediated peptide assembly.…”

“…Whilst in the context of the M5-6 + M3a + M4 pathway this could possibly be explained by other as yet unidentied intermodule interactions, in the experiments where synthetic tripeptide was loaded on M3 and incubated with M5 the evidence for N-terminal extension appears unambiguous. Whilst unexpected, it should be noted that there is a general lack of structural information concerning the presentation of acceptor substrates within Cdomains, 5,41,[43][44][45] and that coupled with the reported exibility of the NRPS assembly line (even within fused modules), 25,41,42,46 there is no evidence that the attack of an acceptor peptide onto a donor amino acid is explicitly prevented. This intriguing result highlights the importance of obtaining further structural snapshots of the NRPS C-domain in relevant catalytic states, and is further underlined by the diverse range of catalytic activities performed by domains derived from Cdomains.…”

Understanding condensation domain selectivity in non-ribosomal peptide biosynthesis: structural characterization of the acceptor bound state

“…The structural analysis of key domains, complexes and complete modules has made major contributions to our understanding of how selectivity is achieved by NRPS assembly lines. 26 Whilst the structures of isolated modules are incredibly informative, NRPSs are highly exible 7,13,27,28 and the interactions between individual domains change during the process of chain assembly. Structural characterization of key domain-domain complexes is thus an important goal to improve our understanding of NRPS selectivity.…”