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

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 reaction steps. Only little is known about the existence and relevance of a higher-order architecture in these mega-enzymes, for which contacts between non-neighboring domains in three-dimensional space would be characteristic. Similarly poorly understood is the structure of communication-mediating (COM) domains that facilitate NRPS subunit docking at the boundaries between epimerization and condensation domains. We investigated a COM domain pair in a minimal two module NRPS using genetically encoded photo-crosslinking moieties in the N-terminal acceptor COM domain. Crosslinks into the C-terminal donor COM domain of the partner module resulted in protein products with the expected migration behavior on SDS-PAGE gels corresponding to the added molecular weight of the proteins. Additionally, an unexpected apparent high-molecular weight crosslink product was revealed by mass spectrometric analysis to represent a T-form isomer with branched connectivity of the two polypeptide chains. Synthesis of the linear L-form and branched T-form isomers by click chemistry confirmed this designation. Our data revealed a surprising spatial proximity between the acceptor COM domain and the functionally unrelated small subdomain of the preceding adenylation domain. These findings provide an insight into three-dimensional domain arrangements in NRPSs in solution and suggest the described photo-crosslinking approach as a promising tool for the systematic investigation of their higher-order architecture.

FRET monitoring of a nonribosomal peptide synthetase.

Nonribosomal peptide synthetases (NRPSs) are multidomain enzyme templates for the synthesis of bioactive peptides. Large-scale conformational changes during peptide assembly are obvious from crystal structures, yet their dynamics and coupling to catalysis are poorly understood. We have designed an NRPS FRET sensor to monitor, in solution and in real time, the adoption of the productive transfer conformation between phenylalanine-binding adenylation (A) and peptidyl-carrier-protein domains of gramicidin synthetase I from Aneurinibacillus migulanus. The presence of ligands, substrates or intermediates induced a distinct fluorescence resonance energy transfer (FRET) readout, which was pinpointed to the population of specific conformations or, in two cases, mixtures of conformations. A pyrophosphate switch and lysine charge sensors control the domain alternation of the A domain. The phenylalanine-thioester and phenylalanine-AMP products constitute a mechanism of product inhibition and release that is involved in ordered assembly-line peptide biosynthesis. Our results represent insights from solution measurements into the conformational dynamics of the catalytic cycle of NRPSs.

Mapping of the Communication-Mediating Interface in Nonribosomal Peptide Synthetases Using a Genetically Encoded Photocrosslinker Supports an Upside-Down Helix-Hand Motif.

Nonribosomal peptide synthetases (NRPSs) are large modular protein templates that assemble bioactive peptides, many of which possess therapeutic importance. Protein-protein interactions between subunits of bacterial NRPSs are essential for proper template formation. The structural basis of the typical subunit interface between epimerization (E) and condensation domains is only poorly understood. Conflicting helix-helix and helix-hand models were previously proposed. Here, the genetically encoded photocrosslinker p-benzoylphenylalanine (BpF) was incorporated into the C-terminal communication-mediating domain (COM) of GrsA. Using the partner elongation module TycB1 to form a dipeptide product, we could correlate the ability to form covalent crosslinks with the functional module interaction. Perturbation of the module interaction with the large side chain of BpF in a scan at 19 positions demonstrated the importance of three hydrophobic residues in an α-helical arrangement. Mapping of covalent crosslinks using tandem mass spectrometry revealed the residues from the interior of the condensation domain as part of the protein interface; a finding not predicted by the helix-helix model. The epimerization domain of GrsA was found to be important for the interaction. Together with multiple sequence analyses and structural modeling, our results suggest an upside-down helix-hand model in which the C-terminal COM-helix is embedded in a hand motif with a hydrophobic core in a reversed orientation compared to a previous proposal. Our results provide a more detailed and the first direct structural understanding of the COM domain interaction and will contribute to successful biocombinatorial engineering attempts in the design of artificial NRPS templates.