ABSTRACT
Nonribosomal peptide synthetases (NRPSs) are large, multifunctional enzymes that facilitate the stepwise synthesis of modified peptides, many of which serve as important pharmaceutical products. Typically, NRPSs contain one module for the incorporation of one amino acid into the growing peptide chain. A module consists of the domains required for activation, covalent binding, condensation, termination, and optionally modification of the aminoacyl or peptidyl moiety. We here describe a protocol using genetically encoded photo-cross-linking amino acids to probe the 3D architecture of NRPSs by determining spatial proximity constraints. p-benzoyl-L-phenylalanine (BpF) is incorporated at positions of presumed contact interfaces between domains. The covalent cross-link products are visualized by SDS-PAGE-based methods and precisely mapped by tandem mass spectrometry. Originally intended to study the communication (COM) domains, a special pair of docking domains of unknown structure between two interacting subunits of one NRPS system, this cross-linking approach was also found to be useful to interrogate the spatial proximity of domains that are not connected on the level of the primary structure. The presented photo-cross-linking technique thus provides structural insights complementary to those obtained by protein crystallography and reports on the protein in solution.
Subject(s)
Peptide Synthases , Peptides , Peptides/genetics , Peptide Synthases/chemistry , Amino Acids/chemistry , Genetic CodeABSTRACT
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.
ABSTRACT
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.
