title: Mapping of the Communication-Mediating Interface in Nonribosomal Peptide Synthetases Using a Genetically Encoded Photocrosslinker Supports an Upside-Down Helix-Hand Motif.
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.
authors.affiliation: Institute of Biochemistry, University of Muenster, Wilhelm-Klemm-Straße 2, D-48149 Münster, Germany.
authors.forename: Eva
authors.initials: E
authors.lastname: Dehling
authors.affiliation: Institute of Biochemistry, University of Muenster, Wilhelm-Klemm-Straße 2, D-48149 Münster, Germany.
authors.forename: Gerrit
authors.initials: G
authors.lastname: Volkmann
authors.affiliation: Institute of Biochemistry, University of Muenster, Wilhelm-Klemm-Straße 2, D-48149 Münster, Germany.
authors.forename: Julian C J
authors.initials: JC
authors.lastname: Matern
authors.affiliation: Institute of Biochemistry, University of Muenster, Wilhelm-Klemm-Straße 2, D-48149 Münster, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Dörner
authors.affiliation: Institute of Biochemistry, University of Muenster, Wilhelm-Klemm-Straße 2, D-48149 Münster, Germany.
authors.forename: Jonas
authors.initials: J
authors.lastname: Alfermann
authors.affiliation: Institute of Biochemistry, University of Muenster, Wilhelm-Klemm-Straße 2, D-48149 Münster, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Diecker
authors.affiliation: Institute of Biochemistry, University of Muenster, Wilhelm-Klemm-Straße 2, D-48149 Münster, Germany. Electronic address: henning.mootz@uni-muenster.de.
authors.forename: Henning D
authors.initials: HD
authors.lastname: Mootz
mesh_terms: D020816:Amino Acid Motifs; D013058:Mass Spectrometry; D008958:Models, Molecular; D008968:Molecular Conformation; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D000072417:Protein Domains; D025941:Protein Interaction Mapping; D055503:Protein Multimerization
chemical_list: D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
keywords: mass spectrometry; peptide antibiotic; peptide mapping; protein–protein interaction; unnatural amino acid