Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 428, Issue 21, Pages 4345-4360Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2016.09.007
Keywords
protein-protein interaction; peptide antibiotic; unnatural amino acid; mass spectrometry; peptide mapping
Categories
Funding
- DFG [SFB858]
- Human Frontiers Science Program (HFSP) [RGP0031/2010]
Ask authors/readers for more resources
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 a-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. (C) 2016 Elsevier Ltd. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available