Non-ribosomal peptide synthetases (NRPSs) are enzymatic assembly lines that produce a wealth of natural products in bacteria and fungi. These products confer virulence to pathogens and often are valuable therapeutics, including antibiotics (penicillin, bacitracin), antitumor agents (bleomycin, epothilone), and immunosuppressants (rapamycin). NRPSs use multiple domains, organized in contiguous modules, to covalently load, modify, and join substrates in an assembly line fashion. This remarkable organization holds the promise of producing novel pharmaceuticals by swapping domains or modules to reprogram the NRPS assembly line. However, most NRPS domain interactions remain uncharacterized, the structure and mechanism of important domains are unknown, and artificially engineered NRPSs are generally unproductive. This proposal aims to reveal the structural basis for heterocycle formation and alteration of their stereochemistry in NRPSs, and unravel domain communication during related synthesis. We will focus on HMWP2, an NRPS that participates in the synthesis of yersiniabactin (Ybt), a virulence factor found in pathogens such as Yersinia pestis, the causative agent of the bubonic plague, Y. enterocolitica, a food pathogen, and uropathogenic E. coli, responsible for urinary tract infections. Our results will contribute to understanding the molecular logic employed by these pathogens during infections. We will primarily use Nuclear Magnetic Resonance (NMR) because of the transient nature of molecular interactions, as well as the existence of multiple conformers in equilibrium. NMR will be used to determine the structures of cyclization and epimerization domains, identify binding sites of chemical and protein substrates, and characterize dynamics within domains during molecular interactions. In a synergistic approach, we will combine mutagenesis and biochemical assays with NMR experiments to provide an atomic level description of reaction mechanisms. The size of the multi-domain complexes reaches 70 kDa and is a challenge for NMR studies, which are typically limited to 20 kDa. In the past we designed methods to solve structures of 50 kDa proteins and obtain useful data from 800kDa complexes. HMWP2 will provide a model system to further develop new NMR methods for large dynamic proteins and to understand conformational rearrangements during protein interactions in general. Our research will simultaneously enable us to push the frontier in NMR studies of larger proteins, help understand the function of protein dynamics in biological systems, reveal the structure of critical domains, and provide a basis for efficient reprogramming of NRPS assembly lines to produce new pharmaceuticals.

Public Health Relevance

This research provides a structural basis for the production of altered natural products to improve or provide pharmaceutical applications. Examples include antibiotics, modified to overcome bacterial resistance, or anti-cancer agents and immunosuppressants. This research will also provide a basis for structure-based design of drugs against Y. pestis (plague), uropathogenic E. coli (urinary tract infections), V. cholera (cholera), M. tuberculosis (tuberculosis), and others.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function E Study Section (MSFE)
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Fabian, Miles
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Johns Hopkins University
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Harden, Bradley J; Frueh, Dominique P (2018) Covariance NMR Processing and Analysis for Protein Assignment. Methods Mol Biol 1688:353-373
Kancherla, Aswani K; Frueh, Dominique P (2017) Covariance nuclear magnetic resonance methods for obtaining protein assignments and novel correlations. Concepts Magn Reson Part A Bridg Educ Res 46A:
Harden, Bradley J; Frueh, Dominique P (2017) Molecular Cross-Talk between Nonribosomal Peptide Synthetase Carrier Proteins and Unstructured Linker Regions. Chembiochem 18:629-632
Goodrich, Andrew C; Meyers, David J; Frueh, Dominique P (2017) Molecular impact of covalent modifications on nonribosomal peptide synthetase carrier protein communication. J Biol Chem 292:10002-10013
Goodrich, Andrew C; Frueh, Dominique P (2015) A nuclear magnetic resonance method for probing molecular influences of substrate loading in nonribosomal peptide synthetase carrier proteins. Biochemistry 54:1154-6
Mishra, Subrata H; Frueh, Dominique P (2015) Assignment of methyl NMR resonances of a 52 kDa protein with residue-specific 4D correlation maps. J Biomol NMR 62:281-90
Harden, Bradley J; Mishra, Subrata H; Frueh, Dominique P (2015) Effortless assignment with 4D covariance sequential correlation maps. J Magn Reson 260:83-8
Frueh, Dominique P (2014) Practical aspects of NMR signal assignment in larger and challenging proteins. Prog Nucl Magn Reson Spectrosc 78:47-75
Harden, Bradley J; Frueh, Dominique P (2014) SARA: a software environment for the analysis of relaxation data acquired with accordion spectroscopy. J Biomol NMR 58:83-99
Mishra, Subrata H; Harden, Bradley J; Frueh, Dominique P (2014) A 3D time-shared NOESY experiment designed to provide optimal resolution for accurate assignment of NMR distance restraints in large proteins. J Biomol NMR 60:265-74

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