Biological activity, ranging from gene activation to enzyme regulation, occurs through molecular interactions, and its regulation can be described as a redistribution of intermolecular interactions through chemical modifications or ligand binding. Unfortunately, when a protein interacts with two partners through remote binding sites, molecular mechanisms that would explain how changes within proteins alter the communication between proteins are often elusive. This challenge limits designing drugs that could alter interactions to rescue abnormal biological activity. The conundrum also applies to microbial enzymatic factories called nonribosomal peptide synthetases (NRPSs). NRPSs use contiguous protein domains to incorporate and assemble simple substrates into complex products in an assembly line fashion. The products are often valuable therapeutics, including antibiotics (bacitracin), antitumor agents (bleomycin), and immunosuppressants (rapamycin), but others confer virulence to pathogens (E. coli, V. cholerae, Y. pestis). NRPSs are the focus of much interest because engineering them to incorporate different substrates could produce novel pharmaceuticals. However, like assembly lines in factories, NRPSs are not static, and their domains interact transiently in a dynamic architecture. Thus, understanding the molecular mechanisms of NRPSs, and potentially engineering them, is tantamount to solving a dynamic, multi-dimensional puzzle. Notably, it is unknown how substrates interact with some domains, and how these interactions, in turn, promote communication between several partner domains, which is the situation we described above for proteins. We found that structural dynamics within domains respond to substrates to promote interactions between domains, and that they couple remote binding sites and enzymatic active sites. That is, dynamics contain keys to understanding both substrate recognition and remote communication. This proposal aims to provide a molecular description of the dynamics within critical NRPS domains and reveal its function in substrate and partner domain recognition. We will use nuclear magnetic resonance, which can describe experimentally dynamics at the atomic-level, to describe dynamic responses when domains interact with each other, and with substrates as they do during synthesis. The studies are supplemented with functional assays, computational methods, and crystallography, and will answer longstanding questions about protein communication, enzyme mechanisms, and remote communication within proteins. The results will provide a basis to engineer exogenous substrate recognition into NRPSs, a condition for producing new pharmaceuticals through NRPS reprogramming.

Public Health Relevance

The research focuses on determining molecular mechanisms of dynamic enzymatic systems involved in the biosynthesis of microbial natural products. It will provide a basis for developing improved therapeutics such as antibiotics, immunosuppressants, or anti-cancer agents. The research will also provide a rationale for combating virulence of Y. pestis (plague), uropathogenic E. coli (urinary tract infections), and indirectly V. cholera (cholera), M. tuberculosis (tuberculosis), and others.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
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Barski, Oleg
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Johns Hopkins University
Schools of Medicine
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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
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
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:
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
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
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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