# Dynamic allosteric communication within nonribosomal peptide synthetase cyclization domains

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2020 · $343,875

## Abstract

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.

## Key facts

- **NIH application ID:** 9971653
- **Project number:** 2R01GM104257-06
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Dominique Pascal Frueh
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $343,875
- **Award type:** 2
- **Project period:** 2013-06-01 → 2024-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9971653

## Citation

> US National Institutes of Health, RePORTER application 9971653, Dynamic allosteric communication within nonribosomal peptide synthetase cyclization domains (2R01GM104257-06). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9971653. Licensed CC0.

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