This program investigates the role of protein-protein interactions in natural product modular synthases, a group of biosynthetic enzymes of three types: polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), and fatty acid synthase (FAS). Many of these natural products serve as anti-cancer agents or antibiotics, while others are pathogenic toxins. Recent evidence indicates that protein-protein interactions between the carrier protein domain and catalytic partner proteins of these pathways are key to proper catalysis and processivity. These critical interactions are transient in nature and remain largely uncharacterized. We have developed tools that allow synthetic modification of carrier protein domains, and here we leverage these tools to increase the lifetime of these protein-protein interactions using substrate mimics and covalent cross-linking inhibitors. These tools will allow us to visualize the molecular details by which carrier proteins bind tethered substrates prior to catalysis and how they interact with cognate catalytic partners. Through these studies, we intend to gain a more complete understanding of the molecular interactions of these catalytic events. We will develop new crosslinking probes to trap carrier proteins and their catalytic partners into their bound state. Subsequent structural studies through analysis of solution phase NMR spectra and X-ray crystallography will be used to visualize the crosslinked species. In addition, NMR titration experiments in the presence of partner proteins will further allow us to accurately pinpoint the residues involved in substrate sequestration and protein-protein interaction. We will further validate our findings through site-directed mutagenesis, crosslinking studies, and other biophysical methods. Finally, we will use molecular dynamics computation along with the data from structural biology to design a novel metabolic pathway through modification of protein-protein interactions between a FAS and NRPS pathway. A deeper understanding of these protein-protein interactions and how to control them will affect emerging fields in drug discovery. Using these tools to guide new interactions will enable combinatorial biosynthesis of new pharmacophores, while new drug targets may be discovered through abrogating these interactions in pathogen biosynthesis.
Secondary metabolites including natural products continue to provide vital clinical entries for a wide range of human ailments. In this continuation, we develop new tools to understand how interactions between enzymes guide the natural product biosynthesis. These studies provide an important next step for drug discovery, and hence benefit human health, by enabling manipulation of these pathways to enable access new therapeutic leads and the biosynthetic systems that make them.
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