The goal of component 2 is to study the specificity of protein-protein interactions and substrate recognition processes involved in the biosynthesis of natural products by applying NMR spectroscopy. The overwhelming majority of antibiotics and chemotherapeutics are natural products, many of which are biosynthesized by Non-Ribosomal Peptide Synthetases (NRPSs). These assembly line protein clusters can reach up to mega-Dalton size. NRPS products include antibiotics, antitumor agents, antiviral and immunosuppressive drugs, and fungal or bacterial toxins. In contrast to ribosomal protein synthesis, little is known about the mechanisms by which NRPSs assemble their products. Structural data on isolated domains become increasingly available but little is known about mechanisms of protein interactions or substrate recognition, which must involve precise recognition of activated substrates, growing chains, and final hydrolysis and release from the assembly line. All substrate recognition and protein interaction processes must be mediated by the terniary structures of the domains and modules of the synthetases, since no coding is available as it is for ribosomal peptide biosynthesis. Basic mechanisms ofthe recognition are widely unknown. We propose to study the processes of domain interactions and the specificity of substrate recognition in this enterobactin synthetase NRPS cluster. The assembly line for the iron chelator enterobactin consists ofthe short one module NRPS EntF, and a split module formed by the isolated enzyme EntE and the di-domainal EntB. Since enterobactin synthetases are widely conserved in enterobacteria they are emerging targets for development of new antibacterial drugs.
Our specific aims are: 1: Structures of holo-EntF T-TE di-domain and ofthe condensation domain EntF C 2: Structure, dynamics and interactions between the EntF A and T domains. 3: Complex formation and dynamic interactions of full-length type I NRPS EntF. 4: Substrate localization and mechanistic studies of the Ent NRPS assembly. 5: Structure-based design of inhibitors to support anti-microbial therapies.

Public Health Relevance

Understanding the structural principles by which substrates are specifically recognized and incorporated may allow the exploration of genetically encoded clusters for the biosynthesis or engineering of new assembly lines for production of novel molecules that could alleviate the emergence of multiple and extreme drug resistance against the majority of established antibiotics including last resort treatments.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Harvard Medical School
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Hyberts, Sven G; Robson, Scott A; Wagner, Gerhard (2017) Interpolating and extrapolating with hmsIST: seeking a tmax for optimal sensitivity, resolution and frequency accuracy. J Biomol NMR 68:139-154
Nasr, Mahmoud L; Baptista, Diego; Strauss, Mike et al. (2017) Covalently circularized nanodiscs for studying membrane proteins and viral entry. Nat Methods 14:49-52
Coote, Paul; Anklin, Clemens; Massefski, Walter et al. (2017) Rapid convergence of optimal control in NMR using numerically-constructed toggling frames. J Magn Reson 281:94-103
Sekiyama, Naotaka; Boeszoermenyi, Andras; Arthanari, Haribabu et al. (2017) 1H, 13C, and 15N backbone chemical shift assignments of 4E-BP144-87 and 4E-BP144-87 bound to eIF4E. Biomol NMR Assign 11:187-191
Obayashi, Eiji; Luna, Rafael E; Nagata, Takashi et al. (2017) Molecular Landscape of the Ribosome Pre-initiation Complex during mRNA Scanning: Structural Role for eIF3c and Its Control by eIF5. Cell Rep 18:2651-2663
Das, Dibyendu Kumar; Mallis, Robert J; Duke-Cohan, Jonathan S et al. (2016) Pre-T Cell Receptors (Pre-TCRs) Leverage V? Complementarity Determining Regions (CDRs) and Hydrophobic Patch in Mechanosensing Thymic Self-ligands. J Biol Chem 291:25292-25305
Sun, Zhen-Yu J; Bhanu, Meera K; Allan, Martin G et al. (2016) Solution Structure of the Cuz1 AN1 Zinc Finger Domain: An Exposed LDFLP Motif Defines a Subfamily of AN1 Proteins. PLoS One 11:e0163660
Salvi, Nicola; Papadopoulos, Evangelos; Blackledge, Martin et al. (2016) The Role of Dynamics and Allostery in the Inhibition of the eIF4E/eIF4G Translation Initiation Factor Complex. Angew Chem Int Ed Engl 55:7176-9
Ilic, Stefan; Akabayov, Sabine R; Arthanari, Haribabu et al. (2016) Identification of DNA primase inhibitors via a combined fragment-based and virtual screening. Sci Rep 6:36322
Kozel, Caitlin; Thompson, Brytteny; Hustak, Samantha et al. (2016) Overexpression of eIF5 or its protein mimic 5MP perturbs eIF2 function and induces ATF4 translation through delayed re-initiation. Nucleic Acids Res 44:8704-8713

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