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.
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.
|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|
|Takeuchi, Koh; Arthanari, Haribabu; Imai, Misaki et al. (2016) Nitrogen-detected TROSY yields comparable sensitivity to proton-detected TROSY for non-deuterated, large proteins under physiological salt conditions. J Biomol NMR 64:143-51|
|Viegas, Aldino; Viennet, Thibault; Yu, Tsyr-Yan et al. (2016) UTOPIA NMR: activating unexploited magnetization using interleaved low-gamma detection. J Biomol NMR 64:9-15|
|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|
|Nishikawa, Joy L; Boeszoermenyi, Andras; Vale-Silva, Luis A et al. (2016) Inhibiting fungal multidrug resistance by disrupting an activator-Mediator interaction. Nature 530:485-9|
|Goricanec, David; Stehle, Ralf; Egloff, Pascal et al. (2016) Conformational dynamics of a G-protein Î± subunit is tightly regulated by nucleotide binding. Proc Natl Acad Sci U S A 113:E3629-38|
|Mallis, Robert J; Reinherz, Ellis L; Wagner, Gerhard et al. (2016) Backbone resonance assignment of N15, N30 and D10 T cell receptor Î² subunits. Biomol NMR Assign 10:35-9|
|Imai, Shunsuke; Kumar, Parimal; Hellen, Christopher U T et al. (2016) An accurately preorganized IRES RNA structure enables eIF4G capture for initiation of viral translation. Nat Struct Mol Biol 23:859-64|
|Coote, Paul; Bermel, Wolfgang; Wagner, Gerhard et al. (2016) Analytical optimization of active bandwidth and quality factor for TOCSY experiments in NMR spectroscopy. J Biomol NMR 66:9-20|
|Takeuchi, Koh; Arthanari, Haribabu; Shimada, Ichio et al. (2015) Nitrogen detected TROSY at high field yields high resolution and sensitivity for protein NMR. J Biomol NMR 63:323-31|
Showing the most recent 10 out of 226 publications