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Structural and Biochemical Studies of Protein-Ligand Interactions: Insights for Drug Development

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2013, Doctor of Philosophy, University of Toledo, Chemistry.

Protein ligand interactions play a key role in the majority of all biological processes. Proteins display unique binding sites that are recognized by specific ligand through distinct interactions. Characterization of these interactions provides insights that are exploitable for designing drugs. Here we study enzyme-substrate interactions in a protein encoded by Helicobacter pylori (H. pylori), which is associated with gastric and duodenal ulcers.

H. pylori MTAN (HpMTAN) catalyzes the hydrolysis of N-ribosidic bonds of at least four different adenosine based substrates; S-adenosylhomocysteine (SAH), 5'-methylthioadenosine (MTA), 5'-deoxyadeosine (5'-DOA) and 6-amino-6-deoxyfutalosine. This hydrolytic activity places MTAN at the hub of at least seven fundamental metabolic pathways: the purine salvage pathway, the methionine salvage pathway, S-adenosylmethionine (SAM)-dependent methylation pathways, polyamine biosynthesis, the production of quorum sensing molecules and menaquinone biosynthesis. Campylobacter and Helicobacter are dependent on MTAN for menaquinone synthesis, an essential metabolite for bacterial viability, making MTAN an excellent target for the development of new treatments for Helicobacter infections. To structurally characterize the interactions between MTAN and its various substrates, complexes of an inactive mutant of the HpMTAN with two known substrates, 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) were formed and crystallized. The crystal structures of mutant HpMTAN complexed with SAH and MTA were solved to 1.2 and 1.6 A respectively. The HpMTAN-SAH co-crystal structure represents the first visualization of interactions between the homocysteine moiety of SAH and the 5'-alkylthiol-binding subsite of the MTAN active site. The co-crystal structure of wild-type MTAN with products, adenine and S-ribosylhomocysteine, was determined to 1.54 A resolution. The similarities and differences in these three structures highlight features that can be exploited to design H. pylori specific drugs. Additionally, a high-throughput fluorescence polarization assay was developed and optimized that will afford the identification of new drug scaffolds that bind to the H. pylori MTAN active site.

Additionally, we studied the I1 protein that is essential for the assembly of vaccinia virus. I1 is a telomere binding protein. We performed structural and biochemical characterizations to understand the mechanism of protein-DNA interaction.

Finally, we studied a protein involved in the folate biosynthetic pathway for drug discovery purpose against Mycobacterium tuberculosis.

Donald Ronning, PhD (Committee Chair)
John Bellizzi, PhD (Committee Member)
Steve Sucheck, PhD (Committee Member)
Constance Schall, PhD (Committee Member)
130 p.

Recommended Citations

Citations

  • Mishra, V. (2013). Structural and Biochemical Studies of Protein-Ligand Interactions: Insights for Drug Development [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1384637704

    APA Style (7th edition)

  • Mishra, Vidhi. Structural and Biochemical Studies of Protein-Ligand Interactions: Insights for Drug Development. 2013. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1384637704.

    MLA Style (8th edition)

  • Mishra, Vidhi. "Structural and Biochemical Studies of Protein-Ligand Interactions: Insights for Drug Development." Doctoral dissertation, University of Toledo, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1384637704

    Chicago Manual of Style (17th edition)