NMR Detected Hydrogen-Deuterium Exchange Reveals Differential Dynamics of Antibiotic and Nucleotide Bound Aminoglycoside Phosphotransferase 3′-IIIa Adrianne.

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Presentation transcript:

NMR Detected Hydrogen-Deuterium Exchange Reveals Differential Dynamics of Antibiotic and Nucleotide Bound Aminoglycoside Phosphotransferase 3′-IIIa Adrianne Norris Department of Biochemistry, Cellular and Molecular Biology Thesis Advisor: Dr. Engin Serpersu

Introduction: Aminoglycoside Antibiotics Kanamycins Neomycins Broad spectrum Meningitis Tuberculosis Diverse size/structure + ribosome Translation Inhibited Cell death Aminoglycoside Mechanism of Action

Introduction: Antibiotic Resistance Enzyme catalyzed covalent modification Aminoglycoside Phosphotransferase (3′)-IIIa (APH) targets at least 10 different aminoglycosides of various size/structure APH ATP ADP OPO3

How is APH so promiscuous? Research Goals Obtain a better understanding of protein-antibiotic interactions More intelligent foundation for drug design to combat resistance How is APH so promiscuous? How is APH affected when interacting with different antibiotics?

How is APH so promiscuous? Front Back Need structural information in solution to determine the mechanism of broad substrate selectivity – NMR! No significant change in structure from apo to antibiotic bound?

How is APH so promiscuous? NMR detected Hydrogen-Deuterium Exchange = In solution dynamics Apo: H2O Apo: ~20hrs in D2O Conclusion: Flexibility of APH allows modification of structurally diverse antibiotics.

How is APH so promiscuous? Nuclear Magnetic Resonance (NMR) Apo-APH APH-Antibiotic Suggests: A flexible apo-enzyme is the secret!

How is APH affected when interacting with different antibiotics? Kanamycin Little change in XL structures of APH-neomycin and APH-kanamycin complexes. Neomycin Back Front

How is APH affected when interacting with different antibiotics? NMR Kanamycin Neomycin > 40 amino acids with different environments

How is APH affected when interacting with different antibiotics? NMR Hydrogen-Deuterium Exchange Kanamycin Neomycin Neomycin induces greater solvent protection of APH than kanamycin.

How is APH affected when interacting with different antibiotics? nucleotide Green: Different Chemical Environment Yellow: Different Solvent Exchange Properties Conclusion: Neomycin Induces Greater Structural/Dynamic Stability than Kanamycin

Neomycin creates greater stability in APH than kanamycin Summary The broad substrate selectivity of APH is due to structural flexibility. Neomycin creates greater stability in APH than kanamycin Future Directions Neutron scattering experiments to determine differences in the radii of gyration of APH in various complexes – complementary to NMR Application of this type of analysis for AAC, aminoglycoside acetyltransferase Testing of synthetic inhibitor molecules.

Dr. Engin Serpersu – Thesis advisor Dr. Dan Roberts Dr. Nitin Jain Acknowledgements Dr. Engin Serpersu – Thesis advisor Dr. Dan Roberts Dr. Nitin Jain Dr. David Baker Dr. Jeremy Smith Can Ozen BCMB Department