1. 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

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

3. 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

4. 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?

5. 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?

6. 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.

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

8. 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

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

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

11. 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

12. 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.

13. 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