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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
• Broad spectrum
• Meningitis
• Tuberculosis
• Diverse size/structure
Kanamycins
Mechanism of Action
Aminoglycoside + ribosome
Translation Inhibited
Cell death
Neomycins
Introduction: Antibiotic Resistance
 Enzyme catalyzed covalent modification
 Aminoglycoside Phosphotransferase (3′)IIIa (APH)
 targets at least 10 different
aminoglycosides of various
size/structure
OPO3
APH
ATP
ADP
Research Goals
Obtain a better understanding of protein-antibiotic interactions
More intelligent foundation for drug design to combat resistance
1) How is APH so promiscuous?
2) How is APH affected when interacting with
different antibiotics?
How is APH so promiscuous?
Front
No significant change in structure
from apo to antibiotic bound?
Back
Need structural information in
solution to determine the
mechanism of broad substrate
selectivity – NMR!
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
Neomycin
Back
Little change in XL
structures of APHneomycin and APHkanamycin complexes.
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?
antibiotic
Green: Different Chemical
Environment
Yellow: Different Solvent
Exchange Properties
nucleotide
Conclusion: Neomycin
Induces Greater
Structural/Dynamic
Stability 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.
Acknowledgements
Dr. Engin Serpersu – Thesis advisor
Dr. Dan Roberts
Dr. Nitin Jain
Dr. David Baker
Dr. Jeremy Smith
Can Ozen
BCMB Department