nmr detected hydrogen-deuterium exchange reveals differential dynamics of antibiotic and nucleotide...
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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
+ ribosome
Translation Inhibited
Cell death
Aminoglycoside
Mechanism of Action
Kanamycins
Neomycins
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
Research Goals
1) How is APH so promiscuous?
2) How is APH affected when interacting with different antibiotics?
Obtain a better understanding of protein-antibiotic interactions
More intelligent foundation for drug design to combat resistance
No significant change in structure from apo to antibiotic bound?
Need structural information in solution to determine the mechanism of broad substrate selectivity – NMR!
How is APH so promiscuous?
Front Back
How is APH so promiscuous?
NMR detected Hydrogen-Deuterium Exchange = In solution dynamics
Conclusion: Flexibility of APH allows modification of structurally diverse antibiotics.
Apo: H2O Apo: ~20hrs in D2O
Apo-APH APH-Antibiotic
Suggests: A flexible apo-enzyme is the secret!
How is APH so promiscuous?
Nuclear Magnetic Resonance (NMR)
How is APH affected when interacting with different antibiotics?
Little change in XL structures of APH-neomycin and APH-kanamycin complexes.
Kanamycin
Neomycin
Back
Front
How is APH affected when interacting with different antibiotics?
Kanamycin
Neomycin
> 40 amino acids with different
environments
NMR
Kanamycin Neomycin
Neomycin induces greater solvent protection of APH than kanamycin.
How is APH affected when interacting with different antibiotics?
NMR Hydrogen-Deuterium Exchange
Green: Different Chemical Environment
Yellow: Different Solvent Exchange Properties
Conclusion: Neomycin Induces Greater Structural/Dynamic Stability than Kanamycin
How is APH affected when interacting with different antibiotics?
antibiotic
nucleotide
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
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