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