NMR-based method of small changes reveals how DNA mutator APOBEC3A interacts with its single-stranded DNA substrate

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• APOBEC3 proteins are crucial innate immune enzymes that deaminate DNA.
• Dysregulation of APOBEC3 activity is implicated in cancer and drug resistance.
• Understanding APOBEC3-DNA interactions is key to targeting these processes.

Purpose of the Study:

• To develop a novel method for modeling DNA-protein complexes in solution.
• To elucidate the structural basis of APOBEC3A interaction with single-stranded DNA.

Main Methods:

• Utilized the 'method of small changes' employing 2D 15N-1H NMR spectroscopy.
• Analyzed chemical shift perturbations of APOBEC3A mutants upon binding to diverse DNA substrates.
• Integrated NMR-derived distance restraints with molecular dynamics simulations.

Main Results:

• Generated a 3-D molecular model of the APOBEC3A-single-stranded DNA complex.
• Identified significant conformational changes in APOBEC3A loops 1 and 7 during substrate binding.
• Demonstrated the dynamic nature of protein-DNA interactions.

Conclusions:

• The 'method of small changes' provides a viable approach for studying challenging DNA-protein interactions.
• Protein-DNA dynamics play a critical role in APOBEC3A substrate recognition.
• This method offers an alternative to crystallography and full NMR structure calculations for similar systems.