title: NMR-based method of small changes reveals how DNA mutator APOBEC3A interacts with its single-stranded DNA substrate.
abstract: APOBEC3 proteins are double-edged swords. They deaminate cytosine to uracil in single-stranded DNA and provide protection, as part of our innate immune system, against viruses and retrotransposons, but they are also involved in cancer evolution and development of drug resistance. We report a solution-state model of APOBEC3A interaction with its single-stranded DNA substrate obtained with the 'method of small changes'. This method compares pairwise the 2D 15N-1H NMR spectra of APOBEC3A bearing a deactivating mutation E72A in the presence of 36 slightly different DNA substrates. From changes in chemical shifts of peptide N-H moieties, the positions of each nucleotide relative to the protein can be identified. This provided distance restraints for molecular-dynamic simulations to derive a 3-D molecular model of the APOBEC3A-ssDNA complex. The model reveals that loops 1 and 7 of APOBEC3A move to accommodate substrate binding, indicating an important role for protein-DNA dynamics. Overall, our method may prove useful to study other DNA-protein complexes where crystallographic techniques or full NMR structure calculations are hindered by weak binding or other problems. Subsequent to submission, an APOBEC3A structure with a bound DNA oligomer was published and coordinates released, which has provided an unbiased validation of the 'method of small changes'.
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand.
authors.forename: Stefan
authors.initials: S
authors.lastname: Harjes
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand.
authors.forename: Geoffrey B
authors.initials: GB
authors.lastname: Jameson
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand.
authors.forename: Vyacheslav V
authors.initials: VV
authors.lastname: Filichev
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand.
authors.forename: Patrick J B
authors.initials: PJB
authors.lastname: Edwards
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand.
authors.forename: Elena
authors.initials: E
authors.lastname: Harjes
mesh_terms: D003564:Cytidine Deaminase / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D056004:Molecular Dynamics Simulation; D009154:Mutation / Q000235:genetics*; D009841:Oligonucleotides / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D011506:Proteins; C121399:APOBEC3A protein, human; D003564:Cytidine Deaminase