Atomic force microscopy studies provide direct evidence for dimerization of the HIV restriction factor APOBEC3G.
APOBEC3G (A3G) is an antiviral protein that binds RNA and single-stranded DNA (ssDNA). The oligomerization state of A3G is likely to be influenced by these nucleic acid interactions. We applied the power of nanoimaging atomic force microscopy technology to characterize the role of ssDNA in A3G oligomerization. We used recombinant human A3G prepared from HEK-293 cells and specially designed DNA substrates that enable free A3G to be distinguished unambiguously from DNA-bound protein complexes. This DNA substrate can be likened to a molecular ruler because it consists of a 235-bp double-stranded DNA visual tag spliced to a 69-nucleotide ssDNA substrate. This hybrid substrate enabled us to use volume measurements to determine A3G stoichiometry in both free and ssDNA-bound states. We observed that free A3G is primarily monomeric, whereas ssDNA-complexed A3G is mostly dimeric. A3G stoichiometry increased slightly with the addition of Mg(2+), but dimers still predominated when Mg(2+) was depleted. A His-248/His-250 Zn(2+)-mediated intermolecular bridge was observed in a catalytic domain crystal structure (Protein Data Bank code 3IR2); however, atomic force microscopy analyses showed that the stoichiometry of the A3G-ssDNA complexes changed insignificantly when these residues were mutated to Ala. We conclude that A3G exchanges between oligomeric forms in solution with monomers predominating and that this equilibrium shifts toward dimerization upon binding ssDNA.
Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
Luda S
LS
Shlyakhtenko
Alexander Y
AY
Lushnikov
Ming
M
Li
Lela
L
Lackey
Reuben S
RS
Harris
Yuri L
YL
Lyubchenko
D000071480:APOBEC-3G Deaminase; D003564:Cytidine Deaminase / Q000737:chemistry* / Q000378:metabolism; D015342:DNA Probes / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D018625:Microscopy, Atomic Force / Q000379:methods*; D011485:Protein Binding; D055503:Protein Multimerization; D016601:RNA-Binding Proteins
D015342:DNA Probes; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D016601:RNA-Binding Proteins; D000071480:APOBEC-3G Deaminase; C464924:APOBEC3G protein, human; D003564:Cytidine Deaminase; D008274:Magnesium