Atomic force microscopy studies provide direct evidence for dimerization of the HIV restriction factor APOBEC3G - PubMed (original) (raw)

Atomic force microscopy studies provide direct evidence for dimerization of the HIV restriction factor APOBEC3G

Luda S Shlyakhtenko et al. J Biol Chem. 2011.

Abstract

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.

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Figures

FIGURE 1.

FIGURE 1.

APOBEC3G purification and activity. A, image of Coomassie Blue-stained A3G and A3G-H248A/H250A-Myc-His proteins purified from HEK-293T cells. A BSA standard is shown for comparison. B, DNA deamination activity of the indicated proteins (arbitrary fluorescent units). C, EMSA for the indicated proteins bound to a 43-nt ssDNA.

FIGURE 2.

FIGURE 2.

Novel AFM approach for studying protein-DNA interactions. A, a hybrid DNA substrate consisting of a dsDNA region (thick black line) and a ssDNA tail (thin grey line) can be complexed with A3G or SSB and imaged by AFM. B, AFM image of the hybrid DNA substrate. Single-stranded regions are indicated with arrows. Numbers 1, 2, and 3 point to the morphologies of these regions in extended (1), compact (2), and globular (3) forms (1 is enlarged in the inset), respectively. Z-scale is 1.5 nm. C, AFM image of hybrid DNA substrate complexed with E. coli SSB. Complexes appear as clearly identified bright blobs attached to the single-stranded end of the hybrid DNA substrate. The yield of complexes is 100%, reflecting the high affinity of SSB for ssDNA. Z-scale is 1.5 nm. D, volume measurements of SSB-ssDNA complexes. The histogram width is narrow, indicating the formation of homogeneous complexes (n = 77).

FIGURE 3.

FIGURE 3.

AFM images of wild-type A3G bound to single-stranded DNA. A, AFM image of the complex of the DNA ruler with A3G protein. Black and blue arrows indicate complexes with dimers and monomers of A3G, respectively. Green arrowheads point to free protein. B, histogram summarizing the volume measurements for A3G-ssDNA complexes (n = 143). C, histogram summarizing the volume measurements for unbound (free) A3G (n = 112).

FIGURE 4.

FIGURE 4.

AFM images of A3G-H248A/H250A bound to ssDNA. A, representative AFM image. The end-bound complexes are indicated with arrows (image size, 600 nm). B, histogram of the measured volumes of the A3G-H248A/H250A-ssDNA complexes. Vertical arrows indicate the expected volume for the dimer (2-mer) and tetramer (4-mer) (n = 150).

FIGURE 5.

FIGURE 5.

AFM results for volume measurements of the complexes formed in the presence of 25 mm Zn2+ cations by WT A3G (A) and the A3G-H248 mutant (B). The number of complexes analyzed were n = 71 (A) and n = 78 (B).

FIGURE 6.

FIGURE 6.

AFM data for the complexes formed in the absence of divalent cations by WT A3G (A) and the A3G-H248 mutant (B). 105 complexes were analyzed for both samples.

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