Atomic Force Microscopy Studies Provide Direct Evidence for Dimerization of the HIV Restriction Factor APOBEC3G

Yuri L. Lyubchenko,Lela Lackey,Luda S. Shlyakhtenko,Ming Li,Alexander Y. Lushnikov,Reuben S. Harris

doi:10.1074/jbc.m110.195685

Yuri L. Lyubchenko, Lela Lackey + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110.195685

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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 Mg2+, but dimers still predominated when Mg2+ was depleted. A His-248/His-250 Zn2+-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.

Highlights

Sine deaminase that is known for its capacity to restrict the replication of Vif-deficient HIV-1
H250A mutant predicted to lack a zinc-mediated intermolecular bridge observed recently in a crystal structure (Protein Data Bank code 3IR2 [19]) was primarily dimeric in single-stranded DNA (ssDNA) complexes with fewer higher order oligomers. These findings suggest a mechanism for A3G assembly on ssDNA and clarify the model HIV restriction by A3G in which dimeric A3G is the form of the protein that deanimates viral cDNA and results in guanine-to-adenine hypermutations
These two enzymes had indistinguishable activity profiles using a fluorescence-based cytosine-touracil DNA deaminase assay (Fig. 1B). Both enzymes were capable of binding a 43-nucleotide ssDNA, as evidenced by similar mobility shift patterns during native polyacrylamide gel electrophoresis (Fig. 1C). These activity and substrate binding data are consistent with prior functional studies showing that His-248 and His-250 are dispensable for A3G to restrict Vif-deficient HIV-1 [19]

Summary

Introduction

Sine deaminase that is known for its capacity to restrict the replication of Vif-deficient HIV-1 (reviewed recently in Refs. 1–3). We use AFM and a hybrid DNA substrate (a molecular ruler) to clearly differentiate between free A3G and ssDNA-bound complexes. H250A mutant predicted to lack a zinc-mediated intermolecular bridge observed recently in a crystal structure (Protein Data Bank code 3IR2 [19]) was primarily dimeric in ssDNA complexes with fewer higher order oligomers. These findings suggest a mechanism for A3G assembly on ssDNA and clarify the model HIV restriction by A3G in which dimeric A3G is the form of the protein that deanimates viral cDNA and results in guanine-to-adenine hypermutations

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