HIV restriction factor APOBEC3G binds in multiple steps and conformations to search and deaminate single-stranded DNA.
ABSTRACT: APOBEC3G (A3G), an enzyme expressed in primates with the potential to inhibit human immunode?ciency virus type 1 (HIV-1) infectivity, is a single-stranded DNA (ssDNA) deoxycytidine deaminase with two domains, a catalytically active, weakly ssDNA binding C-terminal domain (CTD) and a catalytically inactive, strongly ssDNA binding N-terminal domain (NTD). Using optical tweezers, we measure A3G binding a single, long ssDNA substrate under various applied forces to characterize the binding interaction. A3G binds ssDNA in multiple steps and in two distinct conformations, distinguished by degree of ssDNA contraction. A3G stabilizes formation of ssDNA loops, an ability inhibited by A3G oligomerization. Our data suggests A3G securely binds ssDNA through the NTD, while the CTD samples and potentially deaminates the substrate. Oligomerization of A3G stabilizes ssDNA binding but inhibits the CTD's search function. These processes explain A3G's ability to efficiently deaminate numerous sites across a 10,000 base viral genome during the reverse transcription process.
Project description:APOBEC3G (A3G) is a single-stranded DNA (ssDNA) cytosine deaminase that can restrict HIV-1 infection by mutating the viral genome. A3G consists of a non-catalytic N-terminal domain (NTD) and a catalytic C-terminal domain (CTD) connected by a short linker. While the CTD catalyzes cytosine deamination, the NTD is believed to provide additional affinity for ssDNA. Structures of both A3G domains have been solved individually; however, a full-length A3G structure has been challenging. Recently, crystal structures of full-length rhesus macaque A3G variants were solved which suggested dimerization mechanisms and RNA binding surfaces, whereas the dimerization appeared to compromise catalytic activity. We determined the crystal structure of a soluble variant of human A3G (sA3G) at 2.5 Å and from these data generated a model structure of wild-type A3G. This model demonstrated that the NTD was rotated 90° relative to the CTD along the major axis of the molecule, an orientation that forms a positively charged channel connected to the CTD catalytic site, consisting of NTD loop-1 and CTD loop-3. Structure-based mutations, in vitro deamination and DNA binding assays, and HIV-1 restriction assays identify R24, located in the NTD loop-1, as essential to a critical interaction with ssDNA. Furthermore, sA3G was shown to bind a deoxy-cytidine dinucleotide near the catalytic Zn<sup>2+</sup>, yet not in the catalytic position, where the interactions between deoxy-cytidines and CTD loop-1 and loop-7 residues were different from those formed with substrate. These new interactions suggest a mechanism explaining why A3G exhibits a 3' to 5' directional preference in processive deamination.
Project description:APOBEC3G (A3G) is a single-stranded DNA (ssDNA) binding protein that restricts the HIV virus by deamination of dC to dU during reverse transcription of the viral genome. A3G has two zing-binding domains: the N-terminal domain (NTD), which efficiently binds ssDNA, and the C-terminal catalytic domain (CTD), which supports deaminase activity of A3G. Until now, structural information on A3G has lacked, preventing elucidation of the molecular mechanisms underlying its interaction with ssDNA and deaminase activity. We have recently built a computational model for the full-length A3G monomer and validated its structure by data obtained from time-lapse High-Speed Atomic Force Microscopy (HS AFM). Here time-lapse HS AFM was applied to directly visualize the structure and dynamics of A3G in complexes with ssDNA. Our results demonstrate a highly dynamic structure of A3G, where two domains of the protein fluctuate between compact globular and extended dumbbell structures. Quantitative analysis of our data revealed a substantial increase in the number of A3G dumbbell structures in the presence of the DNA substrate, suggesting the interaction of A3G with the ssDNA substrate stabilizes this dumbbell structure. Based on these data, we proposed a model explaining the interaction of globular and dumbbell structures of A3G with ssDNA and suggested a possible role of the dumbbell structure in A3G function.
Project description:APOBEC3G (A3G) protein has antiviral activity against HIV and other pathogenic retroviruses. A3G has two domains: a catalytic C-terminal domain (CTD) that deaminates cytidine, and a N-terminal domain (NTD) that binds to ssDNA. Although abundant information exists about the biological activities of A3G protein, the interplay between sequence specific deaminase activity and A3G binding to ssDNA remains controversial. We used the topographic imaging and force spectroscopy modalities of Atomic Force Spectroscopy (AFM) to characterize the interaction of A3G protein with deaminase specific and nonspecific ssDNA substrates. AFM imaging demonstrated that A3G has elevated affinity for deaminase specific ssDNA than for nonspecific ssDNA. AFM force spectroscopy revealed two distinct binding modes by which A3G interacts with ssDNA. One mode requires sequence specificity, as demonstrated by stronger and more stable complexes with deaminase specific ssDNA than with nonspecific ssDNA. Overall these observations enforce prior studies suggesting that both domains of A3G contribute to the sequence specific binding of ssDNA.
Project description:APOBEC3G (A3G) is a cellular protein that inhibits HIV-1 infection through virion incorporation. The interaction of the A3G N-terminal domain (NTD) with RNA is essential for A3G incorporation in the HIV-1 virion. The interaction between A3G-NTD and RNA is not completely understood. The A3G-NTD is also recognized by HIV-1 Viral infectivity factor (Vif) and A3G-Vif binding leads to A3G degradation. Therefore, the A3G-Vif interaction is a target for the development of antiviral therapies that block HIV-1 replication. However, targeting the A3G-Vif interactions could disrupt the A3G-RNA interactions that are required for A3G's antiviral activity. To better understand A3G-RNA binding, we generated in silico docking models to simulate the RNA-binding propensity of A3G-NTD. We simulated the A3G-NTD residues with high RNA-binding propensity, experimentally validated our prediction by testing A3G-NTD mutations, and identified structural determinants of A3G-RNA binding. In addition, we found a novel amino acid residue, I26 responsible for RNA interaction. The new structural insights provided here will facilitate the design of pharmaceuticals that inhibit A3G-Vif interactions without negatively impacting A3G-RNA interactions.
Project description:Human apolipoprotein-B mRNA-editing catalytic polypeptide-like 3G (A3G) is a cytidine deaminase that restricts retroviruses, endogenous retro-elements and DNA viruses. A3G plays a key role in the anti-HIV-1 innate cellular immunity. The HIV-1 Vif protein counteracts A3G mainly by leading A3G towards the proteosomal machinery and by direct inhibition of its enzymatic activity. Both activities involve direct interaction between Vif and A3G. Disrupting the interaction between A3G and Vif may rescue A3G antiviral activity and inhibit HIV-1 propagation. Here, mapping the interaction sites between A3G and Vif by peptide array screening revealed distinct regions in Vif important for A3G binding, including the N-terminal domain (NTD), C-terminal domain (CTD) and residues 83-99. The Vif-binding sites in A3G included 12 different peptides that showed strong binding to either full-length Vif, Vif CTD or both. Sequence similarity was found between Vif-binding peptides from the A3G CTD and NTD. A3G peptides were synthesized and tested for their ability to counteract Vif action. A3G 211-225 inhibited HIV-1 replication in cell culture and impaired Vif dependent A3G degradation. In vivo co-localization of full-length Vif with A3G 211-225 was demonstrated by use of FRET. This peptide has the potential to serve as an anti-HIV-1 lead compound. Our results suggest a complex interaction between Vif and A3G that is mediated by discontinuous binding regions with different affinities.
Project description:The DNA cytosine deaminase APOBEC3G (A3G) is a two-domain protein that binds single-stranded DNA (ssDNA) largely through its N-terminal domain and catalyzes deamination using its C-terminal domain. A3G is considered an innate immune effector protein, with a natural capacity to block the replication of retroviruses such as HIV and retrotransposons. However, knowledge about its biophysical properties and mechanism of interaction with DNA are still limited. Oligomerization is one of these unclear issues. What is the stoichiometry of the free protein? What are the factors defining the oligomeric state of the protein? How does the protein oligomerization change upon DNA binding? How stable are protein oligomers? We address these questions here using atomic force microscopy (AFM) to directly image A3G protein in a free-state and in complexes with DNA, and using time-lapse AFM imaging to characterize the dynamics of A3G oligomers. We found that the formation of oligomers is an inherent property of A3G and that the yield of oligomers depends on the protein concentration. Oligomerization of A3G in complexes with ssDNA follows a similar pattern: the higher the protein concentrations the larger oligomers sizes. The specificity of A3G binding to ssDNA does not depend on stoichiometry. The binding of large A3G oligomers requires a longer ssDNA substrate; therefore, much smaller oligomers form complexes with short ssDNA. A3G oligomers dissociate spontaneously into monomers and this process primarily occurs through a monomer dissociation pathway.
Project description: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.
Project description:The human cytidine deaminase family of APOBEC3s (A3s) plays critical roles in both innate immunity and the development of cancers. A3s comprise seven functionally overlapping but distinct members that can be exploited as nucleotide base editors for treating genetic diseases. Although overall structurally similar, A3s have vastly varying deamination activity and substrate preferences. Recent crystal structures of ssDNA-bound A3s together with experimental studies have provided some insights into distinct substrate specificities among the family members. However, the molecular interactions responsible for their distinct biological functions and how structure regulates substrate specificity are not clear. In this study, we identified the structural basis of substrate specificities in three catalytically active A3 domains whose crystal structures have been previously characterized: A3A, A3B- CTD, and A3G-CTD. Through molecular modeling and dynamic simulations, we found an interdependency between ssDNA substrate binding conformation and nucleotide sequence specificity. In addition to the U-shaped conformation seen in the crystal structure with the CTC<sub>0</sub> motif, A3A can accommodate the CCC<sub>0</sub> motif when ssDNA is in a more linear (L) conformation. A3B can also bind both U- and L-shaped ssDNA, unlike A3G, which can stably recognize only linear ssDNA. These varied conformations are stabilized by sequence-specific interactions with active site loops 1 and 7, which are highly variable among A3s. Our results explain the molecular basis of previously observed substrate specificities in A3s and have implications for designing A3-specific inhibitors for cancer therapy as well as engineering base-editing systems for gene therapy.
Project description:APOBEC3G (A3G) is a potent restriction factor of HIV-1. The N-terminal domain of A3G (A3G-CD1) is responsible for oligomerization and nucleic acid binding, both of which are essential for anti-HIV activity. As a countermeasure, HIV-1 viral infectivity factor (Vif) binds A3G-CD1 to mediate A3G degradation. The structural basis for the functions of A3G-CD1 remains elusive. Here, we report the crystal structures of a primate A3G-CD1 (rA3G-CD1) alone and in complex with single-stranded DNA (ssDNA). rA3G-CD1 shares a conserved core structure with the previously determined catalytic APOBECs, but displays unique features for surface charge, dimerization and nucleic acid binding. Its co-crystal structure with ssDNA reveals how the conformations of loops and residues surrounding the Zn-coordinated centre (Zn-centre) change upon DNA binding. The dimerization interface of rA3G-CD1 is important for oligomerization, nucleic acid binding and Vif-mediated degradation. These findings elucidate the molecular basis of antiviral mechanism and HIV-Vif targeting of A3G.
Project description:APOBEC3G (A3G), a host protein that inhibits HIV-1 reverse transcription and replication in the absence of Vif, displays cytidine deaminase and single-stranded (ss) nucleic acid binding activities. HIV-1 nucleocapsid protein (NC) also binds nucleic acids and has a unique property, nucleic acid chaperone activity, which is crucial for efficient reverse transcription. Here we report the interplay between A3G, NC and reverse transcriptase (RT) and the effect of highly purified A3G on individual reactions that occur during reverse transcription. We find that A3G did not affect the kinetics of NC-mediated annealing reactions, nor did it inhibit RNase H cleavage. In sharp contrast, A3G significantly inhibited all RT-catalyzed DNA elongation reactions with or without NC. In the case of (-) strong-stop DNA synthesis, the inhibition was independent of A3G's catalytic activity. Fluorescence anisotropy and single molecule DNA stretching analyses indicated that NC has a higher nucleic acid binding affinity than A3G, but more importantly, displays faster association/disassociation kinetics. RT binds to ssDNA with a much lower affinity than either NC or A3G. These data support a novel mechanism for deaminase-independent inhibition of reverse transcription that is determined by critical differences in the nucleic acid binding properties of A3G, NC and RT.