Insights into DNA substrate selection by APOBEC3G from structural, biochemical, and functional studies

<div><p>Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3 (A3) proteins are a family of cytidine deaminases that catalyze the conversion of deoxycytidine (dC) to deoxyuridine (dU) in single-stranded DNA (ssDNA). A3 proteins act in the innate immune response to viral infection by mutating the viral ssDNA. One of the most well-studied human A3 family members is A3G, which is a potent inhibitor of HIV-1. Each A3 protein prefers a specific substrate sequence for catalysis—for example, A3G deaminates the third dC in the CC<u><b>C</b></u>A sequence motif. However, the interaction between A3G and ssDNA is difficult to characterize due to poor solution behavior of the full-length protein and loss of DNA affinity of the truncated protein. Here, we present a novel DNA-anchoring fusion strategy using the protection of telomeres protein 1 (Pot1) which has nanomolar affinity for ssDNA, with which we captured an A3G-ssDNA interaction. We crystallized a non-preferred adenine in the -1 nucleotide-binding pocket of A3G. The structure reveals a unique conformation of the catalytic site loops that sheds light onto how the enzyme scans substrate in the -1 pocket. Furthermore, our biochemistry and virology studies provide evidence that the nucleotide-binding pockets on A3G influence each other in selecting the preferred DNA substrate. Together, the results provide insights into the mechanism by which A3G selects and deaminates its preferred substrates and help define how A3 proteins are tailored to recognize specific DNA sequences. This knowledge contributes to a better understanding of the mechanism of DNA substrate selection by A3G, as well as A3G antiviral activity against HIV-1.</p></div

P210 is important for selection of the -2 and +1 nucleotides at the A3G deamination sites.

<p>A) Relative single-cycle infectivity of VSV-G-psuedotyped HIV-1Δ<i>vif</i> viruses produced in the presence or absence of A3G-WT or A3G-P210R. Mean of two independent experiments done in triplicates are shown relative to the “no A3G” control (set to 100%); error bars, standard deviation. B and C) Effect of A3G-P210R substitution on the relative mutation frequencies at the +1 position nucleotide (b) and the -2 position nucleotide (c) with the preferred C at the -1 position (5’C<u><b>C</b></u>) or the non-preferred T at the -1 (5’T<u><b>C</b></u>) position. B) In the + 1 position nucleotide, mutation frequencies for the preferred sites with a C at -1 position (5’C<u><b>C</b></u>), A3G-WT prefers C<u><b>C</b></u>A or C<u><b>C</b></u>G over C<u><b>C</b></u>T and C<u><b>C</b></u>C; A3G-P210R prefers C<u><b>C</b></u>C over C<u><b>C</b></u>A, C<u><b>C</b></u>G, and C<u><b>C</b></u>T, but has no significant difference in preference between C<u><b>C</b></u>A and C<u><b>C</b></u>T. For the non-preferred sites with a T at -1 position (5’T<u><b>C</b></u>), A3G-WT and A3G-P210R both prefer T<u><b>C</b></u>A over T<u><b>C</b></u>T, T<u><b>C</b></u>G, or T<u><b>C</b></u>C. C) In the -2 position nucleotide, mutation frequencies for the preferred sites with a C at -1 position (5’C<u><b>C</b></u>), both A3G-WT and A3G-P210R prefer CC<u><b>C</b></u> over TC<u><b>C</b></u>, AC<u><b>C</b></u>, or GC<u><b>C</b></u>. For the non-preferred sites with a T at the -1 position (5’T<u><b>C</b></u>), A3G-WT prefers CT<u><b>C</b></u> over TT<u><b>C</b></u>, AT<u><b>C</b></u>, or GT<u><b>C</b></u>, whereas A3G-P210R prefers TT<u><b>C</b></u> or GT<u><b>C</b></u> over AT<u><b>C</b></u> and CT<u><b>C</b></u>. The significantly preferred nucleotide in the -2 or +1 positions are indicated (*<i>P <</i> 0.001). The number of sites, number of mutations, and relative mutation frequencies are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195048#pone.0195048.s002" target="_blank">S1 Table</a>.</p

Structure of Pot1A3G_CTD with ssDNA.

<p>A) Schematic of the Pot1A3G_CTD fusion protein design. Pot1 (pink) is fused directly to the N-terminus of A3G_CTD (blue). The ssDNA contains both Pot1 and A3G binding sites: the Pot1 site in dark gray and the A3G hotspot in light gray with the linker sequence in smaller font. The resolved adenine in the -1 pocket is colored orange and the expected deaminated cytidine is blue. B) Size exclusion binding test shows that Pot1A3G_CTD binds to the ssDNA substrate. Pot1A3G_CTD alone is in black, the ssDNA is in gray, and the mixture of the two is in red. C) Deamination activity using a UDG-dependent cleavage assay. The Pot1A3G_CTD fusion protein has the same deamination activity as that of A3G_CTD. D) Schematic and structure of the Pot1A3G_CTD in complex with DNA as observed in the crystal. The dA nucleotide bound to the -1 pocket is shown in orange. Two copies of the complex observed in the asymmetric unit are shown in blue (A3G), pink (Pot1), and grey/orange (DNA). The red star (schematic) and red sphere (structure) represent the zinc ion found in the catalytic site. The inset shows the 2Fo-Fc density (1σ contour level) observed for the adenine in the -1 nucleotide-binding pocket.</p

Schematic for DNA selection and nucleotide pocket communication.

<p>A) When A3G (represented by a teal oval) encounters a hotspot (preferred nucleotides represented by orange circles), the A3G is active and the cytidine in the 0 position is deaminated, resulting in a uridine at the 0 position (orange star). B) When A3G (teal oval) encounters non-preferred nucleotides flanking a cytidine (purple circles), it adapts an unfavorable conformation (orange trapezoid) at the catalytic site and no deamination occurs.</p

Structural analysis of loop 7 in A3G-ssDNA interaction.

<p>A) Overview of the A3G_CTD, shown in gray surface representation, bound to an adenine in the -1 nucleotide-binding pocket, shown in light blue sticks. Selected A3G_CTD residues in the pocket are shown in teal sticks. B) The backbones of I314 and P210 form hydrogen bonds with the adenosine (shown in light blue) in the -1 nucleotide pocket. W211, W285, and Y315 stack with the nucleotide to stabilize it in the pocket. C) Structural alignment of the A3G_CTD (teal) to A3A (magenta, PDBID 5SWW) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195048#pone.0195048.ref031" target="_blank">31</a>], shows that conserved residues W285, I314, and Y315 of the -1 nucleotide-binding pocket are held in similar positions. D) Comparison of the A3G_CTD, teal, to the A3A-DNA complex, magenta (PDBID 5SWW) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195048#pone.0195048.ref031" target="_blank">31</a>]. In the A3A structure, D131 forms a hydrogen bond with the Watson-Crick edge of the hotspot nucleotide thymidine (light pink). In the A3G_CTD structure, D316 and D317 are flipped 180 degrees to avoid clashing with the non-preferred adenine (light blue). The flipped conformation is similar to that of the apo structure of the A3G_CTD, green (PDBID 3IR2) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195048#pone.0195048.ref024" target="_blank">24</a>].</p

Mutating residues in loop 1 results in change of preference for +1 nucleotide.

<p>A) Schematic of the nucleotide binding sites of A3A (PDBID 5SWW) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195048#pone.0195048.ref031" target="_blank">31</a>] that are spatially close to one another (marked by pink ovals). A3A is in surface presentation and the backbone of the bound ssDNA is shown as a pink coil. Right panel: sequence alignment of the proteins from the A3 superfamily. Conserved residues are in bold. Residues involved in hydrogen bonding with the nucleotide at the -1 position during scanning are shaded in magenta, and the other residues forming the -1 nucleotide pocket are shaded in teal. The arrow marks the A3G P210 corresponding position. B) Frequency (%) of the preference for each nucleotide at the -2, -1, and +1 positions for A3B, A3F, A3G (* data from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195048#pone.0195048.ref002" target="_blank">2</a>]) and AID (** data from[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195048#pone.0195048.ref038" target="_blank">38</a>]). C) Deaminase activity assay shows that the P210R mutation does not disrupt deaminase function when compared to WT A3G_CTD. D) A3G_CTD catalytic parameter measurements and sequence preference as determined by a UDG-dependent cleavage assay (graphs shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195048#pone.0195048.s001" target="_blank">S1 Fig</a>). Error values are based on fits to the hyperbolic K_d curve, <i>k</i>_<i>obs</i> <i>= (k</i>_<i>chem</i><i>*[E])/(K</i>_<i>d</i><i>+[E])</i>. The errors represent standard errors of the parameters. † Efficiency = k_chem/K_d, ‡ Preference = Efficiency(CC<u><b>C</b></u>A)/Efficiency(CC<u><b>C</b></u>T/G).</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

A3G_CTD loop 1 is important for substrate recognition.

<p>A) Loop 1 in A3G_CTD-DNA complex (teal, coil representation) moves 3Å compared to apo A3G_CTD (PDBID 3IR2, green) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195048#pone.0195048.ref024" target="_blank">24</a>] to enclose the adenine in the -1 pocket. B) Comparison of the A3G_CTD apo crystal structure (green; PDB ID 3IR2) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195048#pone.0195048.ref024" target="_blank">24</a>] to the A3G_CTD-DNA structure (teal). Residue W211 flips in to stack with the nucleotide and residue P210 moves toward the nucleotide as compared to the apo structures, while W285 and Y315 remain static. C) Deaminase activity assays on A3G_CTD mutants show that mutating residues on loop 1 can disrupt the deaminase activity of A3G completely in the case of W211A or partially in the case of P210G.</p