Structural determinants of human APOBEC3A enzymatic and nucleic acid binding properties

Abstract

Human APOBEC3A (A3A) is a single-domain cytidine deaminase that converts deoxycytidine residues to deoxyuridine in single-stranded DNA (ssDNA). It inhibits a wide range of viruses and endogenous retroelements such as LINE-1, but it can also edit genomic DNA, which may play a role in carcinogenesis. Here, we extend our recent findings on the NMR structure of A3A and report structural, biochemical and cell-based mutagenesis studies to further characterize A3A's deaminase and nucleic acid binding activities. We find that A3A binds ssRNA, but the RNA and DNA binding interfaces differ and no deamination of ssRNA is detected. Surprisingly, with only one exception (G105A), alanine substitution mutants with changes in residues affected by specific ssDNA binding retain deaminase activity. Furthermore, A3A binds and deaminates ssDNA in a length-dependent manner. Using catalytically active and inactive A3A mutants, we show that the determinants of A3A deaminase activity and anti-LINE-1 activity are not the same. Finally, we demonstrate A3A's potential to mutate genomic DNA during transient strand separation and show that this process could be counteracted by ssDNA binding proteins. Taken together, our studies provide new insights into the molecular properties of A3A and its role in multiple cellular and antiviral functions.

Figure 1.

(A) Sequence alignment of A3A (residues 1–199) with A3G-CD2 (residues 194–384). The Zn-binding residues and the catalytic glutamic acid residue are highlighted in blue and the loop 7 region (residues 127–135) of A3A is marked. The A3A residues mutated in this study are labeled in green asterisks. The sequence alignment was performed using Lasergene software (DNASTAR, Inc., Madison, WI, USA). (B) and (C) A3A ribbon structures with residues that were mutated and tested (Figures 3B, 5 and 6C) highlighted in green. The coordinated Zn²⁺ ion is shown as a brown ball.

Figure 2.

Deamination and binding assays with A3A and a 9-nt ssRNA. (A) Deamination of the single cytosine in the ssRNA oligonucleotide, AUUUCAUUU (JL1181, Table 1), was monitored by following the ¹³C-5-¹H resonances of cytosine and uracil in 2D ¹H-¹³C HSQC spectra. The total reaction time is indicated at the top right. Binding isotherms for representative HN resonances (B) and mapping of the binding site onto the A3A structure (C) for ssRNA (AUUUCAUUU) interacting with A3A, monitored by ¹H-¹⁵N HSQC spectroscopy. (D) Binding site of ssDNA ATTTCATTT mapped onto the A3A structure (16) for comparison. In (C) and (D), A3A residues whose resonances exhibit large ¹H,¹⁵N-combined chemical shift changes on nucleotide addition are colored red (> 0.05 p.p.m.) and orange (0.028–0.050 p.p.m.).

Figure 3.

Deaminase activity of A3A mutants. Residues were selected for mutagenesis based on our earlier NMR results (16). (A) Deaminase activity. (B) Expression of A3A WT and mutants in 293T cells detected by western blot analysis, as described in ‘Materials and Methods’.

Figure 4.

A3A EMSA and deaminase assays with ssDNAs of different lengths. (A) Binding of A3A to TTCA-containing DNA oligonucleotides of varying lengths (20 nM) (Table 1): 80 nt (JL988); 70 nt (JL987); 60 nt (JL986); 40 nt (JL895); 30 nt (JL974); and 20 nt (JL975). Lanes: 1, 6, 11, 16, 21, 26, no A3A; 2, 7, 12, 17, 22, 27, 25 µM; 3, 8, 13, 18, 23, 28, 50 µM; 4, 9, 14, 19, 24, 29, 75 µM; 5, 10, 15, 20, 25, 30, 100 µM. The percentage of oligonucleotide bound to A3A is indicated at the bottom of the gel image as percent shifted. The positions of free oligonucleotide and shifted bands (A3A-bound nucleic acid) are also indicated. The calculations were performed as described in ‘Materials and Methods’. (B) Deaminase assays using 100 nM and 200 nM purified WT A3A and ssDNAs with different lengths (180 nM) (Table 1): JL1152 (20 nt, white bar), JL913 (40 nt, black bar) and JL1153 (60 nt, gray bar). (C) Binding of an 80-nt TTCA-containing ssDNA (JL988) by 60 μM (lanes 2, 4, 6, 8 and 10) or 80 μM (lanes 3, 5, 7, 9 and 11) A3A. Lanes: 1 and 12, no A3A; 2, 3, 6 and 7, WT A3A; 4 and 5, E72Q; 8 and 9, Y130F; 10 and 11, D131E. The positions of free and protein-bound DNA and the percent shifted are indicated.

Figure 5.

Deaminase and retrotransposition activities of A3A WT and active site mutants. (A) Deaminase assays using 293T cell extracts expressing WT and active site mutants of A3A. (B) Retrotransposition assays to monitor the effect of active site mutations of A3A on LINE-1 activity. A description of the assays is given in ‘Materials and Methods’. The gray and black bars represent transfection of 0.1 and 0.5 μg of A3A WT or mutant plasmids, respectively. (C) Western blot analysis of A3A WT and active site mutant expression in 293T cells.

Figure 6.

Deaminase and retrotransposition activities of A3A WT and non-active site mutants. (A and D) Deaminase and (B and E) retrotransposition assays were performed as described in ‘Materials and Methods’. (C and F) Western blot analysis of A3A WT and non-active site mutant expression in 293T cells. In the Loop 7 mutant, A3A residues were changed to the corresponding residues in A3G: Y132D/D133Q/P134G/L135R (Figure 1).

Figure 7.

Effect of A3A on HIV-1 RT-catalyzed extension of a (−) SSDNA oligonucleotide. The bar graph shows the percent of DNA extension product without (−) or with (+) RT and/or A3A. The positive control reaction with RT only is shown as a white bar. Reactions with RT contain either 5 µM (black bar) or 10 µM (gray bar) A3A (WT and E72Q mutant).

Figure 8.

Deamination of dC in ss regions of a 40-bp DNA duplex and the effect of SSB proteins. (A) Schematic representation of a series of TBs in a ds nucleic acid. Unpaired bases are located in the center of a 40-bp DNA duplex that contains the TTCA sequence in the ss region of one strand. (B) Deaminase assay performed using duplexes (40 bp) containing TBs with different lengths of unpaired bases (1–9 nt). These duplexes were generated by heat annealing the ssDNA substrate (JL913) to oligonucleotides containing 1 nt (JL1088; TB-1), 3 (JL1089; TB-3), 5 (JL1090; TB-5) and 9 (JL1091; TB-9) that are not complementary to the corresponding residues in the other DNA strand. (C) Deaminase assay using the ssDNA substrate (JL913; 180 nM) after preincubation with SSB proteins (HIV-1 NC, T4 Gene 32 or E. coli SSB; each protein at 500 nM) for 15 min at 37°C before addition of A3A and incubation for 1 h. Bars: 1, 5 and 9, no proteins (negative control); 2, 6 and 10, A3A only (positive control); 3, 7, 11 and 4, 8, 12, A3A and ssDNA preincubated with 2.5 or 5 µM SSB protein, respectively.