Nuclear Magnetic Resonance Structure of the APOBEC3B Catalytic Domain: Structural Basis for Substrate Binding and DNA Deaminase Activity

Abstract

Human APOBEC3B (A3B) is a member of the APOBEC3 (A3) family of cytidine deaminases, which function as DNA mutators and restrict viral pathogens and endogenous retrotransposons. Recently, A3B was identified as a major source of genetic heterogeneity in several human cancers. Here, we determined the solution nuclear magnetic resonance structure of the catalytically active C-terminal domain (CTD) of A3B and performed detailed analyses of its deaminase activity. The core of the structure comprises a central five-stranded β-sheet with six surrounding helices, common to all A3 proteins. The structural fold is most similar to that of A3A and A3G-CTD, with the most prominent difference being found in loop 1. The catalytic activity of A3B-CTD is ∼15-fold lower than that of A3A, although both exhibit a similar pH dependence. Interestingly, A3B-CTD with an A3A loop 1 substitution had significantly increased deaminase activity, while a single-residue change (H29R) in A3A loop 1 reduced A3A activity to the level seen with A3B-CTD. This establishes that loop 1 plays an important role in A3-catalyzed deamination by precisely positioning the deamination-targeted C into the active site. Overall, our data provide important insights into the determinants of the activities of individual A3 proteins and facilitate understanding of their biological function.

Figure 1

Deaminase activities and sequence specificity of FL A3B and A3B-CTD. (A) Western blot analysis showing FL A3B, FL A3B E255Q, and A3B-CTD protein levels in 293T cell extracts. Transfection of the empty vector and E255Q were used as the negative controls. Note that the lack of A3B expression with the empty vector indicates that no endogenous A3B could be detected in the extracts. The tubulin signal served as a loading control. (B) The percent (%) deamination was calculated as described under Materials and Methods and was plotted against increasing amounts of total protein (μg) added. (C) Deaminase activity measured for cell extracts, visualized by gel electrophoresis. A 40-nt ssDNA containing the TTCA deaminase motif was used as the substrate. The oligonucleotide was incubated in a series of reactions with increasing amounts of each extract (1, 2, 3, and 5 μg of protein). Arrows to the right of the gel indicate the positions of the 40-nt ssDNA substrate and the deamination product. Lanes 1, 6, 11, and 16, no protein control; lanes 2 to 5, FL A3B; lanes 7 to 10, E255Q; lanes 12 to 15, A3B-CTD; lanes 17 to 20, empty vector. (D and E) Deaminase substrate sequence specificity of FL A3B (D) and A3B-CTD (E). Reactions contained 40-nt substrates with one of the following deaminase motifs: TTCA, TTCT, TTCG, TGCA, and ACCCA . The data were analyzed and plotted as described in Materials and Methods. Note that the values for deamination of the TTCA substrate with the FL (D) and CTD (E) A3B proteins were taken from the data shown in (B).

Figure 2

800 MHz ¹H-¹⁵N HSQC NMR spectrum of 76 μM ¹³C/¹⁵N-labeled A3B-CTD in 25 mM sodium phosphate, pH 6.9, 25° C. Assignments are indicated by residue name and number. An expansion of the boxed region is provided in the inset in the upper left corner. SEC-MALS data are shown in the inset in the lower left corner, with the elution profile indicated with black circles and the estimated molecular mass across the peak with blue triangles.

Figure 3

A3B-CTD NMR solution structure. (A) Stereo-view (defocused) of the backbone (N, Cα, C′) atoms of the final 30-conformer ensemble. Regions of helical and beta sheet structures are colored magenta and blue, respectively, and the remainder of the structure is colored grey. The Zn²⁺ ion is shown as a brown ball. (B) Ribbon representation of the lowest energy structure of the ensemble, using the same color scheme as in (A). Secondary structure elements are labeled. (C) Stereo-view (defocused) of the superposition of the active site regions of the current A3B-CTD NMR and the A3A NMR (PDB: 2M65) structures. Secondary structure elements of the A3B-CTD structure are colored using the same color scheme as in (A) and (B) and those of A3A are colored in pink (helices), light blue (beta strands), and khaki (loops). The active site residues (H253, E255, C284, and C289 in A3B-CTD and H70, E72, C101, and C106 in A3A) and the Zn²⁺ ions are shown in ball and stick representation with carbon, nitrogen, oxygen, sulfur, and zinc atoms in green, blue, red, yellow, and brown, respectively, for A3B-CTD, or in pale green, cyan, red, yellow, and orange, respectively, for A3A. The active site residues are labeled only in the A3B-CTD structure. (D) Amino acid sequence alignment of loops 1, 3, 5, and 7 for A3B-CTD and A3A. The entire sequence alignment is given in Supplementary Figure S4. Identical residues are highlighted in yellow; residues in the loop 1 region that were changed to construct the A3B-CTD L1 mutant are enclosed in a black rectangle. Residues R212 (A3B-CTD) and H29 (A3A) are highlighted in magenta.

Figure 4

Structural mapping of 5′-ATTTUATTT-3′ binding to A3B-CTD. (A) Residues whose resonances experience significant ¹H,¹⁵N chemical shift changes upon binding are colored red (>0.050 p.p.m.) or orange (0.028–0.050 p.p.m.). ¹H,¹⁵N-combined chemical shift changes were calculated using $\sqrt{\mathrm{\Delta }{{\delta }_{HN}}^2+{(\mathrm{\Delta }{\delta }_N/6)}^2}$, with Δδ_HN and Δδ_N representing ¹HN and ¹⁵N chemical shift differences, respectively. (B) and (C) Binding isotherms for representative ¹HN resonances of A3B-CTD (B) and A3A (C).

Figure 5

A3B-CTD and A3A catalyzed deamination reactions and pH dependence. A series of 1D ¹H NMR spectra of the 15-nt ssDNA (5′-ATTATTTCATTTATT-3′) substrate after addition of A3B-CTD at pH 7.1 (A) or A3A at pH 6.9 (B) were recorded at 25 °C. The time for each 1D measurement was ~1.8 min. The intensities of the ¹H-5 resonances in the 1D spectra of the substrate (dC) and product (dU) were measured for calculation of unreacted substrate concentrations, which were plotted as a function of reaction time. Representative 1D ¹H NMR spectra recorded at the indicated reaction times are shown in the insets. Spectra at different pH values for A3B-CTD at the 3 h time point (C, left panel) and A3A at the 0.2 h time point (D, left panel) and initial rates of deamination for A3B-CTD (C, right panel) and A3A (D, right panel) with 5′-ATTATTTCATTTATT-3′ and 5′-ATTTCATTT-3′, respectively, as substrates. Using the 9- and 15-nt substrates for activity comparisons is valid, since the dC in these substrates is deaminated at essentially identical rates by A3A as well as by A3B-CTD (data not shown). The initial deamination rates were calculated as in (A) and (B). The error bars represent the SD obtained from two to four independent measurements. Concentrations of ssDNA and enzyme were ~1 mM and ~0.2 μM, respectively.

Figure 6

pH dependence of the deamination rates of selected A3 proteins. (A) A3B-CTD WT; (B) A3A WT; (C) A3B-CTD L1 mutant; (D) A3A H29R mutant; (E) A3G-CTD; and (F) A3C. The following substrates were used: 5′-ATCCCATTT-3′, 5′-ATTTCATTT-3′, 5′-ATATCATTT-3′, and 5′-ATTATTTCATTTATT-3′. In the insets in (A), (D), (E), and (F), the y-axes showing the initial rates are expanded. The values for A3B-CTD WT in (A) with the 5′-ATTATTTCATTTATT-3′ substrate (empty circles) and for A3A in (B) with the 5′-ATTTCATTT-3′ substrate (filled circles) were taken from the data shown in Figure 5C and 5D (right panels), respectively. Concentrations of ssDNA and enzymes were ~1 mM and ~0.2 or ~2 μM, respectively. The data were normalized to an enzyme concentration of 0.2 μM and the error bars represent the S.D. from two to four independent measurements.

Figure 7

Comparison of the active sites in the NMR solution structures of A3B-CTD and A3A. Pockets in (A) A3B-CTD WT (this study) and (B) A3A WT (PDB: 2M65) are shown as grey meshes.