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Structural basis for targeted DNA cytosine deamination and mutagenesis by APOBEC3A and APOBEC3B


APOBEC-catalyzed cytosine-to-uracil deamination of single-stranded DNA (ssDNA) has beneficial functions in immunity and detrimental effects in cancer. APOBEC enzymes have intrinsic dinucleotide specificities that impart hallmark mutation signatures. Although numerous structures have been solved, mechanisms for global ssDNA recognition and local target-sequence selection remain unclear. Here we report crystal structures of human APOBEC3A and a chimera of human APOBEC3B and APOBEC3A bound to ssDNA at 3.1-Å and 1.7-Å resolution, respectively. These structures reveal a U-shaped DNA conformation, with the specificity-conferring −1 thymine flipped out and the target cytosine inserted deep into the zinc-coordinating active site pocket. The −1 thymine base fits into a groove between flexible loops and makes direct hydrogen bonds with the protein, accounting for the strong 5′-TC preference. These findings explain both conserved and unique properties among APOBEC family members, and they provide a basis for the rational design of inhibitors to impede the evolvability of viruses and tumors.

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Figure 1: Deep-deamination approach for determining an optimal human A3A substrate.
Figure 2: Crystal structure of human A3A bound to ssDNA with preferred 5′-TCG deamination target motif.
Figure 3: Crystal structure of a variant of the human A3B catalytic domain bound to ssDNA with a 5′-TCA deamination target motif.
Figure 4: Comparison between apo-enzyme and ssDNA-bound A3A and A3B structures.
Figure 5: Corroborating biochemical data for human A3A.
Figure 6: Human A3A and Staphylococcus aureus TadA have similar U-shaped polynucleotide-binding conformations.
Figure 7: Structural comparison of the active sites of A3B and distantly related deaminase family members.

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We thank D. Largaespada and D. Yee for insightful comments, R. Moorthy for oligonucleotide sample preparations, and J. Stivers (Pharmacology and Molecular Sciences Department, Johns Hopkins University, Baltimore, Maryland, USA) for providing the human UNG2 expression construct and purification protocol. This work was supported by grants from the US National Institutes of Health (NIGMS R01-GM118000 to R.S.H. and H.A., NIGMS R35-GM118047 to H.A., NIGMS R01-GM110129 to D.A.H., NCI R21-CA206309 to R.S.H., and DP2-OD007237 and NIGMS P41-GM103426 to R.E.A.), the NSF (CHE060073N to R.E.A.), the Prospect Creek Foundation (R.S.H. and D.A.H.), and the University of Minnesota Masonic Cancer Center (Spore-Program-Project-Planning Seed Grant to R.S.H.). This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the US National Institutes of Health (NIGMS P41-GM103403). The Pilatus 6M detector on the 24-ID-C beamline is funded by an NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. R.S.H. is supported as the Margaret Harvey Schering Land Grant Chair for Cancer Research and as an Investigator of the Howard Hughes Medical Institute.

Author information




R.S.H. and H.A. conceived and designed the studies. K.S., N.M.S., K.K., J.V.D., and H.A. purified proteins and established crystallization conditions. S.B. collected X-ray diffraction data. K.S. solved the crystal structures. M.A.C., D.J.S., J.L.M., and G.J.S. performed the deep-deamination studies. M.A.C. performed biochemical experiments. D.A.H. designed modified DNA substrates. O.D. and R.E.A. provided computational and structural insights. K.S., M.A.C., N.M.S., R.S.H., and H.A. drafted the manuscript, and all authors contributed to revisions and figure preparation.

Corresponding authors

Correspondence to Reuben S Harris or Hideki Aihara.

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Competing interests

R.S.H. and D.A.H. are cofounders, shareholders, and consultants of ApoGen Biotechnologies Inc. H.A. and R.E.A. are consultants for ApoGen Biotechnologies Inc. R.E.A. is a cofounder of Actavalon Inc. The other authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Protein sequence alignment of human A3A and the C-terminal catalytic domain of human A3B.

Clustal Omega amino acid alignment of human A3A 13-199 and A3B 193-382 (catalytic domain) showing secondary structures (α-helices in red, β-strands in yellow) and loop regions. Blue boxes indicate amino acids that were altered in order to obtain soluble proteins for structural studies and, in the case of the catalytic glutamate, to prevent genotoxicity to E. coli during protein expression and substrate turnover during crystallization.

Supplementary Figure 2 Additional representations of A3A–ssDNA complexes.

(a) Overlay of the 4 distinct A3A-ssDNA complexes in the asymmetric unit of the crystal.(b) Composite omit 2Fo-Fc map contoured at 1.0σ for the 4 complexes in the asymmetric unit (protein electron density is shown in green, and ssDNA in blue).(c) Enlarged view of the composite omit 2Fo-Fc map contoured at 1.0σ (blue mesh) for the ssDNA oligonucleotides bound in the 4 distinct A3A molecules (differently colored) captured in the asymmetric unit of the crystal. The orange mesh (composite omit 2Fo-Fc map contoured at 8.0σ) represents the position of the single zinc atom (gray sphere shown at 0.6x scale of the van der Waals radius).

Supplementary Figure 3 Additional representations of A3Bctd*–ssDNA complexes.

(a) Composite omit 2Fo-Fc map for the single A3Bctd*-ssDNA complex contoured at 1.0σ in the asymmetric unit (electron density for protein and ssDNA is shown in green and blue, respectively).(b) Enlarged view of the composite omit 2Fo-Fc map contoured at 1.0σ (blue mesh) for the ssDNA oligonucleotide (yellow sticks) bound to the active site of A3B (magenta). The orange mesh (composite omit 2Fo-Fc map contoured at 8.0σ) represents the position of the single zinc atom (gray sphere, shown at 0.6x scale of the van der Waals radius).

Supplementary Figure 4 Human A3A deaminates ssDNA containing –1 Super-T.

(a) Letter format of the single-stranded DNA sequence modeled in panels b and c with 5-hydroxybutynl-2'-deoxyuridine (Super T) at the -1 position relative to the target cytidine.(b) Chemical structures of normal deoxy-thymidine and Super T differing only at the 5 position of the cytosine ring.(c) Predicted conformation of ssDNA containing -1 Super T bound to human A3A. A semi-transparent molecular surface is shown for the protein.(d) Raw dose response data for human A3A and ssDNA substrates with a normal T or Super T at the -1 position relative to the target cytosine (0.1 - 100 nM A3A with 100 nM A3A-E72A and no enzyme reactions shown as negative controls). The wild-type A3A data are identical to those in Supplementary Fig. 5 to facilitate cross-comparisons.(e) A plot quantifying product accumulation for the experiment shown in panel d. These data indicate that the methyl group at the 5-position of the thymine ring is solvent exposed and unlikely to be involved in an interaction with the enzyme.

Supplementary Figure 5 Hydrogen-bonding potential of ssDNA nucleobases +1 to +3.

(a) A schematic showing a ssDNA substrate containing an optimal A3A target site (5'-ATCGGG) and a derivative substrate with 5-nitroindole bases substituted at the +1 to +3 positions.(b) Representative endpoint data for human A3A showing catalytic activity with normal or 5-nitroindole substituted ssDNA substrates (S, substrate; P, product).(c) Raw dose response data for human A3A and ssDNA substrates with normal GGG or XXX at the +1 to +3 positions relative to the target cytosine (0.1 - 100 nM A3A with 100 nM A3A-E72A and no enzyme reactions shown as a negative controls). The wild-type A3A data are identical to those in Supplementary Fig. 4 to facilitate cross-comparisons.(d) A plot quantifying product accumulation for the experiment shown in panel c. A3A shows a modest 2-fold preference for normal ssDNA substrate in comparison to the 5-nitroindole substituted ssDNA substrate. The data in panels b-d combine to suggest that base stacking of the +1 to +3 nucleotides may be more relevant for the ssDNA deamination mechanism than nucleobase hydrogen-bonding with enzyme.

Supplementary Figure 6 Comparison of A3A–ssDNA and A3Gntd–poly dT structures.

(a) Ribbon schematics of A3A-ssDNA (this study) and A3Gntd-poly dT (pdb 5K83) with active site regions positioned at similar angles to facilitate comparisons.(b) Superposition of A3A-ssDNA and A3Gntd-poly dT structures showing a lack of congruency in the binding conformations. A3A-bound ssDNA is shown in yellow.

Supplementary Figure 7 APOBEC3 subfamily superposition.

(a) Superposition of ribbon schematics of crystal structures for A3A in cyan (pdb 4XXO), A3Bctd in magenta (pdb 5CQH), A3C in yellow (pdb 3VOW), A3Fctd in gray and green (pdb 5HX5 and pdb 3WUS), and A3Gctd in salmon (pdb 3V4K).(b) A superposition of key active site amino acid residues. The zinc-coordinating residues and those that line the active site, including the Trp-Ser-Pro-Cys-X2-4-Cys motif and Thr31 (Thr214) that directly interact with the target cytosine, show high structural conservation. In contrast, Tyr130 (Tyr313) from loop 7 and Asn57 (Asn240) preceding loop 3, which both make critical ssDNA backbone contacts, can adopt more variable conformations. A3A residue numbers are indicated and those for A3B are shown in parentheses.

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Supplementary Data Set 1

Raw gel images for data in Figures 3e and 5a,b (PDF 8507 kb)

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Shi, K., Carpenter, M., Banerjee, S. et al. Structural basis for targeted DNA cytosine deamination and mutagenesis by APOBEC3A and APOBEC3B. Nat Struct Mol Biol 24, 131–139 (2017).

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