Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implications - PubMed (original) (raw)

Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implications

Lauren G Holden et al. Nature. 2008.

Abstract

The APOBEC family members are involved in diverse biological functions. APOBEC3G restricts the replication of human immunodeficiency virus (HIV), hepatitis B virus and retroelements by cytidine deamination on single-stranded DNA or by RNA binding. Here we report the high-resolution crystal structure of the carboxy-terminal deaminase domain of APOBEC3G (APOBEC3G-CD2) purified from Escherichia coli. The APOBEC3G-CD2 structure has a five-stranded beta-sheet core that is common to all known deaminase structures and closely resembles the structure of another APOBEC protein, APOBEC2 (ref. 5). A comparison of APOBEC3G-CD2 with other deaminase structures shows a structural conservation of the active-site loops that are directly involved in substrate binding. In the X-ray structure, these APOBEC3G active-site loops form a continuous 'substrate groove' around the active centre. The orientation of this putative substrate groove differs markedly (by 90 degrees) from the groove predicted by the NMR structure. We have introduced mutations around the groove, and have identified residues involved in substrate specificity, single-stranded DNA binding and deaminase activity. These results provide a basis for understanding the underlying mechanisms of substrate specificity for the APOBEC family.

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Figures

Figure 1

Figure 1. The X-ray structure of enzymatically active APOBEC3G-CD2

a, Analysis of the deamination activity for full-length GST–APOBEC3G (GST–A3G) and GST–APOBEC3G-CD2 (GST–CD2) and APOBEC3G-CD2 (CD2). The 32-nucleotide (nt) band indicates deamination activity. b, APOBEC3G processivity and the 3′to 5′deamination bias was characterized on ssDNA with two CCC motifs. Single deaminations of the 5′C and the 3′C appear as 67- and 48-nucleotide fragments, respectively; deamination of both the 5′C and the 3′C results in a 30-nucleotide fragment (see Methods) c, d, Two views of the APOBEC3G-CD2 domain rotated 90° showing the five-stranded β-sheet core surrounded by six helices. The zinc is represented as a red sphere.

Figure 2

Figure 2. Structural comparison of APOBEC3G-CD2 with APOBEC2

a, Core structures of APOBEC3G-CD2 (yellow) and APOBEC2 (cyan) superimposed. The red sphere represents zinc. b, c, The superposition of APOBEC3G-CD2 and an APOBEC2 monomer, with the AC loop 1 collapsed over the active site (conformation 1, b) or forming an α-hairpin (conformation 2, c). d, In APOBEC3G-CD2, the AC loop 1 R215 residue forms hydrogen bonds (green dashes) with F204, E211, N207, E209 and W285 (pink). The R215 aliphatic chain hydrophobically packs with F204, R313 and W285. e, The APOBEC3G-CD2 AC loop 3 residues, R256, F252, G251, H248 and G244 (pink), form main-chain hydrogen bonds (green dashes). The conserved N244 is shown in cyan. The active site residues are H257, C288 and C291 (wheat).

Figure 3

Figure 3. Predicted substrate groove and deamination activity of APOBEC3G mutants

a, The active site residues of APOBEC3G-CD2. The water (W) and zinc molecules are cyan and red spheres, respectively. b, Superposition of APOBEC3G-CD2 (A3G-CD2, yellow) and TadA (light blue, PDB accession 2B3J). c, Superposition of APOBEC3G-CD2 (yellow) and human CDA (pink). d, Surface representation of APOBEC3G-CD2, showing a horizontal groove with residues (magenta) predicted to interact with ssDNA. ssDNA is represented by a green line. f, Mutational data of APOBEC3G purified from Sf9 (left) or from E. coli (right) are shown. The right inset shows the relative deamination of the 3′C (5′CCC) or the middle C (5′CCC) on a ssDNA substrate. Error bars represent the s.d.

References

    1. Peled JU, et al. The biochemistry of somatic hypermutation. Annu Rev Immunol. 2008;26:481–511. - PubMed
    1. Goodman MF, Scharff MD, Romesberg FE. AID-initiated purposeful mutations in immunoglobulin genes. Adv Immunol. 2007;94:127–155. - PubMed
    1. Conticello SG, Langlois MA, Yang Z, Neuberger MS. DNA deamination in immunity: AID in the context of its APOBEC relatives. Adv Immunol. 2007;94:37–73. - PubMed
    1. Chiu YL, Greene WC. The APOBEC3 cytidine deaminases: an innate defensive network opposing exogenous retroviruses and endogenous retroelements. Annu Rev Immunol. 2008;26:317–353. - PubMed
    1. Prochnow C, Bransteitter R, Klein MG, Goodman MF, Chen XS. The APOBEC-2 crystal structure and functional implications for the deaminase AID. Nature. 2007;445:447–451. - PubMed

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