Structure, interaction and real-time monitoring of the enzymatic reaction of wild-type APOBEC3G - PubMed (original) (raw)

Structure, interaction and real-time monitoring of the enzymatic reaction of wild-type APOBEC3G

Ayako Furukawa et al. EMBO J. 2009.

Abstract

Human APOBEC3G exhibits anti-human immunodeficiency virus-1 (HIV-1) activity by deaminating cytidines of the minus strand of HIV-1. Here, we report a solution structure of the C-terminal deaminase domain of wild-type APOBEC3G. The interaction with DNA was examined. Many differences in the interaction were found between the wild type and recently studied mutant APOBEC3Gs. The position of the substrate cytidine, together with that of a DNA chain, in the complex, was deduced. Interestingly, the deamination reaction of APOBEC3G was successfully monitored using NMR signals in real time. Real-time monitoring has revealed that the third cytidine of the d(CCCA) segment is deaminated at an early stage and that then the second one is deaminated at a late stage, the first one not being deaminated at all. This indicates that the deamination is carried out in a strict 3' --> 5' order. Virus infectivity factor (Vif) of HIV-1 counteracts the anti-HIV-1 activity of APOBEC3G. The structure of the N-terminal domain of APOBEC3G, with which Vif interacts, was constructed with homology modelling. The structure implies the mechanism of species-specific sensitivity of APOBEC3G to Vif action.

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Figures

Figure 1

SDS–polyacrylamide gel electrophoresis, HSQC spectrum and structure of the deaminase domain of the wild-type APOBEC3G. (A) SDS–polyacrylamide gel electrophoresis of the purified APOBEC3G. (B) 1H–15N HSQC spectrum. (C) A stereo view of superposition of the main chains of 10 final structures. N and C indicate L193 and N384, respectively. α-helices and β-strands are coloured red and blue, respectively. (D) The main chain of a representative structure with the lowest energy. α-helices and β-strands are coloured red/yellow and blue, respectively.

Figure 2

Comparison of the structures of the deaminase domain of APOBEC3G. (A) The wild type in solution by NMR (this study). (B) The mutant carrying five substitutions in solution by NMR (Chen et al, 2008). (C) The wild type in crystal by X-ray (Holden et al, 2008).

Figure 3

Gel retardation experiments indicating binding of the wild-type deaminase domain to a short ssDNA comprising 10 residues. 32P-labelled 10-mer DNA (200 nM), d(ATTCCCAATT), was incubated with 0, 40 and 80 μM of the deaminase domain of the wild-type APOBEC3G (lanes 1–3). The mixtures were run on a polyacrylamide gel and then exposed.

Figure 4

Mapping of chemical shift perturbations upon binding of DNA, surface electrostatic potential and proposed position of a substrate cytidine. (A) Chemical shift perturbations observed for the wild-type deaminase domain upon binding of 10-mer DNA. The residues exhibiting combined chemical shift perturbations as to HN and N of >0.03 p.p.m. and 0.02–0.03 p.p.m. are coloured red and yellow, respectively. The residues whose 1H–15N correlation peaks either disappeared or became notably weak, the relative intensity in a free state to that in a complex state being greater than 1.2, are coloured blue. (B) Chemical shift perturbations observed for the mutant deaminase domain upon binding of 21-mer DNA. The residues exhibiting combined chemical shift perturbations of >0.03 p.p.m. and 0.02–0.03 p.p.m. are coloured red and yellow, respectively. The structure and perturbation data reported for the mutant deaminase domain (Chen et al, 2008) were used to make this figure. (C) Left, the perturbations for the wild-type deaminase domain mapped on the surface representation; right, positive and negative surface potentials of the wild-type deaminase domain represented in blue and red, respectively. (D) A close-up of the deduced key interactive region of the wild-type APOBEC3G deaminase domain, with the proposed position of a substrate cytidine indicated by a dashed circle, viewed from two different angles. (E) Three possible positions of ssDNA relative to the APOBEC3G deaminase domain. Right, the position proposed by Chen et al, 2008 (dashed vertical line in blue) and that by Holden et al, 2008 (dashed kinked horizontal line in green); left, the third possible position.

Figure 5

1H–13C HSQC spectra indicating cytidine to uridine conversion for 10-mer DNA through the deamination reaction in an NMR tube. The pyrimidine (either cytidine or uridine residues) H5–C5 region of the 1H–13C HSQC spectrum of 10-mer DNA, d(ATTCCCAATT) (A), and that recorded 24 h after the addition of the wild-type deaminase domain to the NMR tube (B). The same regions of mutant 10-mer DNAs with the C4C5U6 (C), C4U5U6 (D) and C4U5C6 (E) segments, respectively, for reference.

Figure 6

Time chase of 1H NMR spectra indicating deamination of two cytidine residues in a strict 3′ → 5′ order. 1H NMR spectrum of 10-mer DNA (A), and ones recorded 0.5 h (B), 1.5 h (C), 4.5 h (D) and 24 h (E) after the addition of the wild-type deaminase domain to the NMR tube. 1H NMR spectra of mutant 10-mer DNAs with the C4C5U6 (F), C4U5U6 (G) and C4U5C6 (H) segments, respectively, for reference.

Figure 7

The structures of the N-terminal domains (residues 10–192) that interact with Vif. The structures of the N-terminal domains of human (A) and African green monkey (B) APOBEC3Gs, constructed with homology modelling on the basis of the structure of the C-terminal deaminase domain of human APOBEC3G. The surface potentials of the N-terminal domains of human (C) and African green monkey (D) APOBEC3Gs. The residue at the position 128 is indicated with a dashed circle.

References

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Structure, interaction and real-time monitoring of the enzymatic reaction of wild-type APOBEC3G - PubMed (original) (raw)