An extended structure of the APOBEC3G catalytic domain suggests a unique holoenzyme model

Abstract

Human APOBEC3G (A3G) belongs to a family of polynucleotide cytidine deaminases. This family includes APOBEC1 and AID, which edit APOB mRNA and antibody gene DNA, respectively. A3G deaminates cytidines to uridines in single-strand DNA and inhibits the replication of human immunodeficiency virus-1, other retroviruses, and retrotransposons. Although the mechanism of A3G-catalyzed DNA deamination has been investigated genetically and biochemically, atomic details are just starting to emerge. Here, we compare the DNA cytidine deaminase activities and NMR structures of two A3G catalytic domain constructs. The longer A3G191-384 protein is considerably more active than the shorter A3G198-384 variant. The longer structure has an alpha1-helix (residues 201-206) that was not apparent in the shorter protein, and it contributes to catalytic activity through interactions with hydrophobic core structures (beta1, beta3, alpha5, and alpha6). Both A3G catalytic domain solution structures have a discontinuous beta2 region that is clearly different from the continuous beta2 strand of another family member, APOBEC2. In addition, the longer A3G191-384 structure revealed part of the N-terminal pseudo-catalytic domain, including the interdomain linker and some of the last alpha-helix. These structured residues (residues 191-196) enabled a novel full-length A3G model by providing physical overlap between the N-terminal pseudo-catalytic domain and the new C-terminal catalytic domain structure. Contrary to predictions, this structurally constrained model suggested that the two domains are tethered by structured residues and that the N- and C-terminal beta2 regions are too distant from each other to participate in this interaction.

Fig. 1

E. coli-based DNA cytidine deaminase activity of A3G catalytic domain constructs. (a) The capacity of untagged A3G198-384, A3G191-384 and the indicated mutant derivatives to trigger Rif^R mutations in E. coli growing under non-inducing conditions. Each X represents the mutation frequency of an independent culture and the median values are indicated. VEC and WT represent empty vector control and the indicated construct with no additional mutations, respectively. A representative anti-A3G immunoblot is shown and non-specific (NS) bands provide loading controls. 2K3A-derivates migrate slightly slower. (b) A representative Rif^R mutation experiment with the same constructs as in panel (a), except this was performed under IPTG-induced expression conditions. The Y-axis values are different. (c) A representative Rif^R mutation experiment with the indicated GST-tagged constructs performed under non-inducing expression conditions. The Y-axis values are different. The complementary IPTG-induced experiment could not be done with the GST expression constructs because induction causes cell death.

Fig. 2

In vitro DNA cytidine deaminase activity. (a) A schematic view of the major steps of the in vitro deamination assay. See the text and methods for details. (b) In vitro DNA cytidine deaminase activity of A3G191-384-2K3A and A3G198-384-2K3A. The migration positions of a positive control (PC) and a negative control (NC) are indicated. The controls were processed in parallel with the experimental reactions and analyzed as part of the same gel, but non-relevant intervening lanes were removed for presentation. The percentage of deaminated substrate (uncut PCR product) from three independent experiments is shown below each lane.

Fig. 3

NMR structure of A3G191-384-2K3A (PDB ID code 2kem). (a) A superimposition of 10 NMR structures showing α-helices in red, β-sheets in yellow and Zn²⁺ in purple. Residues 195–200 are colored blue to highlight their well-defined structure. (b) A ribbon diagram of the NMR structure shown in A from the same angle. Since our prior work had detected only five out of six α-helices, the helices were renumbered α1 (201–206), α2 (258–269), α3 (289–301), α4 (321–330), α5 (340–351) and α6 (363–380). (c) A schematic of the hydrophobic contacts involving F202, F206 and F350. The side-chains of E259 and G355 are also labeled. (d) ¹H-¹⁵N correlation NMR signals of G355 and E259 of A3G191-384-2K3A in red and A3G198-384-2K3A in black.

Fig. 4

A structural comparison A3G191-384-2K3A (PDB ID code 2kem; this study), A3G193-384 [2kbo;20], A3G197-380 [3e1u;22] and APOBEC2 [2nyt;23]. (a, b, c and d)α-carbon traced ribbon schematics for the aforementioned structures. The β2 regions are highlighted by blue ovals and colored green in panels (b), (c) and (d). The inter-domain linker region in panel (a) is indicated by an arrow. (e, f, g, h) Polypeptide backbone and chemical contact schematics for the β2 and β1 regions of A3G191-384-2K3A, A3G193-384, A3G197-380 and A2, respectively. Amino acid positions are numbered and, if the residue is conserved, colored red. In panel (e) and (f), observed NOEs are indicated by arrows. In panels (g) and (h), predicted hydrogen bonds are indicated by dashed lines and labels corresponding to distances between the amide nitrogen and the carbonyl oxygen atoms.

Fig. 5

A model for full-length A3G. (a) Amino acid alignments of A3G1-196, A3G197-384 and APOBEC2. The NCBI accession numbers for these proteins are NP_068594 and NP_006780.Identity is indicated by light blue and similarity by gray shading, and the actual secondary structure elements for A3G191-384-2K3A are illustrated above the alignments. (b, c) Two different angles of a full-length A3G structural model. In (b) the catalytic domain is viewed with the same angle of Fig. 3b, which clearly shows the superposition N-α6 region. The model structure of the N-terminal pseudo-catalytic domain is colored gray. Helices and β-strands within the C-terminal catalytic domain are colored red and yellow, respectively. N-β2 and N-α6 are labeled and highlighted in blue and red, respectively. The β2 region of the pseudo-catalytic domain modeled as a continuous strand and, unlike prior A3G catalytic domain structures, the pseudo-catalytic domain has an N-terminal extension that modeled as an alpha helix.

Fig. 5

A model for full-length A3G. (a) Amino acid alignments of A3G1-196, A3G197-384 and APOBEC2. The NCBI accession numbers for these proteins are NP_068594 and NP_006780.Identity is indicated by light blue and similarity by gray shading, and the actual secondary structure elements for A3G191-384-2K3A are illustrated above the alignments. (b, c) Two different angles of a full-length A3G structural model. In (b) the catalytic domain is viewed with the same angle of Fig. 3b, which clearly shows the superposition N-α6 region. The model structure of the N-terminal pseudo-catalytic domain is colored gray. Helices and β-strands within the C-terminal catalytic domain are colored red and yellow, respectively. N-β2 and N-α6 are labeled and highlighted in blue and red, respectively. The β2 region of the pseudo-catalytic domain modeled as a continuous strand and, unlike prior A3G catalytic domain structures, the pseudo-catalytic domain has an N-terminal extension that modeled as an alpha helix.

Fig. 6

E. coli-based DNA cytidine deaminase activity of full-length A3G amino acid substitution mutants. (a) Mutator phenotype of untagged A3G1-384 alanine substitution mutants. The histogram bars show the relative Rif^R mutation frequencies of cells expressing the vector control (−), A3G1-384 (+) or the indicated alanine substitution derivatives. Each histogram bar reports the average and SEM of the median mutation frequency from multiple independent experiments (each with 6–8 cultures per condition). Residue 189 was not examined due to construction difficulties. The primary amino acid sequence is indicated under the histogram. The positioning of N-α6 and C-α1 is indicated by solid red lines and the predicted part of N-α6 by a dashed line. (b) Mutator phenotype of three M197 variants. The experimental conditions were identical to those described in (a), but the Y-axis was adjusted to represent lower maximal mutation values.

Fig. 7

HIV restriction activity of A3G-2K3A. (a) A histogram reporting the infectivity of Vif-deficient HIV-1 produced in the presence of a GFP control expression vector, wild-type A3G-GFP or full-length A3G-2K3A-GFP. (b) Representative immunoblots of virus particles (unti-A3G, anti-p24) and cellular lysates (anti-A3G and anti-tubulin).