Crystal Structure of DNA Cytidine Deaminase ABOBEC3G Catalytic Deamination Domain Suggests a Binding Mode of Full-length Enzyme to Single-stranded DNA

Background: The mechanism for DNA cytidine deaminase APOBEC3G (A3G) interacting with single-stranded DNA (ssDNA) is not well characterized.

Results: The crystal structure of a head-to-tail dimer of the A3G catalytic deamination domain (A3G-CD2) was obtained.

Conclusion: The dimer structure of A3G-CD2 suggests a binding mode of full-length A3G to ssDNA.

Significance: The dimer structure of A3G-CD2 may represent a structural model of full-length A3G.

Abstract

APOBEC3G (A3G) is a DNA cytidine deaminase (CD) that demonstrates antiviral activity against human immunodeficiency virus 1 (HIV-1) and other pathogenic virus. It has an inactive N-terminal CD1 virus infectivity factor (Vif) protein binding domain (A3G-CD1) and an actively catalytic C-terminal CD2 deamination domain (A3G-CD2). Although many studies on the structure of A3G-CD2 and enzymatic properties of full-length A3G have been reported, the mechanism of how A3G interacts with HIV-1 single-stranded DNA (ssDNA) is still not well characterized. Here, we reported a crystal structure of a novel A3G-CD2 head-to-tail dimer (in which the N terminus of the monomer H (head) interacts with the C terminus of monomer T (tail)), where a continuous DNA binding groove was observed. By constructing the A3G-CD1 structural model, we found that its overall fold was almost identical to that of A3G-CD2. We mutated the residues located in or along the groove in monomer H and the residues in A3G-CD1 that correspond to those seated in or along the groove in monomer T. Then, by performing enzymatic assays, we confirmed the reported key elements and the residues in A3G necessary to the catalytic deamination. Moreover, we identified more than 10 residues in A3G essential to DNA binding and deamination reaction. Therefore, this dimer structure may represent a structural model of full-length A3G, which indicates a possible binding mode of A3G to HIV-1 ssDNA.

Introduction

The APOBEC protein family are activation-induced deaminases, including APOBEC1, APOBEC2, APOBEC3, and APOBEC4 (1–8). Among them, the APOBEC3 subfamily consists of cellular proteins that prevent the mobilization of diverse retroviruses, retrotransposons, and other viral pathogens (4). In humans, seven members of this subfamily, APOBEC3A (A3A),² APOBEC3B (A3B), APOBEC3C (A3C), APOBEC3D (A3D), APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H (A3H), are encoded in a tandem array on chromosome 22 (9). These proteins are characterized by the presence of one or two conserved zinc-coordinated domains consisting of HXEX_23–28CXXC motifs (10). Human A3G is a single-stranded DNA (ssDNA) cytidine deaminase that restricts replication of HIV-1 in strains lacking the virus infectivity factor protein (Vif) (11). HIV-1 Vif protein facilitates polyubiquitination of A3G, leading to proteasomal degradation (12–17). Without Vif, A3G is encapsulated in an HIV virus particle through interaction with the nucleocapsid portion of Gag and HIV RNA or 7SL RNA (18–27). A3G suppresses HIV-1 infectivity by deaminating the viral cDNA cytidine to uridine during the reverse transcription (25–27). Subsequent replication of the cDNA generates the hallmark G to A hypermutations, causing proviral inactivation (25–27).

Human A3G exists as monomer, dimer, and tetramer, depending on the DNA substrate and salt concentration. It possesses two homologous deaminase domains, an inactive N-terminal CD1 domain (i.e. A3G-CD1) required for Vif, DNA, and RNA binding and an active C-terminal CD2 domain (i.e. A3G-CD2) required for catalysis and motif specificity (28–30). The CD1 domain is also suggested to be required for the incorporation of A3G into virions (29). A3G deaminates cytidine processively 3′ → 5′ on ssDNA (31, 32). The processive deamination reactions have been decided in a non-random way (31, 32). To date, the three-dimensional structure of the free A3G-CD2 domain has been determined by NMR (33–35) and x-ray crystallography (36–38). The three-dimensional structures of free APOBEC2 (39) and other APOBEC3 subfamily members, such as A3A (40, 41), A3C (42), and A3F (43, 44), have also been reported. The structural basis for Vif hijacking CBF-β and CUL5 E3 ligase was recently revealed too (45). However, the mechanism of how A3G-CD2 or full-length A3G and other members of its family interact with ssDNA is still not well understood.

To address how A3G-CD2 interacts with HIV-1 ssDNA, we crystallized A3G-CD2 in the presence of the ssDNA containing one target motif sequence 5′-CCC-3′. A novel head-to-tail dimer structure of A3G-CD2 was obtained. In its surface, a continuous groove for ssDNA binding was found. Our structural analysis and biochemical assays suggest that this dimer structure may represent a structural model of full-length A3G. From this model, we identified more than 10 new residues in both A3G-CD1 and A3G-CD2 critical to the deamination catalysis reaction.

EXPERIMENTAL PROCEDURES

Expression and Purification of A3G-CD2 and Its Variants

The DNA corresponding to gene of wild-type (WT) A3G-CD2 (residues 193–384) or its variant was cloned into a pET28a vector containing an N-terminal His tag and thrombin cleavage sites. A3G-CD2 and its variants were expressed in Rosetta (DE3)plys Escherichia coli cells. Cell cultures were grown to A₆₀₀ value equal to about 0.8 and induced with a final concentration of 0.5 mm isopropyl 1-thio-β-d-galactopyranoside for 20 h at 18 °C. Cells were resuspended in nickel-binding buffer (20 mm Tris-HCl, pH 7.5, 500 mm NaCl, 10 μm ZnCl₂, and 0.5 mm dithiothreitol (DTT)) with protease inhibitor (DNase I) and lysed at 15,000 p.s.i. using a hydraulic cell disruption system (constant System JINBO Benchtop) (Guangzhou Juneng Biology and Technology Co., Ltd., Guangzhou, China). The lysate was centrifuged at 12,000 rpm and 4 °C for 55 min to remove cellular debris prior to loading into a nickel-nitrilotriacetic acid resin (GE Healthcare). The protein was washed with nickel-binding buffer 20 mm imidazole and then eluted by a five-step gradient of nickel-binding buffer with 40 mm imidazole, 100 mm imidazole, 250 mm imidazole, and 500 mm imidazole, respectively. Fractions containing A3G-CD2 or its variants were concentrated and purified on a Superdex75 16/600 GL column (GE Healthcare) previously equilibrated with buffer B (50 mm HEPES, pH 7.5, 50 mm NaCl, 50 μm ZnCl₂, 5 mm DTT).

X-ray Crystallization Screening and Data Collection

Purified A3G-CD2 and its D370A variant were quantified by A280 and then mixed with ssDNA (it was commercially synthesized at an HPLC grade from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China) with a sequence 5′-TTAACCCTTA- 3′) at a molar ratio of 1:1.2 (protein/ssDNA) and concentrated to final concentrations of 10 and 20 mg/ml for crystallization. At 18 °C, the crystals of the WT A3G-CD2 protein were grown by sitting-drop vapor diffusion against a reservoir containing 0.04 m citric acid, 0.06 m Bistris propane, pH 6.4, 20% (w/v) polyethylene glycol 3350, whereas the crystals of D370A were grown by sitting-drop vapor diffusion against a reservoir containing 0.1 m sodium citrate tribasic dehydrate, pH 5.6, 20% (v/v) 2-propanol, 20% (w/v) polyethylene glycol 4000. The crystals were flash-frozen in Paratone-N. X-ray diffraction data were collected at beamline BL17U of the Shanghai Synchrotron Radiation Facility using a MAR CCD MX-225 detector. The wavelength of the radiation was 0.9792 Å, and the distance between the crystal and the detector was 300 nm. The exposure time for each frame was 1 s with a 1º oscillation, and 360 frames were collected. The data were indexed, integrated, and scaled using the HKL-2000 program suite (46).

Structural Determination

The A3G-CD2 and D370A structures were determined by molecular replacement, using the program PHENIX-Auto MR (47) and the structure of the A3G-CD2 2K3A variant (PDB code 3IR2) as the search model (37). Iterative rounds of model rebuilding and simulated annealing torsion angle refinement were performed using the programs Coot (48) and PHENIX Refine (47). Identification of proper sequence registry was confirmed with the location of the catalytic zinc site and the presence of bulky aromatic residues. Ramachandran plot analysis revealed that 93.7 and 6.7% of the residues of WT A3G-CD2 protein and 91.8 and 8.2% of the residues of A3G-CD2 D370A variant were in most favored and allowed regions, respectively. The final model of WT protein contains residues 194–381 in monomer H and residues 196–246, 253–316, and 321–381 in monomer T. Weak electron density was observed for residues 246–253 and 316–321 in monomer T, whereas the final model of the D370A variant contains residues 193–381.

Real-time Studies of A3G-CD2-catalyzed Deamination by NMR

A series of one-dimensional ¹H NMR spectra of the reported HIV-1 virus ssDNA with the sequence 5′-ATTCCCAATT-3′ (34) were acquired as a function of time at 20 °C in NMR buffer (50 mm Na₂HPO₄, 50 mm NaCl, pH 7.5, 50 μm ZnCl₂, 2 mm DTT), after adding concentrated A3G-CD2 solution. To accurately assign uridine NMR signals, three ssDNAs with sequences of 5′-ATTCCUAATT-3′, 5′-ATTCUCAATT-3′, and 5′-ATTCUUAATT-3′ were used as controls. Concentrations of A3G-CD2 and its variants were fixed at 9.4 μm. The intensities of the ¹H-NMR signal belonging to U⁶ was used for quantification. Real-time monitoring of A3G-CD2 catalyzed deamination reaction by NMR was performed to extract initial rates (<5% dC → dU conversion) for a series of substrate concentration (37.5, 56.3, 93.8, 117.2, and 140.6 μm). K_m values were obtained using Michaelis-Menten module with the software Prism 5 (GraphPad Inc.).

E. coli-based Deaminase Activity Assays

The intrinsic DNA cytidine deaminase activity of full-length A3G and its variants was measured by expressing these proteins in ung-deficient E. coli BW310 and by quantifying the frequency of Rif^R-conferring rpoB mutations (33, 49), as described in the previous report (35). A lot of single-base mutations in rpoB lead to active site amino acid replacements that confer Rif^R. In each case, five single colonies were grown at 37 °C in LB medium containing 100 μg/ml ampicillin and induced overnight by 1 mm isopropyl 1-thio-β-d-galactopyranoside at 18 °C. Then appropriate volumes of cells were spread on plates containing 100 μg/ml rifampicin to select for Rif^R mutants and to plates containing 100 μg/ml ampicillin to determine the number of viable cells. Colonies were allowed to form overnight at 37 °C and then counted manually.

E. coli Immunoblots

The full-length A3G and its variant constructs were expressed in E. coli strain BW310. Proteins were generated by overnight expression at 18 °C in LB medium containing 100 μg/ml ampicillin. To induce expression, cells were diluted 1:10 in LB medium containing 100 μg/ml ampicillin and 1 mm isopropyl 1-thio-β-d-galactopyranoside and grown for 1 h at 37 °C. Cells were pelleted and resuspended in SDS gel loading buffer (50 mm Tris-Cl, pH 6.8, 100 mm β-mercaptoethanol, 2% SDS (v/v) glycerol). Lysates were heated at 95 °C for 5 min and fractionated by SDS-PAGE. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and probed with a rabbit anti-A3G polyclonal serum. The primary antibody was detected by incubation with donkey peroxidase-conjugated anti-rabbit IgG (Shanghai Sangon Biotech Co. Ltd.), followed by chemiluminescent imaging.

Size Exclusion Chromatography Assay

Size exclusion chromatography assays were performed on a Superdex 75 column (10/300 GL) (GE Healthcare), previously equilibrated with buffer B (50 mm HEPES, pH 7.5, 50 mm NaCl, 50 μm ZnCl₂, 5 mm DTT).

RESULTS

Overall Fold of Novel Head-to-tail A3G-CD2 Dimer Crystal Structure

In the presence of ssDNA during crystallization, two monomers of human A3G-CD2 domain (residues 193–384) occupy one asymmetric unit. The final crystal structure was determined at 1.8 Å resolution and solved by molecular replacement, using the structure of the A3G-CD2 2K3A variant (residues 191–384; PDB code 3IR2) (37) as a model. The final refinement statistics are summarized in Table 1. Different from the previously reported tail-to-tail or head-to-head conformation (PDB code 3IR2) (37), the crystal structure demonstrates a new head-to-tail dimer conformation (to simplify discussion, the two monomers are referred to here as monomer H (head) and monomer T (tail), respectively), which means that the N terminus of the monomer H interacts with the C terminus of the monomer T, as shown in Fig. 1. The two monomers are identical to each other with a root mean square deviation value of 0.19 Å for backbone C_α atoms in the secondary structural regions. They contain a core sandwich-like α-β-α fold, consistent with the reported cytidine deaminases (33–36, 39–41, 43), in which the monomer structure has five β strands encircled by six α helices on both sides (Fig. 1). The catalytic zinc ion in each monomer was coordinated directly by the side chains of residues His-257, Cys-288, and Cys-291 and indirectly by catalytic center Glu-259 via a water molecule. The secondary structural elements are numbered after the x-ray crystal structures of WT A3G-CD2 (residues 197–380, PDB codes 3E1U and 3IQS) (36) and of its 2K3A variant (PDB code 3IR2) (37). Different from structures 3E1U and 3IQS (refined from 3E1U) but similar to the reported structures of A3G-CD2 (PDB codes 3IR2, 2KEM, 2JYW, and 2KBO) (33–35, 37), the second β strand in the current dimer structure is discontinuous. Loop 3 (residues 246–253) and loop 7 (residues 316–322) are missed in monomer T, which is also distinct from the head-to-head or tail-to-tail dimer conformation (PDB code 3IR2). Thus, on the whole, in terms of the overall fold, this structure is similar to the previously reported structures, but many key differences were still observed.

TABLE 1.

Summary of diffraction data and structure refinement statistics

	A3G-CD2	D370A
Data collection
Wavelength (Å)	0.9792	0.9792
Space group	P212121	P22121
Resolution (Å)	50.00–1.86	50.00–1.7
	(1.86–1.80)a	(1.76–1.70)
Cell parameters
a (Å)	72.4	35.3
b (Å)	76.5	70.0
c (Å)	80.5	82.3
Observed reflections	504,022	137,969
Unique reflections (I/σ(I) > 0)	43,059	22,865
Average redundancy	11.7 (11.2)	6.0 (4.7)
Average I/σ(I)	33.2 (6.8)	29.2 (5.8)
Completeness (%)	99.7 (100.0)	98.8 (97.2)
Rmerge (%)b	7.6 (41.7)	5.5 (25.6)
Refinement
Resolution (Å)	50.0–1.80	50.0–1.70
Reflections (Fo ≥0σ(Fo))
Working set/test set	37,800/2,119	20,479/1,168
Rwork/Rfree (%)c	17.3/21.9	14.7/18.8
No. of protein atoms	2,983	1,591
No. of water atoms	441	190
Average B factor of all atoms (Å2)
All atoms	29.2	21.4
Protein	27.4	20.0
Water	40.9	32.6
Root mean square deviation
Bond lengths (Å)	0.007	0.007
Bond angles (degrees)	1.09	1.05
Ramachandran plot (%)
Most favored regions	93.7	91.8
Allowed regions regions	6.3	8.2
Generously allowed regions	0.0	0.0
Disallowed regions	0.0	0.0

^a Numbers in parentheses refer to the highest resolution shell.

^b R_merge = Σ_hklΣ_i|I_i(hkl)_i − 〈I(hkl)〉|/Σ_hklΣ_iI_i(hkl).

^c R factor = ‖F_o| − |F_c‖/|F_o|.

FIGURE 1.

The overall fold of a novel head-to-tail A3G-CD2 dimer. A, ribbon representation of the two monomers (monomer H (gray) and monomer T (green)). B, the secondary structure elements of A3G-CD2 in this dimer, represented in ribbon mode. C and D, ribbon representation of the tail-to-tail and the head-to-head A3G-CD2 dimer conformations, respectively. E, the superimposition of one monomer (gray) in the head-to-tail dimer with one monomer (orange) in the tail-to-tail or head-to-head dimer. The spheres represent zinc ions in the three-dimensional structures.

Differences between the Crystal Structures

The reported crystal structures of A3G-CD2 (residues 197–380) with PDB codes 3E1U and 3IQS have a continuous β2 sheet (36), significantly differing from those (discontinuous β2/β2′ sheets) in three NMR structures (PDB codes 2YJW, 2KBO, and 2KEM) (33–35), the x-ray crystallographic tail-to-tail or head-to-head dimer structure (3IR2) (37), and the current dimer structure. The different regions include β1-β2, β2′-α2, and α2-β3, which are due to the ambiguity in electron density. The bulge between the β2-β2′ strands in the NMR structures and 3IR2 is obviously an intrinsic feature of A3G-CD2 structure rather than an experimental artifact (37). Thus, for correct comparison, monomer H in the current A3G-CD2 dimer was just overlapped with one monomer of the tail-to-tail A3G-CD2 2K3A variant dimer (Fig. 1), which produces root mean square deviation values of 1.64 Å for all of the backbone C_α atoms in the region of residues from 193 to 384 and of 0.30 Å for all of the backbone C_α atoms in the secondary structure regions. This observation indicates that the monomer conformation of the current A3G-CD2 dimer is almost identical to that of 3IR2. The main differences are located in loop 1 (residues 206–215), loop 3 (residues 245–256), and loop 7 (residues 315–320), which are all involved in ssDNA binding and stabilize the active deamination center. The orientations of the side chains of the residues in sequence P²¹⁰WVR²¹³ in loop 1; of the residues His-248, His-249, Phe-252, and Glu-254 in loop 3; and of the residues in sequence Y³¹⁵DDQ³¹⁸ in loop 7 are apparently distinct from those in PDB entry 3IR2 (Fig. 2, A–C). Among these residues, Trp-211 (37), Arg-213 (33, 34, 36, 37), Tyr-315 (36), and Asp-316 and -317 (36) have been confirmed to be crucial for the deaminase activities. Moreover, it has been suggested that the sequence Y³¹⁵DDQ³¹⁸ in loop 7 specifically recognizes the second cytosine in the target motif sequence of ssDNA (5′-CCC-3′), due to its polarity, whereas the sequence Y³⁰⁷YFW³¹⁰ in loop 7 of A3F specifically identifies G or T in the ssDNA sequence 5′-(G/T)C-3′ (36, 43). Analysis on the surface of 3IR2 and the current structure indicates that the conformational changes in loops 1, 3, and 7 result in a bigger ssDNA-binding groove in the monomer of 3IR2 than that in the current dimer structure (Fig. 2, D and E), thus probably enhancing DNA binding. Moreover, the positively charged side chain of Arg-213 points to the ssDNA binding groove in 3IR2 structure, strengthening DNA binding, but it deviates from the ssDNA binding groove in the current structure. These observations may account for the fact that the A3G-CD2 2K3A variant has deaminase activities ∼2.7-fold more than its WT protein (33).

FIGURE 2.

A–E, the differences in the side chain orientations of the residues in loop 1 (A), loop 3 (B), and loop 7 (C) of the current head-to-tail dimer (orange) and tail-to-tail dimer (green) result in the conformational changes in the ssDNA binding groove in the monomer of the 2K3A variant (PDB code 3IR2) (D) and of the current head-to-tail dimer (E), respectively. F, the mutation from Asp-370 to Ala-370 results in a wider ssDNA binding groove in the surface of the D370A variant than that in the WT protein.

Intermolecular Interfaces in Head-to-tail A3G-CD2 Dimer

The obviously big difference between A3G-CD2 head-to-tail dimer and tail-to-tail dimer (or head-to-head dimer) (PDB code 3IR2) is the intermolecular interface. In monomer H of the current head-to-tail dimer, helix α2 and loop 3 form an L-shaped hook, which stabilizes the dimer conformation by interacting with helix α6′ and loop 1′ in monomer T. Thus, the current head-to-tail dimer contains three main interfaces, which are between loop 3 and loop 1′, between helix α2 and helix α6′, and between loop 3 and helix α6′ with surface areas of 223, 374, and 317 Ų, respectively (Fig. 3A). The total surface area is 1032 Ų, much larger than those in the tail-to-tail dimer conformation (901 Ų) and in the head-to-head dimer conformation (604 Ų), respectively. This indicates that the head-to-tail dimer conformation might be more stable than those of the tail-to-tail dimer and head-to-head dimer.

FIGURE 3.

The interactions in three unique interfaces in the head-to-tail A3G-CD2 dimer conformation. A, the secondary structural elements involved in the interactions in the interfaces. Shown are detailed interactions in the first interface (B–D), the second interface (E–G), and the third interface (H), respectively. The salt bridge between side chains of the residues Asp-316 and Arg-374 was observed in the head-to-tail dimer conformer (K) but not found in D370A variant structure (L). The salt bridge between Arg-374 and Glu-209′ was observed in the tail-to-tail dimer conformer. The numbers in each panel indicate the distances between the specific atoms connected by dotted lines. The double-headed arrows indicate the hydrophobic interactions between the related groups. The red spheres represent water molecules.

In the head-to-tail dimer conformation, the helix α2 in monomer H is almost perpendicular to the helix α6′ in monomer T, making up the largest interface. Residues Gly-373′, Ala-377′, and Gln-380′ in the C terminus of helix α6′ in monomer T form hydrogen bond nets by interacting with residues Leu-263 and Asp-264 in helix α2 in monomer H through several water molecules (Fig. 3, B and C). The side chain of Arg-376′ in monomer T has hydrophobic interactions with the side chain of Phe-268 in monomer H (Fig. 3D). To estimate the functional significance of this interface, four variants (D264A, F268A, R376A, and Q380A) were designed to disrupt the observed interactions. An NMR enzymatic assay was performed to measure the catalytic efficiency on the deamination at base C⁶ in the reported sequence of 5′-ATTC⁴C⁵C⁶AATT-3′ (34). These variants show decreased DNA deaminase activity in vitro (Table 2), especially for the D264A variant. Compared with the WT A3G-CD2 (K_cat^WT-CD2 = 0.11 ± 0.00049 min⁻¹, K_m^WT-CD2 = 20.42 ± 0.34 μm, K_cat^WT-CD2/K_m^WT-CD2 = 5.42 × 10⁻³ min⁻¹ μm⁻¹), the catalytic efficiency K_cat^D264A/K_m^D264A of the D264A variant is decreased about 20-fold (K_cat^D264A = 0.11 ± 0.02 min⁻¹, K_m^D264A = 394.40 ± 85.43 μm, K_cat^D264A/K_m^D264A = 2.78 × 10⁻⁴ min⁻¹ μm⁻¹). In the structure 3E1U, an intramolecular salt bridge was observed between the side chains of residues Asp-264 and Arg-256 in loop 3 (33)(Fig. 3E), which obviously stabilizes the conformation of loop 3. Therefore, the mutation from Asp-264 to Ala-264 not only disrupts the interactions between loop 3 in monomer H and the helix α6′ in monomer T but also makes loop 3 more flexible, destabilizing the ssDNA binding center and thus further resulting in the reduced catalytic efficiency of the D264A variant. The mutation from Arg-376 to Ala-376 keeps its hydrophobic interactions with the side chain of Phe-268 (Fig. 3D) but destroys the hydrogen bond nets that stabilize the interactions between the helix α2 in monomer H and the helix α6′ in monomer T. Thus, the R376A variant has only 15% of the catalytic efficiency of the WT protein (K_cat^R376A = 0.051 ± 0.00039 min⁻¹, decreased 2 fold; K_m^R376A = 63.23 ± 0.97 μm, decreased about 3-fold; K_cat^R376A/K_m^R376A = 8.10 × 10⁻⁴ min⁻¹ μm⁻¹, reduced 6.7-fold), consistent with the previous observation that Arg-376 was involved in the ssDNA binding activity and catalytic reaction (36, 37).

TABLE 2.

The catalytic efficiency of A3G-CD2 and its variants measured by NMR enzymatic assay

Protein	Kcat	Km	Kcat/Km	Activity (% of WT)
	min−1	μm	min−1 μm−1	%
A3G-CD2	0.11 ± 0.00049	20.42 ± 0.34	5.42E − 3	100
P210A	0.0061 ± 0.00078	81.88 ± 23.34	7.45E − 5	1.37
P210G	0.0099 ± 0.00064	18.46 ± 4.89	5.37E − 4	9.91
Q245A	0.083 ± 0.019	278.5 ± 81.36	2.99E − 4	5.52
H248G	0.28 ± 0.0011	33.06 ± 0.37	8.55E − 3	158
H250A	0.48 ± 0.00094	33.25 ± 0.18	1.44E − 2	266
R256A	0.012 ± 0.00018	163.90 ± 4.17	7.20E − 5	1.33
D264A	0.11 ± 0.020	394.40 ± 85.43	2.78E − 4	5.13
F268A	0.031 ± 0.0020	22.52 ± 5.28	1.37E − 3	25.21
D370A	0.010 ± 0.00038	11.89 ± 2.49	8.58E − 4	15.83
R374A	0.0050 ± 0.00043	27.05 ± 7.48	1.85E − 4	3.41
R376A	0.051 ± 0.00039	63.23 ± 0.97	8.10E − 4	14.94
Q380A	0.060 ± 0.0025	16.15 ± 3.02	3.71E − 3	68.45

In the second interface, loop 3 in monomer H acts as a hook to catch the helix α6′ in monomer T. The side chains of the residues Gln-245 and Arg-256 and the backbone oxygen atoms of the residues His-250 and Gly-251 in monomer H form a hydrogen bond network through water molecules with the charged side chains of Asp-370′ and Arg-374′ in monomer T (Fig. 3, F and G). To evaluate the contributions of these residues to the catalytic deamination, we replaced Gln-245, Arg-256, Asp-370′, and Arg-374′ with alanine. Compared with the WT protein, the Q245A and R256A variants nearly abolish the catalytic efficiency (for Q245A variant, K_cat^Q245A = 0.083 ± 0.019 min⁻¹, K_m^Q245A = 278.5 ± 81.36 μm, K_cat^Q245A/K_m^Q245A = 2.99 × 10⁻⁴ min⁻¹ μm⁻¹, decreased about 20-fold; for R256A variant, K_cat^R256A = 0.012 ± 0.00018 min⁻¹, K_m^R256A = 163.90 ± 4.17 μm, K_cat^R256A/K_m^R256A = 7.20 × 10⁻⁵ min⁻¹μm⁻¹, reduced 75-fold). Obviously, these mutations destroy the hydrogen bond interactions observed above and thus account for the changes in the catalytic efficiency. These results are different from those observed in structure 3IR2, where a side chain of Gln-245 was coordinated to the zinc ion in the dimer interface (37).

The D370A variant has smaller K_cat and K_m values (K_m^D370A = 11.89 ± 2.49 μm, K_cat^D370A = 0.010 ± 0.00038 min⁻¹) than the WT protein, with only about 16% of the catalytic efficiency of the WT protein (K_cat^D370A/K_m^D370A = 8.58 × 10⁻⁴ min⁻¹ μm⁻¹), suggesting that the D370A variant might have a stronger binding affinity to ssDNA than the WT protein (the DNA binding groove in the structure of the D370A variant becomes wider, as shown in Fig. 2F). We tried to crystallize the complex of D370A with ssDNA. Different from the WT protein, in the presence of ssDNA during crystallization, one asymmetric unit contains a D370A molecule. No electronic density was observed for ssDNA either. Its final crystal structure was determined at 1.7 Å resolution. One molecule of D370A forms a dimer with another molecule in an adjacent asymmetric unit in a head-to-tail way. The structure of the D370A variant reveals that the mutation from Asp-370 to Ala-370 not only directly results in the breakage of the hydrogen bond between Asp-370′ (in loop 7) and Gln-245 (in loop 3) (Fig. 3F) but also indirectly impairs the hydrophobic interaction between His-248 (in loop 3) and Trp-211′ (in loop 1) (Fig. 3H), and the intramolecular salt bridge interaction between Arg-374 (in helix α6) and Asp-316 (in loop 7) (the distances between the side chains of Arg-374 and Asp-316 become bigger in D370A than those in the WT protein, as shown in Fig. 3, K and L). Thus, the decrease in the catalytic efficiency of the A3G-CD2 D370A variant further reveals that the stability of the active center is important to the catalytic deamination reaction.

To assess the importance of Arg-374 in the cytidine deamination, we replaced Arg-374 by Ala-374. Compared with the WT protein, the R374A variant has a K_m^R374A value almost equal to that of WT A3G-CD2 (K_m^R374A = 27.05 ± 7.48 μm, K_m^WT-CD2 = 20.42 ± 0.34 μm), but its K_cat^R374A = 0.005 ± 0.00043 min⁻¹ is much less than that of WT protein (K_cat^WT-CD2 = 0.11 ± 0.00049 min⁻¹), resulting in its catalytic efficiency (K_cat^R374A/K_m^R374A = 1.85 × 10⁻⁴ min⁻¹μm⁻¹) being decreased about 35-fold, consistent with previous observations (36, 37). On one hand, we think that the mutation from Arg-374 to Ala-374 may directly destroy the intramolecular salt bridge between Arg-374 (in helix α6) and Asp-316 (in loop 7) (Fig. 3K) and the intermolecular salt bridge between Arg-374 and Glu-209′ (in loop 1) (which was observed in the tail-to-tail dimer conformation; Fig. 3M), both interactions making loops 1 and 7 more flexible. On the other hand, the replacement of Arg-374 by Ala-374 disrupts additional hydrogen bonds between the Arg-374 side chain and backbone oxygen atoms of residues Gly-251 and His-250 in loop 3 (Fig. 3G), which results in loop 3 being more flexible. Thus, like the mutation from Asp-370 to Ala-370, the mutation from Arg-374 to Ala-374 destabilizes the active deamination center of A3G-CD2 by changing the conformations of loops 1, 3, and 7.

The interaction between loop 3 in monomer H and loop 1′ in monomer T composes the third interface, in which the side chains of the residues His-250 and His-248 in loop 3 have weak hydrophobic interactions with the residues Pro-210′ and Trp-211′ in loop 1′ as well as hydrogen bond interactions between the His-250 side chain nitrogen atom and the Pro-210 backbone oxygen atom through one water molecule (Fig. 3H). These interactions enhance the stability of the dimer conformation. To examine the significance of this interface, four variants (H248G, H250A, P210A, and P210G) of A3G-CD2 were designed. Beyond our expectations, compared with the WT protein, the catalytic efficiency values (K_cat/K_m)of the H248G and H250A variants are improved about 1.6- and 2.6-fold, respectively (K_cat^H248G = 0.28 ± 0.0011 min⁻¹, K_m^H248G = 33.06 ± 0.37 μm, K_cat^H248G/K_m^H248G = 8.55 × 10⁻³ min⁻¹ μm⁻¹, K_cat^H250A = 0.48 ± 0. 00094 min⁻¹, K_m^H250A = 33.25 ± 0.18 μm, K_cat^H250A/K_m^H250A = 1.44 × 10⁻² min⁻¹ μm⁻¹). These observations are distinct from the previous studies on the structure 3IR2 (33, 37), where the side chains of His-248 and His-250 were ligated to zinc ion, the decrease in DNA deaminase activity of the trisite H248A/H250A/C261A variant of A3G-CD2 was thought to be attributable to C261A alone, and the mutations on His-248 and His-250 had no impact on the DNA deamination. The P210A and P210G variants were devised to decrease hydrophobic interaction with the side chain of His-250; their catalytic efficiency K_cat/K_m was thus nearly ablated (for the P210A variant, K_cat^P210A = 0.0061 ± 0.00078 min⁻¹, K_m^P210A = 81.88 ± 23.34 μm, K_cat^P210A/K_m^P210A = 7.45 × 10⁻⁵ min⁻¹ μm⁻¹, decreased about 100-fold). For the P210G variant, K_cat^P210G = 0.0099 min⁻¹, K_m^P210G = 18.46 ± 4.89 μm, K_cat^P210G/K_m^P210G = 5.37 × 10⁻⁴ min⁻¹ μm⁻¹, decreased about 10-fold).

Before measuring the catalytic efficiency of each variant of A3G-CD2, we tested their aggregation states by running an analytic Superdex^TM G75 column (10/300) (Fig. 4). The results suggest that all mutations on the residues mentioned above have no effects on the aggregation state of A3G-CD2. Therefore, we can exclude the possibility that the differences in the catalytic efficiency of A3G-CD2 variants resulted from A3G-CD2 aggregation state changes.

FIGURE 4.

The aggregation state of wild-type A3G-CD2 and its variants determined by size exclusion chromatography assay.

We further investigated the contributions of the residues in the interface in the head-to-tail A3G-CD2 dimer to the deaminase activities of full-length A3G through an E. coli-based deaminase activity assay (Fig. 5). The expression of the full-length A3G and its variants was confirmed by running E. coli immunoblots (Fig. 5). The variants, including P210A, P210G, Q245A, R256A, D264A, D370A, R374A, R376A, and Q380A, demonstrate weaker deaminase activities. In addition, the residue Phe-252 (in loop 3) is not located in the interface, but it has hydrophobic interactions with the side chain of residue Arg-256 (in loop 3) within one monomer (Fig. 3E) of the current head-to-tail dimer conformation (this interaction was also observed in the x-ray structure 3E1U) (33), which may stabilize the active center by fixing the conformation of loop 3. Thus, the mutation from Phe-252 to Ala-252 impairs this hydrophobic interaction and decreases the activities of HIV-1 virus ssDNA deamination by A3G-CD2. The H248G, H250A, and H250G variants have higher deaminase activities than WT protein, consistent with in vitro enzymatic results from A3G-CD2 H248G, H250A, and H250G variants.

FIGURE 5.

E. coli-based deaminase assay. The results of E. coli immunoblots (input) confirmed expression of the proteins. Error bars, S.E.

In summary, the interfaces in the current head-to-tail dimer conformation present new insights into the residues involved in HIV-1 virus ssDNA binding and the catalytic deamination. Mutagenesis studies on those residues further confirm that the stability of the active center is extremely important to catalytic C → U deamination. Nine new residues (Pro-210, Gln-245, His-248, His-250, Phe-252, Asp-264, Phe-268, Asp-370, and Gln-380) in the A3G-CD2 domain necessary for HIV-1 ssDNA binding and the catalytic deamination reaction were observed from this new dimer conformation.

DISCUSSION

The Head-to-tail Dimer Conformer of A3G-CD2 Reveals the Mode of Full-length A3G Binding to ssDNA

To well understand how the A3G CD1 and CD2 domains work together to facilitate cytidine deamination, a holoenzyme structure is a prerequisite. Although the three-dimensional structure of A3G-CD1 domain is not available, different models of full-length A3G were previously constructed. Two of them were predicted based on the APOBEC2 structure because APOBEC2 has amino acid sequence identical to A3G-CD1 (24%) and A3G-CD2 (31%) domains. One model, where A3G-CD1 and A3G-CD2 are tethered through the interactions between their β-strands, was successfully used to identify three residues (Arg-122, Trp-127, and Asp-128) important for packaging A3G into virions (50). The other model, however, produced from the extended NMR structure of A3G-CD2 domain (PDB code 2KEM) (35), suggested that the β-strands in A3G-CD1 and A3G-CD2 were distant. Instead, an N-terminal pseudocatalytic domain, including the interdomain linker and some of helix α6 of A3G-CD1, packs A3G-CD1 and A3G-CD2 together. Unfortunately, in this model, the catalytic deaminase domains (i.e. A3G-CD2 and N-terminal pseudocatalytic domain) point away from each other and from the nucleic acid binding site, creating a topological dilemma. The third model, the tail-to-tail dimerization model of the full-length A3G-DR (A3G was treated with RNase), and tetramer model of the full-length A3G-D (A3G was not treated with RNase) were generated by small angle x-ray scattering and shape reconstruction methods. This model implied that the full-length A3G in either low molecular mass or high molecular mass is symmetrically associated (51). This model was further refined into the fourth model after the A3G-CD2 three-dimensional structure was reported, through an in-cell quenched fluorescence resonance energy transfer (FRET) assay, small angle x-ray scattering, and other techniques (52). In this model, A3G was self-associated via its CD2 domain, forming a dimer structure. It seems that this model is more reasonable than any other reported model. Its low resolution, however, limits its usage in the analysis of the biological functions of the full-length A3G.

It is well known that A3G deaminates ssDNA processively with a strong 3′ → 5′ bias (31, 32, 34). When there is more than one target motif in the sequence of ssDNA, A3G deaminates the 5′-CCC target motif 5-fold more rapidly than the 3′-CCC target motif (53). Unlike either WT full-length A3G or its monomer F126A/W127A mutant, the A3G-CD2 alone, which does not oligomerize, catalyzes ssDNA-dependent C → U deamination (33, 36, 49), displaying no deamination polarity and no dead zone. The non-catalytic A3G-CD1 plays an indispensable role in stabilizing ssDNA binding, enhancing the catalysis, and establishing 3′ → 5′ deamination polarity and processivity. To explain the polarity and processivity of the deamination by A3G, Chelico et al. (53) suggested the fifth structural model of the full-length A3G, in which A3G effectively deaminates the ssDNA 5′-proximal CCC motif only upon binding to ssDNA in an active orientation. However, it is still difficult to obtain information about how A3G-CD1 is involved in ssDNA binding from this model.

To address this, we predicted an A3G-CD1 model through the SWISS model server (54–57) on the basis of its high sequence identity (43.68%) to A3C (42) (Fig. 6A), which contains six α-helices and five β-sheets. The β2 sheet is continuous, different from that in the A3G-CD2 structure. This structural model has a root mean square deviation value of 3.27 Å for all backbone atoms upon superimposing with the monomer T in the current head-to-tail A3G-CD2 dimer, indicating that its overall fold is almost identical with that of the A3G-CD2 domain (Fig. 6B). Thus, we raise a question of whether the current head-to-tail A3G-CD2 dimer conformation is a potential three-dimensional structural model of the full-length A3G.

FIGURE 6.

The structural model of full-length A3G. A, sequence alignment of A3G-CD1, A3G-CD2, and A3C. B, ribbon representations of the A3G-CD1 structural model (blue) overlapped with the head-to-tail dimer conformer (gray, similar orientation to Fig. 1A); the green spheres represent zinc ions. C, the continuous DNA binding groove in head-to-tail dimer conformation. Residues in or along the groove in monomers H and T are highlighted. D, the residues highlighted in A3G-CD1 (pink) correspond to those located in or along the groove in A3G-CD2 monomer T. In both C and D, the docked cytidine is highlighted in blue. E, electrostatic potential surface of A3G-CD2 monomer H plus predicted A3G-CD1 structural model (similar orientation to Fig. 5B) indicates possible interactions between full-length A3G and ssDNA.

To answer this, first, we probed the surface of head-to-tail dimer conformation, where an obviously continuous groove was observed (Fig. 6C). Part of this groove is composed of the reported main ssDNA binding elements in A3G-CD2, such as loop 1 (33, 35–37), loop 3 (33, 36), loop 7 (33, 36), and helix α6 (36, 37). The residues located in or along this continuous groove in monomer H are involved in ssDNA binding, including the mostly reported side chain charged amino acids, such as Arg-213 (33, 36), Arg-215 (33, 36), Arg-256 (36), Arg-313 (33), Asp-316 (36, 43), Asp-317 (36, 43), Arg-320 (33), Arg-374 (36, 37), and Arg-376 (36, 37), and hydrophobic residues, such as Phe-202 (35), Phe-206 (35), Trp-211 (37), His-216 (36), His-257 (33, 36), Trp-269 (36), Leu-271 (36), Trp-285 (33, 36), Phe-289 (36), and Tyr-315 (36), as well as Asn-208 (35) and Asn-244 (36). The new active sites, including the residues Pro-210, Gln-245, His-248, His-250, Phe-252, Asp-264, Phe-268, Asp-370, and Gln-380, discussed above are also situated in or along this groove.

Second, the ssDNA binding sites in A3G-CD1 have not been completely identified. We thus tried to obtain the binding information basically from the sequence alignment (Fig. 6A). A3G-CD2 has 33.51% sequence identity to A3G-CD1. We replaced the residues in the A3G-CD1 domain by alanine or glycine, including Pro-25, Leu-27, Glu-61, His-65, Glu-67, Phe-70, His-72, Tyr-91, Trp-94, Arg-122, Tyr-124, Tyr-125, Trp-127, Asp-130, Leu-184, Met-188, and Glu-191 (Fig. 6D), which have positions similar to residues Pro-210, Val-212, Glu-254, His-257 (33, 36), Glu-259, Phe-262, Asp-264, Phe-282, Trp-285 (33, 36), Arg-313 (33), Tyr-315 (36), Asp-316, Asp-317, Arg-320, Asp-370, and Arg-374 (36, 37) seated along or in the groove in monomer T (Fig. 6C), respectively, and tested the deaminase activities of these variants by the E. coli-based deaminase assay. As shown in Fig. 5, the variants E61A, H65G, E67A/D264H (the E67A variant was not successfully obtained), F70A, H72A, Y91A/D264H (the Y91A variant was not successfully obtained), W94A, R122A, Y124A, Y125A, W127A, D130A (and D130R), L184/185A (the L184A variant was not successfully generated), M188A, and E191A demonstrate weaker deaminase activities compared with the WT full-length A3G. These observations are consistent with previous reports that residues in the surface of A3G-CD1, including His-65, Glu-67, Phe-70, Tyr-91, Trp-94, Arg-122, Tyr-124, Trp-127, and Met-188, are critical for A3G antiviral activities (50). Based on superimposing structures (Fig. 6B), we also suppose that the residues Pro-25, Trp-127, Leu-184, Ile-187, Met-188, and Glu-191 are also possibly involved in the interactions between A3G-CD1 and A3G-CD2 domain, as shown in Fig. 7.

FIGURE 7.

Possible interactions between A3G-CD1 (gray) and A3G-CD2 (green) represented in ribbon mode. A, hydrophobic interactions between residues Pro-25 (P25) and Leu-27 in loop 1 of A3G-CD1 and His-248 and His-250 in loop 3 of A3G-CD2; B, hydrophobic interactions between residues Trp-127 in loop 7 of A3G-CD1 and Leu-253 and His-250 in loop 3 of A3G-CD2; C–E, hydrophobic or hydrogen bond interactions between residues Leu-184, Ile-187, and Met-188 in helix α6 of A3G-CD1 and Gln-245, His-250, Gly-251, and Phe-252 in loop 3 of A3G-CD2; F, salt bridge interaction between residues Glu-191 in helix α6 of A3G-CD1 and Arg-256 in loop 3 of A3G-CD2. Residues mentioned above are shown in stick mode. The names of the residues from A3G-CD1 are highlighted in red.

Third, in what orientation does ssDNA upon its bind to full-length A3G? In our current A3G-CD2 head-to-tail dimer conformation, close to the catalytic center of the residue Glu-259 (ligated to Zn²⁺ ion), there is a deep pocket for accommodating the first base C^m in the 5′-proximal -C^m+2C^m+1C^m- target motif in ssDNA (the ssDNA sequence is assumed to be 5′-C^m+2C^m+1C^m-Cⁿ⁺²Cⁿ⁺¹Cⁿ-3′). By overlapping the structure of monomer H with that of the mouse cytidine deaminase in complex with cytidine in the active site (PDB code 2fr6), we found that the hot spot cytidine was really docked into this site near the residues -Y³¹⁵DDQ³¹⁸- in the conserved sequence -R³¹³IYDDQ³¹⁸- in loop 7 (Fig. 6, C and D). This sequence was previously predicted to specifically identify the second base, C^m+1, in the 5′-proximal C^m+2C^m+1C^m target motif in ssDNA, which is different from the residues -Y³⁰⁷Y³⁰⁸FWD³¹¹- in loop 7 of A3F-CD2 (specifically binding to the second base T^m+1 in 5′-T^m+2T^m+1C^mA-3′) (43, 44) and from the residues -Y¹³⁰DYD¹³³-in loop 7 of A3A-CD1 (specifically binding to the second base, T^m+1 or C^m+1, in target motif 5′-T^m+1C^m-3′ or 5′-C^m+1C^m-3′) (40, 41). Thus, upon binding to full-length A3G, the 5′ terminus of ssDNA is located in the A3G-CD2 side, whereas its 3′ terminus interacts with the A3G-CD1 side. In other words, ssDNA binds to full-length A3G in an active orientation, as shown in Fig. 6, C and D, which accords with the previous prediction (53). The ssDNA interaction cavity displays large hydrophobic regions and negatively charged regions in the electrostatic potential surface of A3G-CD2 plus the A3G-CD1 structural model (Fig. 6E). The main hydrophobic regions distribute near the 3′-end of the ssDNA, and the negatively charged regions mainly locate close to the catalytic center.

In conclusion, based on the structural analysis, NMR-based enzymatic assay, and E. coli-based deaminase activity assay, we suggest that the head-to-tail dimer conformation may represent a structural model of full-length A3G. This model indicates that A3G may bind to HIV-1 virus ssDNA in an active orientation. According to this structural model, several new residues in A3G-CD1 (including Pro-25, Glu-61, His-72, Asp-130, and Leu-184) were found to be necessary to ssDNA binding and catalytic deamination.