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. 2016 Feb 23;113(8):2074-9.
doi: 10.1073/pnas.1525616113. Epub 2016 Feb 8.

Structural and biochemical analysis of Bcl-2 interaction with the hepatitis B virus protein HBx

Tianyu Jiang  1 , Minhao Liu  1 , Jianping Wu  1 , Yigong Shi  2
Affiliations

Structural and biochemical analysis of Bcl-2 interaction with the hepatitis B virus protein HBx

Tianyu Jiang et al. Proc Natl Acad Sci U S A. .

Abstract

HBx is a hepatitis B virus protein that is required for viral infectivity and replication. Anti-apoptotic Bcl-2 family members are thought to be among the important host targets of HBx. However, the structure and function of HBx are poorly understood and the molecular mechanism of HBx-induced carcinogenesis remains unknown. In this study, we report biochemical and structural characterization of HBx. The recombinant HBx protein contains metal ions, in particular iron and zinc. A BH3-like motif in HBx (residues 110-135) binds Bcl-2 with a dissociation constant of ∼193 μM, which is drastically lower than that for a canonical BH3 motif from Bim or Bad. Structural analysis reveals that, similar to other BH3 motifs, the BH3-like motif of HBx adopts an amphipathic α-helix and binds the conserved BH3-binding groove on Bcl-2. Unlike the helical Bim or Bad BH3 motif, the C-terminal portion of the bound HBx BH3-like motif has an extended conformation and makes considerably fewer interactions with Bcl-2. These observations suggest that HBx may modulate Bcl-2 function in a way that is different from that of the classical BH3-only proteins.

Keywords: Bcl-2; apoptosis; crystal structure; hepatitis B virus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Biochemical characterization of the hepatitis B virus protein HBx. (A) The primary sequences of HBx. The hydrophilicity plot by the Kyte–Doolittle criteria (48) is shown above the sequences. The 63 hydrophobic amino acids (Ala, Val, Leu, Ile, Phe, Cys, and Met) account for 41% of the total. In addition, there are 15 Pro and 4 Trp residues. The putative BH3-like motif is shown below the sequences in an α-helical conformation. (B) The full-length HBx appears to form large oligomers on gel filtration. The full-length MBP-tagged HBx was eluted from gel filtration with an elution volume of 9.4 mL, close to the void (Left). The peak fractions were visualized on SDS/PAGE (Right). (C) The purified full-length MBP-tagged HBx exhibits a yellow-brownish color. Wavelength scan of this protein, but not the MBP control, shows an absorption peak at 415 nm. (D) Element analysis of the full-length MBP-tagged HBx reveals the presence of metal ions. Both iron and zinc are present in significant quantities with ∼1.9 and 0.4 molar equivalence of the MBP-HBx protein. (E) The HBx fragment (residues 110–154) bound to Bcl-2 exhibits excellent behavior on gel filtration. The fusion protein between the HBx fragment and Bcl-2 (residues 1–50 and 92–207) was purified to homogeneity and separated by proteolysis. The resulting complex was subject to gel filtration analysis (Left). The peak fractions were visualized on SDS/PAGE (Right). (F) The HBx fragment (residues 110–154) bound to Bcl-2 has a yellow-brownish color. Wavelength scan of HBx-Bcl-2 shows an absorption peak at 415 nm.
Fig. 2.
Fig. 2.
Quantification of the interactions between the HBx BH3-like motif and Bcl-2. (A) Sequence alignment of the BH3 domains from the proapoptotic Bcl-2 proteins and HBx. Invariant and conserved residues among the canonical BH3 motifs are shaded black and gray, respectively. (B) Measurement of binding affinities between the HBx BH3-like motif (residues 110–135) and Bcl-2 (residues 1–50 and 92–207). Measurement by ITC and MST reveals dissociation constants of 193 ± 8 and 382 ± 10.8 µM, respectively. (C) Measurement of the binding affinity between the HBx fragment (residues 110–137) and Bcl-2 by ITC. Data fitting reveals a dissociation constant of 435 ± 19 µM. (D) The HBx fragment (residues 110–140) binds to Bcl-2 with a dissociation constant of ∼292 ± 15 µM by ITC.
Fig. 3.
Fig. 3.
Structure of Bcl-2 bound to the HBx BH3-like motif. (A) Overall structure of Bcl-2 (residues 1–50 and 92–207) in complex with the HBx BH3-like motif (residues 110–135). HBx and Bcl-2 are colored pink and slate, respectively. (B) A close-up view of the HBx/Bcl-2 interface. For clarity, the crowded interface (Left) is opened up to show its two components (Middle and Right). The backbones of HBx and Bcl-2 are colored pink and slate, respectively. (C) A close-up view of the hydrogen bonds at the HBx-Bcl-2 interface. Glu125 and Arg128 of HBx each makes a pair of charge-stabilized hydrogen bonds to residues in Bcl-2. Hydrogen bonds are represented by red dashed lines. (D) Measurement of the binding affinity between the HBx BH3-like motif (residues 110–135) and the Bcl-2 variant (R100A/D101A) by ITC. Data fitting reveals a dissociation constant of ∼763 ± 130 µM.
Fig. 4.
Fig. 4.
Structural comparison of the Bcl-2-HBx complex with the Bcl-xL-Bad and Bcl-2A1-Bim complexes. (A) Comparison of the overall structure of the Bcl-2-HBx complex with that of Bcl-xL-Bad [PDB code 1G5J (32)] (Left) and Bcl-2A1-Bim [PDB 2VM6 (34)] (Right). The backbones are shown in ribbon representation. HBx (residues 110–135) and Bcl-2 are colored pink and slate, respectively. Bcl-xL and Bcl-2A1 are colored gray, whereas the Bad and Bim helices are colored green and orange, respectively. (B) A close-up view of the interface comparison between the Bcl-2-HBx and Bcl-xL-Bad complexes. For clarity, only the surface of Bcl-xL is shown in electrostatic potential. Bad and HBx are colored in green and pink, respectively. Hydrophobic and charged side chains of the bound BH3 motifs are shown in the Upper and Lower panels, respectively. (C) A close-up view on the interface comparison between the Bcl-2-HBx and Bcl-2A1-Bim complexes. Bim and HBx are colored in orange and pink, respectively. Hydrophobic and charged side chains of the bound BH3 motifs are shown in the Upper and Lower, respectively. (D) Analysis of Bcl-2 interactions with BH3 motifs by native PAGE. The HBx BH3-like motif and Bcl-2 failed to form a stable complex on native PAGE (lanes 1 and 2). In contrast, the BAD BH3 motif formed a stable complex with Bcl-2, and this complex remained undisturbed by excess amount of HBx BH3-like motif (lanes 7–10). Similarly, the Bim BH3 motif also formed a stable complex with Bcl-2 (lanes 3–6), which also remained undisturbed by the presence of HBx BH3-like motif.
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