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

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.Bcl-2 | hepatitis B virus | apoptosis | crystal structure T he human hepatitis B virus (HBV) is a prototypical hepadnavirus that specifically infects hepatocytes (1, 2). Over 240 million people worldwide are chronically infected by HBV according to the World Health Organization. Persistent HBV infection is one of the major risk factors for the development of hepatocellular carcinoma (HCC), accounting for over 50% of all HCC cases (3, 4). The genome of HBV encodes only four proteins, among which the product of the X gene (HBx) is least understood (1).HBx contains 154 residues and is closely associated with HCC development. The X gene is most frequently integrated and preferentially maintained in HBV-associated HCC, and HBx expression is frequently detected in HCC patients (5, 6). Several studies using transgenic mice and cell culture models yielded contrasting conclusions. Some studies suggest that the development of HCC is closely linked to HBx expression (7-10), although HBx alone may be insufficient for HCC development (11,12). Other results suggest that HBx has the ability to suppress cell transformation through induction of apoptosis (13,14). As such, the molecular mechanism of HBx-induced cellular transformation remains enigmatic.Numerous cellular activities are reported to be modulated by HBx through interactions with various host targets. For example, HBx-mediated transcriptional activation is achieved through its interaction with transcription factors (15, 16) or components involved in the basal transcriptional machinery (17-19). HBx can also modulate cellular proliferation and viability through interactions with p53 and Bcl-2 family members. Reflecting its enigmatic functions, HBx has been demonstrated to either induce apoptosis (20) or prevent apoptosis (21).HBx was recently found to contain a BH3-like motif in its C-terminal sequences (22). Experiments carried out in human hepato...

Novel anionic cecropins from the spruce budworm feature a poly-L-aspartic acid C-terminus

“…3A). HBx contains a BH3-like motif that folds to form an amphipathic α-helix that binds to the conserved BH3-binding groove of Bcl-2, an anti-apoptotic protein (Ma et al, 2008; Jiang et al, 2016). The BH3-like motif may inhibit the anti-apoptotic action of Bcl-2 through interactions with its conserved BH3-binding groove.…”

“…Glu125 and Arg128 of HBx (Fig. 3C) each makes a pair of charge-stabilized hydrogen bonds to residues Arg100 and Asp101 in Bcl-2 (Jiang et al, 2016). The amino acids corresponding to Glu125 and Arg128 in the N-terminus of Cfcec - are Gln9 and Arg12 (Fig.…”

HBV X protein mutations affect HBV transcription and association of histone-modifying enzymes with covalently closed circular DNA

“…The HBx C-terminal has two zinc-finger motif-containing trans-activating regions at aa 58-84 and 120-141 (corresponding to MT3-MT7 and MT20-MT26 in this study), as well as other motifs such as the Bcl-2 homology 3 (BH3)-like motif (aa 110-135) and the H-box motif (aa 88-100; covering MT8-MT12) [9][10][11][12][13][14][15][16] . HBx binds to anti-apoptotic Bcl-2 proteins via the BH3-like motif within HBx (aa 110-135) 17,18 . Mutations within the BH3-like motif at HBx aa 124 and 127 (covering MT21 and MT22) has been reported to prevent its binding to the anti-apoptotic Bcl-2 proteins, thereby abrogating cytosolic calcium elevation, and reducing HBV replication 17,18 .…”

“…HBx binds to anti-apoptotic Bcl-2 proteins via the BH3-like motif within HBx (aa 110-135) 17,18 . Mutations within the BH3-like motif at HBx aa 124 and 127 (covering MT21 and MT22) has been reported to prevent its binding to the anti-apoptotic Bcl-2 proteins, thereby abrogating cytosolic calcium elevation, and reducing HBV replication 17,18 . In another study, Guo et al found that the HBx H-box motif regulates HBV replication both positively and negatively through different pathways 14 .…”

HBV T1719G mutation reduced HBV replication through mutant Enh II and HBx protein in vitro

“…Li et al reported that HBx 111‐117 amino acid residues in c‐terminal played an important role in HBx positioning in the mitochondria, among which aa116 played a decisive role. Replacement of Phe to Cys at aa116 changed HBx binding affinity to Bcl2, which subsequently affected cell apoptosis induced by HBx . Further, cysteine was easy to form disulphide bond, and this amino acid change might disturb HBx protein folding, even impact its transcriptional regulation.…”