Structural and functional analyses of hepatitis B virus X protein BH3-like domain and Bcl-xL interaction

Tian-Ying Zhang,Jia-Li Cao,Ning-Shao Xia,Hong-Ying Chen,Tian-Liang Li,Xiao-Bing Mo,Xiang Zhao,Quan Yuan,Hua-Long Xiong,Xiao-Zhen Kang,Ding Xue,Bo Yin,Cheng-Hao Huang,Jing-Hua Zhao,Y Adam Yuan

doi:10.1038/s41467-019-11173-1

Abstract

Hepatitis B virus (HBV) X protein, HBx, interacts with anti-apoptotic Bcl-2 and Bcl-xL proteins through its BH3-like motif to promote HBV replication and cytotoxicity. Here we report the crystal structure of HBx BH3-like motif in complex with Bcl-xL where the BH3-like motif adopts a short α-helix to snuggle into a hydrophobic pocket in Bcl-xL via its noncanonical Trp120 residue and conserved Leu123 residue. This binding pocket is ~2 Å away from the canonical BH3-only binding pocket in structures of Bcl-xL with proapoptotic BH3-only proteins. Mutations altering Trp120 and Leu123 in HBx impair its binding to Bcl-xL in vitro and HBV replication in vivo, confirming the importance of this motif to HBV. A HBx BH3-like peptide, HBx-aa113-135, restores HBV replication from a HBx-null HBV replicon, while a shorter peptide, HBx-aa118-127, inhibits HBV replication. These results provide crucial structural and functional insights into drug designs for inhibiting HBV replication and treating HBV patients.

Highlights

Hepatitis B virus (HBV) is a small DNA virus with a genome size around 3.2 kb
HBV X protein (HBx) has been shown to interact with damage-specific DNA-binding protein 1 (DDB1), which could redirect the DDB1contaning E3 ubiquitin ligase to target the structural maintenance of chromosome 5/6 complex (Smc5/6) for degradation, unleashing the transcriptional repression by Smc5/6 to increase HBV viral gene expression[6,9]
HBx is found to directly target anti-apoptotic proteins Bcl-2 and Bcl-xL through a Bcl-2 homology region 3 (BH3)-like motif to induce the increase of cytosolic calcium, which is required for HBV viral replication, and induces cytotoxic effects through apoptosis and necrosis, leading to HBV pathogenesis[7,12]

Summary

Introduction

Hepatitis B virus (HBV) is a small DNA virus with a genome size around 3.2 kb. HBV infection stimulates the development of hepatitis and hepatocellular carcinoma (HCC), which is a leading cause of liver cancer worldwide[1,2]. Trp[120] and Leu[123], in the HBx-BH3-like motif closely interact with residues in the Bcl-xL binding pocket via hydrophobic interactions Supporting this structural observation, a peptide comprising the HBx-BH3like motif (HBx-BH3-aa113–135), but not an equivalent peptide harboring the W120A/L123A double mutations, is capable of pulling down the endogenous Bcl-xL protein from HepG2 cells. Our structural and functional analyses of the HBx/Bcl-xL interaction identify key residues and the structural basis that mediate binding of HBx to Bcl-xL and a new and distinct binding pocket in Bcl-xL for the HBx-BH3-like motif and point out the need to employ a new strategy to screen for unique HBx-BH3like mimetics that can inhibit HBV replication and treat HBVrelated liver disease

Methods

Results

Conclusion