Structure And Function Study Of Hepatitis B Virus Protein HBx

Hepatitis B virus is a typical DNA virus that affects liver.According to the statistic data from World Health Organization,about 240 million people are infected around the world.For the most seriously cases,in those who get suffered from liver cancer,nearly 50% result from HBv infection in Asia.There are four genes encoded on the genome of HBv,X gene among them.However,the functions of HBx protein expressed from X gene are messy and poorly understood.Full length HBx protein contains 154 amino acids,only can be detected in infected liver cell.Paradoxically,HBx required for viral infectivity and replication is thought to be highly integrated to carcinogenesis and apoptosis.Meanwhile,numerous target proteins might have interactions with HBx,which is believed to modulate kinds of cellular activities.However,in terms of poor protein behavior,the lack of directly binding evidence in vitro hinders the fully understanding of the molecular mechanism of HBx-induced carcinogenesis.The well-known oncogene B-cell lymphoma-2(bcl-2)plays a centre role in the process of programmed cell death.As other Bcl-2 family gene discovered in succession,these genes are divided into two categories by their function.They are roughly divided into oncogenes and cancer suppressor genes represented by bcl-2 and bax,bak respectively.In the view of structure,Bcl-2 is composed of four relatively conserved alpha helices(BH1-4).The BH3 motif plays a decisive role in controlling cell apoptosis.In the present study,we first attempt to optimize the expression of HBx full length protein,in order to obtain the full length HBx protein with relatively stable properties.Through its biochemical properties and interaction experiments in vitro,we identify a metal ion binding domain at the C terminal of HBx and its potential to interact with the native protein from liver tissue.Secondly,we co-crystallized the BH3 motif(110-135)of HBx with Bcl-2.Via X-ray crystallographic diffraction,the structure with resolution 2.1 ? was successfully resolved.In contrast to the typical BH3 motif,the motif from HBx has an extended conformation.In addition,we further discover that the stability of the complex is drastically lower than that for a canonical BH3 motif from Bim or Bad,after performing gel-filtration,microscale thermophoresis and isothermal titration calorimetry experiment.This coincides with the dual-role of the reported HBx in cell apoptosis.In conclusion,HBx protein with relatively stable properties has been obtained through optimizing purification conditions.Meanwhile,we report biochemical and structural characterization of C-terminal HBx(110-135)binding with Bcl-2.In the view of molecular,we further explain the regulatory role of HBx in the intermediate state of apoptosis,which provides a new direction for the research of HBV.