One protein, at least three structures, and many functions

Summary

Hepatitis B virus core gene products can adopt different conformations to perform their functional roles. In this issue of Structure, DiMattia et al. (2013) show the crystal structure of immuno-modulating HBeAg and thereby reveal the similarities and differences between it and HBcAg, the variant found in virions.

How can one protein generate two distinct immune responses and be endowed with two unique functions? Some proteins achieve multiple functions through intrinsically disordered regions, others by subtle allosteric shifts. The Hepatitis B virus core protein undergoes a radical reorientation of its dimer interface; it is a striking example of Gregorio Weber’s characterization that a protein is a “kicking, screaming, stochastic molecule”.

Hepatitis B virus (HBV) chronically infects 360 million people. It has a unique ability to establish virus-specific immuno-tolerance while continually producing infectious virus particles. The core gene, source of ‘e’ and ‘c’ antigens, plays both sides of this street. The ‘c’ antigen (HBcAg) is a homodimeric protein that self-assembles to package viral RNA and reverse transcriptase; this complex is the HBV core. The ‘e’ antigen (HBeAg) begins translation at an upstream methionine so that it includes a signal sequence which leads it to be secreted after proteolytic processing of the signal and removal of the RNA-binding C-terminus. The resulting HBeAg sequence thus includes a 10-amino acid propeptide. While HBcAg capsids are highly antigenic, HBeAg is implicated in attenuating immune response to chronic infection but is entirely dispensable for virus assembly and replication (Chen et al., 2005; Seeger et al., 2007).

In this issue, DiMattia and colleagues present the structure of HBeAg to explain the basis of the similarities and differences between HBeAg and HBcAg (Dimattia et al., 2013). Both HBcAg and HBeAg have dimeric forms. A monomer is comprised of five helices. The helix 3 - helix 4 hairpin dimerizes with a second monomer to form a central helical bundle (Dimattia et al., 2012; Wynne et al., 1999). This dimer interface is the fulcrum for changes in structure and activity. In the context of a capsid, the dimer interface is symmetrical (rightmost panels); helix 5 and following amino acids (pale red) form interdimer contacts (Wynne et al., 1999). Free HBcAg dimers have a notably different structure where the intradimer helical bundle is distorted, which results in helix 5 adopting a geometry incompatible with forming an icosahedral capsid (Packianathan et al., 2010). It has been hypothesized that the dimer interface of HBcAg undergoes an allosteric change to activate assembly (Bourne et al., 2009). In free and capsid forms, HBcAg can form a Cys61-Cys61 disulfide connecting the two monomers. In the HBeAg structure, the monomer structure is very similar to the HBcAg monomer, but the dimer interface is completely remodeled. The second subunit is rotated by 140° around an axis near the top of helix 3. This structure is stabilized by a novel disulfide between Cys(−7) of the propeptide (magenta, leftmost panels) and Cys61.

Where HBcAg readily assembles into T=4 or T=3 capsids, HBeAg with the Cys61 – Cys(−7) disulfide is locked in a structure that cannot assemble. Also, the major HBcAg epitope, the spike tip formed by the dimer interface, is completely disrupted in the Cys61 – Cys(−7) HBeAg structure. The smaller buried surface of the HBeAg interface suggests that it is a less stable state that is locked down by the disulfide (the melting temperature of HBeAg is 14° C lower than oxidized free HBcAg (Watts et al., 2011)). Fantastically, when the HBeAg intra-monomer disulfide is reduced, the resulting protein readily assembles into morphologically normal capsids indicating HBeAg adopts an HBcAg conformation (Dimattia et al., 2012; Watts et al., 2011).

The ability of HBeAg to flip from its assembly-incompetent form to one that is consistent with HBcAg demonstrates that these are tremendously dynamic structures. To adopt both e and c conformations, the reduced protein must switch between at least these two states. This flexibility is also consistent with allosteric activation of capsid assembly. Supporting this assertion, the F97L mutation at the dimer interface enhances in vitro capsid assembly compared to wild-type protein and leads to defects in virus secretion in cell culture (Ceres et al., 2004; Le Pogam et al., 2000; Yuan et al., 1999).

DiMattia and coworkers’ findings suggest a possible explanation for the conservation of the Cys61 and Cys(−7) in all genotypes of HBV by proposing a dual function for Cys61. It can be hypothesized that formation of the Cys61-Cys61 disulfide bond increases the stability of the HBcAg dimer and/or capsid. While formation of the Cys(−7)-Cys61 disulfide bond prevents assembly of HBeAg creating a structurally distinct molecule that is able to suppress immune response to HBV and provide an evolutionary advantage for virus replication (Chen et al., 2005; Seeger et al., 2007). The dynamic structure of HBeAg/HBcAg not only regulates capsid assembly, but also preserves the different functional roles of the core protein gene products in other steps of the virus life cycle.

Figure. HBV core protein dimers have different structures that reflect their activities.

The monomer structure is very similar in all three known states, with some changes evident in the helices that make the dimer interface (helix 3 (green) and helix 4 (light orange). In HBeAg (3V6Z), a propeptide (magenta spheres) disulfide crosslinks with Cys61 (yellow spheres). In free HBcAg dimer (3KXS) and HBcAg from capsid (1QGT) (assembly-inactive and assembled states, respectively), which lack the propeptide, the second subunit is rotated 140° compared to HBeAg. In HBcAg there is an intradimer Cys61-Cys61 disulfide.