Abstract
The Hepatitis B virus (HBV) X protein (HBx) is a regulatory oncoprotein associated with the development of Hepatocellular Carcinoma (HCC) and cirrhosis (Feitelson and Duan 1997, Neuveut et al., 2010). Regardless of the overabundance of reported functions, the structural and functional characteristics of HBx still remain unknown. In this study, we focused on the characterization of the [Fe-S] cluster, recently discovered by us, and the identification of the protein-based coordinating ligands. Soluble HBx fusion proteins were constructed with two different solubility enhancement tag proteins, namely the maltose binding protein (MBP) and the N-utilization substance protein A (NusA). Single-point substitutions of 7 tentative ligand residues (i.e. C7, C61, C69, C137, H139, C143, and C148) to alanine were performed together with larger sequence truncations of 14 (Δ14) or 27 (Δ27) amino acids (aa) at the C-terminus, which are clinically relevant. The extent of [Fe-S] cluster incorporation in the variant proteins was quantified and compared to that in the wild-type HBx. Only some substitutions (C61A, C69A, and C137A, and H139A) resulted in a lesser yield of the [Fe-S] cluster incorporation. However, none of the single-point substitutions was able to abolish completely [Fe-S] cluster formation. We thus adopted two different strategies; a) generation of C-terminal truncated variants, which lack two and three cysteine residues, respectively and b) generation of double- and triple-point substitutions. In addition, apart from monitoring cluster assembly, we also performed kinetic experiments to investigate the O2-sensitivity of the cofactor and its fate upon oxidation. Intriguingly, our results demonstrate that both clinically relevant variants (Δ14, and Δ27) can assemble the same type of [Fe-S] cluster to similar extents, whereas its O2-sensitivity is hardly affected.