[Fe-S] containing enzymes

Many processes associated with DNA synthesis and repair, involve metal-containing cofactors, and misregulation of these processes can lead to carcinogenesis. However, because of the paucity of biochemical and structural information, knowledge of their modi operandi or the molecular linkage between integrity of the metallocofactor and pathological events remain largely unknown. A particularly important class of metal cofactors is that of iron-sulfur [Fe-S] clusters, perhaps the most ubiquitous prosthetic groups in nature. Their abundance is likely the consequence of their structural and redox plasticity, thereby making them versatile and tunable cofactors for mediating electron transfer. However, [Fe-S]-cluster containing proteins are much more functionally versatile than that originally thought; they mediate and participate in a variety of cellular processes, such as DNA maintenance, amino acid and nucleotide metabolism, ribosome function and tRNA modification. [Fe-S] clusters participate in enzyme catalysis and bind substrates, regulate gene expression, act as sensors of small molecules (i.e. O₂, NO), store iron, serve as sulfur donors during synthesis of lipoic acid and biotin, etc. Thus, despite the simplicity of their chemical ‘make-up’, their chemical and functional diversity is both astounding and complex.

The HBV protein X harbors an unprecedented [Fe-S] cluster

Chronic infection by Hepatitis B viruses (HBVs) is associated with liver disease and hepatocellular carcinoma (HCC). HCC is the third most common cause of cancer mortality and the fifth most common cancer worldwide. HBx is the smallest gene product of HBV and the main etiological agent of virus-mediated liver oncogenesis. Although there are innumerable reported HBx functions and protein binding partners, the molecular mechanisms by which HBx promotes tumorigenesis are still unclear. Despite the multi-decade studies, hardly any biochemical or structural information is known about HBx, which has been the major obstacle for linking its structure and activity to the cascade of cellular processes it modulates.

We have shown that HBx harbors a non-classical, redox-dependent [Fe-S] cofactor that can switch between a [2Fe] and [4Fe] form. It was previously impossible to identify the [Fe-S] cluster based on sequence, because there are no recognizable binding motifs. The redox-induced conversion of the metallocofactor raises a question about whether the protein ligands stem from one or more polypeptides, a fact that would have a consequence for the oligomeric state of the protein. We hypothesize that the metallocofactor is not solely a structural element, but has an active redox and structural role in the biological activity of HBx.

HBx has multiple functions; it acts as a transactivator by interacting with many cellular factors, it has a reactive oxygen species (ROS) generating potential and exhibits ATPase activity. We propose that the novel [Fe-S] cluster is the missing link in elucidating HBx function, both on its own and in the context of target proteins. We have generated highly soluble HBx constructs that permit its structural and biophysical characterization, which was previously hardly accessible. By structurally and biochemically characterizing HBx and the [Fe-S] cluster, we aim to shed the first light on the oncogenic potential of HBx.

HBx is a potential target for the development of anti-cancer drugs; therefore determining the chemical nature of the metallocofactor and the structure/function relationships supporting its role in viral-induced pathogenesis are of great importance. Discovery of an unusual [Fe-S] cluster in HBx sets new grounds for delineating its structure and function, which was previously inaccessible. Our cofactor reconstituted HBx will allow to resolve its purported, and often controversial, roles in oncogenesis, ultimately yielding new insights for antiviral therapies.