The transcriptional repressor Bach2 regulates humoral and cellular immunity, including antibody class switching. It possesses a basic leucine zipper domain that mediates DNA binding. Heme inhibits the DNA-binding activity of Bach2 in vitro and induces the degradation of Bach2 in B cells. However, the structural basis of the heme-Bach2 interaction has not been identified. Spectroscopic analyses revealed that Bach2331-520 is the heme-binding domain, as it includes three Cys-Pro motifs known to be important for heme binding. Heme-titration experiments demonstrated the presence of 5- and 6-coordinated heme-binding modes. Circular dichroism measurements indicated that Bach2331-520 exists mostly in a random-coil conformation. However, dynamic light scattering analyses showed that, upon heme binding to Bach2331-520, this region becomes denatured at a lower temperature, as compared with unbound Bach2331-520. In addition, small-angle X-ray scattering and chemical modification analyses revealed that heme binding induces conformational alterations within the unstructured region. A GAL4-based luciferase assay in 293T cells showed that heme alters the protein interactions mediated by Bach2331-520. These observations suggested that the unstructured region of Bach2 is important for heme binding, and consequently for its functional regulation.
- Chemical modification
- Conformation change
- Heme binding
- Intrinsically disordered protein
- Small-angle X-ray scattering