Heme binds to an intrinsically disordered region of Bach2 and alters its conformation

Abstract

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.