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Heme binds to an intrinsically disordered region of Bach2 and alters its conformation
- Watanabe-Matsui, Miki, Matsumoto, Takashi, Matsui, Toshitaka, Ikeda-Saito, Masao, Muto, Akihiko, Murayama, Kazutaka, Igarashi, Kazuhiko
- Archives of biochemistry and biophysics 2015 v.565 pp. 25-31
- B-lymphocytes, DNA, antibodies, binding properties, cell-mediated immunity, circular dichroism spectroscopy, heme, leucine zipper, light scattering, luciferase, repressor proteins, small-angle X-ray scattering, spectral analysis, temperature
- 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.