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

Archives of biochemistry and biophysics (2014-12-03)
Miki Watanabe-Matsui, Takashi Matsumoto, Toshitaka Matsui, Masao Ikeda-Saito, Akihiko Muto, Kazutaka Murayama, Kazuhiko Igarashi
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 Bach2(331-520) 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 Bach2(331-520) exists mostly in a random-coil conformation. However, dynamic light scattering analyses showed that, upon heme binding to Bach2(331-520), this region becomes denatured at a lower temperature, as compared with unbound Bach2(331-520). 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 Bach2(331-520). These observations suggested that the unstructured region of Bach2 is important for heme binding, and consequently for its functional regulation.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
L-Glutathione reduced, ≥98.0%
Sigma-Aldrich
Hemin, from bovine, ≥90%
Sigma-Aldrich
Hemin, BioXtra, from Porcine, ≥96.0% (HPLC)
Sigma-Aldrich
L-Glutathione reduced, suitable for cell culture, BioReagent, ≥98.0%, powder
Supelco
Glutathione, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
L-Glutathione reduced, BioXtra, ≥98.0%
Glutathione, European Pharmacopoeia (EP) Reference Standard