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bioRxiv · 10.1101/2025.10.07.681048

Regulatory mechanism of heme-regulated inhibitor through autophosphorylation-driven activation and heme-induced deactivation

Oka, T.·Yoshida, H.·Mizutani, K.·Obayashi, E.·Park, S.-Y.·Iwasaki, K.
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Abstract

Heme-regulated inhibitor (HRI) is a key modulator of hemoglobin synthesis, exerting such effects by sensing intracellular heme levels. Under heme-deficient conditions, HRI dissociates from heme, becomes activated, and phosphorylates the translation initiation factor eIF2. However, the precise regulatory mechanisms governing HRI activation remain incompletely understood. In this study, we delineate part of the regulatory mechanism involving autophosphorylation-dependent activation and heme-mediated deactivation of HRI. HRI formed a dimer in solution through its N-terminal domain, irrespective of its phosphorylation state. However, a mutant with deletion of the N-terminal domain retained autophosphorylation activity, indicating that N-terminal domain-mediated dimerization is not essential for activation. Phosphorylated HRI formed a stable complex with eIF2, whereas the dephosphorylated form failed to bind, indicating that autophosphorylation is required for eIF2 recognition. Biochemical analyses, together with modeling based on the predicted structure, revealed that the phosphate groups of Thr488 and Thr493 interact with adjacent basic residues. These interactions enhance eIF2 recognition and phosphorylation. Additionally, heme-induced deactivation selectively targeted the dephosphorylated kinase domain and suppressed autophosphorylation. These findings provide multiple mechanistic insights into regulation of the activity of HRI, highlighting distinct roles for autophosphorylation, structural elements, and heme responsiveness in controlling its function.

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Oka, T., Yoshida, H., Mizutani, K., Obayashi, E., Park, S.-Y., Iwasaki, K.. 2025-10-08. Regulatory mechanism of heme-regulated inhibitor through autophosphorylation-driven activation and heme-induced deactivation. https://doi.org/10.1101/2025.10.07.681048

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