Structure-function studies of HRIKD-{triangleup}KI, a Minimal Kinase Domain of Human Heme-Regulated Inhibitor Kinase

Authors

Rajasekaran, M. B., Booth, J., Crepin, D. F., Roe, S. M., Zhou, L., Gianga, T.-M., Siligardi, G., Gonzalez-Mendez, R., Staikopoulou, M., Hassan, H., Oliver, A., Mancini, E., Spencer, J.

Abstract

EIF2alpha kinase heme-regulated inhibitor (HRI) is a novel target for haematological malignancies with modulators reported to trigger cell death via the HRI-eIF2alpha-ATF4 pathway. We report a protocol for producing the minimal kinase domain of full-length human HRI, termed HRIKD-delta-KI, where the unstructured 140 amino acid (aa) kinase insert (KI) within HRI kinase domain (HRIKD) is replaced with a 2aa glycine/serine (GS) linker. X-ray crystal structures were determined of apo-HRIKD-delta-KI and of its complex with ATP at 2.1 & 2.5 Angstrom resolution respectively. Both structures display a canonical bi-lobal kinase fold. However, they remain in a non-productive state with a displaced C-helix, disassembled R-spine, and a disordered activation segment hindering the substrate site. Biophysical assays (fluorescence based thermal shift & Synchrotron Radiation Circular Dichroism) demonstrate HRIKD-delta-KI retains its functional ligand-binding conformation. All together, these findings define structural and ligand-binding features of HRI to support ongoing drug discovery efforts in blood cancer.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 08 Jul 2026.

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