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

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.

doi:10.64898/2026.07.06.735516

bioRxiv · 10.64898/2026.07.06.735516

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

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.

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Abstract

EIF2 kinase heme-regulated inhibitor (HRI) is a novel target for haematological malignancies with modulators reported to trigger cell death via the HRI-eIF2-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 [A] 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.

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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.. 2026-07-07. Structure-function studies of HRIKD-{triangleup}KI, a Minimal Kinase Domain of Human Heme-Regulated Inhibitor Kinase. https://doi.org/10.64898/2026.07.06.735516

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