Structure-function studies of HRI _KD-ΔKI , a Minimal Kinase Domain of Human Heme-Regulated Inhibitor Kinase

EIF2α kinase heme-regulated inhibitor (HRI) is a novel target for haematological malignancies with modulators reported to trigger cell death via the HRI-eIF2α-ATF4 pathway. We report a protocol for producing the minimal kinase domain of full-length human HRI, termed ‘HRI _KD-ΔKI ,’ where the unstructured 140 amino acid (aa) kinase insert (KI) within HRI kinase domain (HRI _KD ) is replaced with a 2aa glycine/serine (GS) linker. X-ray crystal structures were determined of ‘apo’-HRI _KD-ΔKI and of its complex with ATP at 2.1 & 2.5 Å 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 HRI _KD-ΔKI 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.