Gianga, T.-M. | Search bioRxiv

bioRxiv2026

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

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.

biochemistry↗

bioRxiv2026

Protein disorder controls allostery in DNA

Metabolism, gene expression, and signaling all require the adaptation of protein activity to the mixture of reactants and products in a cell. This trait to adapt, called allostery, is hardwired in the structure of proteins. Binding a ligand at one location in a protein can change distant locations, thus tuning protein activity. How allostery works has been subject of intense research since its discovery sixty years ago. The challenge is to understand the order of events that follow ligand-binding in the three-dimensional architecture of proteins. Here we simplify this task by studying allostery in DNA, a nearly one-dimensional system. DNA can transmit allosteric signals over many nanometers to generate cooperativity in the binding of transcription factors, an archetype of the long-range action of allostery. We found that binding of the transcription factor ComK amplifies intrinsic microsecond structural fluctuations in DNA many nanometers distant from the binding site. Yet, it is not protein binding per se, but the intrinsically disordered region (IDR) of the protein that amplifies these fluctuations. IDR removal does not only rigidify DNA, but it also abolishes allostery. The result is a structurally distorted protein-DNA complex that lost its function. These findings have important implications for our understanding of transcription activation and suggest a new functional role for IDRs in transcription factors.

biophysics↗