Substrate promiscuity of inositol 1,4,5-trisphosphate kinase driven by structurally-modified ligands and active site plasticity

Abstract

Abstractd-myo-inositol 1,4,5-trisphosphate (InsP3) is a fundamental second messenger in cellular Ca2+ mobilization. InsP3 3-kinase, a highly specific enzyme binding InsP3 in just one mode, phosphorylates InsP3 specifically at its secondary 3-hydroxyl group to generate a tetrakisphosphate. Using a chemical biology approach with both synthetised and established ligands, combining synthesis, crystallography, computational docking, HPLC and fluorescence polarization binding assays using fluorescently-tagged InsP3, we have surveyed the limits of InsP3 3-kinase ligand specificity and uncovered surprisingly unforeseen biosynthetic capacity. Structurally-modified ligands exploit active site plasticity generating a helix-tilt. These facilitated uncovering of unexpected substrates phosphorylated at a surrogate extended primary hydroxyl at the inositol pseudo 3-position, applicable even to carbohydrate-based substrates. Crystallization experiments designed to allow reactions to proceed in situ facilitated unequivocal characterization of the atypical tetrakisphosphate products. In summary, we define features of InsP3 3-kinase plasticity and substrate tolerance that may be more widely exploitable.

Arg1 from Cryptococcus neoformans lacks PI3 kinase activity and conveys virulence roles via its IP 3-4 kinase activity

“…IP 3-4 Ks are often referred to as inositol polyphosphate multikinases (IPMK) due to their ability to phosphorylate more than one substrate, i.e., IP 3, IP 4 , and IP 5 ( 19 , 27 , 28 ). The promiscuity in substrate binding observed for IP 3-4 kinases has also recently been shown for IP 3 kinase ( 29 ). In the model fungus, S. cerevisiae , IP 3-4 K is referred to as IPMK, ArgRIII, Ipk2, or Arg82 as in this manuscript.…”

Research advances in huntingtin-associated protein 1 and its application prospects in diseases

“…Similarly, HAP1 interacts with GR and stabilizes GR in the cytoplasm (Chen et al, 2020). HAP1 can also interact with inositol (1,4,5)-triphosphate receptor 1 (InsP3R1), affecting the activities of receptors and neurons (Márquez-Moñino et al, 2024). Htt and HAP1 bind to the C-terminus of InsP3R1 to form a ternary complex (Tang et al, 2003).…”