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

• Published in: Nature communications Vol. 15; no. 1; p. 1502
• Main Authors: Márquez-Moñino, María Ángeles, Ortega-García, Raquel, Whitfield, Hayley, Riley, Andrew M., Infantes, Lourdes, Garrett, Shane W., Shipton, Megan L., Brearley, Charles A., Potter, Barry V. L., González, Beatriz
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 19-02-2024; Nature Publishing Group; Nature Portfolio
• Subjects: 631/45/535; 631/535/1266; 631/92/275; 631/92/507; 631/92/613; 82/16; 82/83; Binding; Calcium - metabolism; Calcium ions; Calcium mobilization; Carbohydrates; Catalytic Domain; Crystallization; Crystallography; Fluorescence polarization; Humanities and Social Sciences; Hydroxyl groups; Inositol 1,4,5-Trisphosphate - metabolism; Inositol 1,4,5-Trisphosphate Receptors - metabolism; Inositol Phosphates - metabolism; Inositol trisphosphate; Inositols; Kinases; Ligands; Liquid chromatography; multidisciplinary; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Plastic properties; Plasticity; Science; Science (multidisciplinary); Substrates
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-45917-5

d
-
myo
-inositol 1,4,5-trisphosphate (InsP
3
) is a fundamental second messenger in cellular Ca
2+
mobilization. InsP
3
3-kinase, a highly specific enzyme binding InsP
3
in just one mode, phosphorylates InsP
3
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 InsP
3
, we have surveyed the limits of InsP
3
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 InsP
3
3-kinase plasticity and substrate tolerance that may be more widely exploitable.
InsP
3
3-kinase phosphorylates 1,4,5-trisphosphate (InsP
3
) specifically at its secondary 3-hydroxyl group to generate a tetrakisphosphate. Here, the authors used a combination of methods to survey InsP
3
3-kinase ligand specificity and determined that IP3K specificity surpasses that of its natural substrate, allowing it to bind diverse ligands with a primary hydroxyl in the reactive position and based on a carbohydrate moiety.