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

María Ángeles Márquez-Moñino, Raquel Ortega-García, Hayley Whitfield, Andrew M. Riley, Lourdes Infantes, Shane W. Garrett, Megan L. Shipton, Charles A. Brearley, Barry V. L. Potter () and Beatriz González ()
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María Ángeles Márquez-Moñino: Institute of Physical-Chemistry Blas Cabrera, CSIC
Raquel Ortega-García: Institute of Physical-Chemistry Blas Cabrera, CSIC
Hayley Whitfield: University of East Anglia, Norwich Research Park
Andrew M. Riley: University of Oxford, Mansfield Road
Lourdes Infantes: Institute of Physical-Chemistry Blas Cabrera, CSIC
Shane W. Garrett: University of Bath
Megan L. Shipton: University of Oxford, Mansfield Road
Charles A. Brearley: University of East Anglia, Norwich Research Park
Barry V. L. Potter: University of Oxford, Mansfield Road
Beatriz González: Institute of Physical-Chemistry Blas Cabrera, CSIC

Nature Communications, 2024, vol. 15, issue 1, 1-16

Abstract: Abstract d-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.

Date: 2024
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DOI: 10.1038/s41467-024-45917-5

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