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

Márquez-Moñino, María Ángeles, Ortega-García, Raquel, Whitfield, Hayley, Riley, Andrew M., Infantes, Lourdes, Garrett, Shane W., Shipton, Megan L., Brearley, Charles A. ORCID:, Potter, Barry V. L. and González, Beatriz (2024) Substrate promiscuity of inositol 1,4,5-trisphosphate kinase driven by structurally-modified ligands and active site plasticity. Nature Communications, 15. ISSN 2041-1723

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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.

Item Type: Article
Additional Information: Data availability statement: The authors declare that the main data supporting the findings of this study are available within the article and its Supplementary Information files. The atomic coordinates and structure factors of all structures generated in this study have been deposited in the PDB database (Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ ( under accession codes 8PP8, 8PP9, 8PPA, 8PPB, 8PPC, 8PPD, 8PPE, 8PPF, 8PPG, 8PPH, 8PPI and 8PPJ. The atomic coordinates used in this study are available in the PDB database under accession codes 1w2c, 1w2d, 1w2f and 5w2i. Source data are provided with this paper. Funding information: This work has been supported by grants PID2020-117400GB-100 and BFU2017-89913-P from the Spanish Ministerio de Ciencia e Innovación and Ministerio de Economía y Competitividad. B.V.L.P is a Wellcome Trust Senior Investigator (grant 101010). This research was supported by The Wellcome Trust. Rights Retention statement: For the purpose of Open Access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission.
Faculty \ School: Faculty of Science > School of Biological Sciences
UEA Research Groups: Faculty of Science > Research Groups > Plant Sciences
Faculty of Science > Research Groups > Molecular Microbiology
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Depositing User: LivePure Connector
Date Deposited: 04 Mar 2024 18:35
Last Modified: 04 Mar 2024 18:35
DOI: 10.1038/s41467-024-45917-5

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