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

Maria Angeles Marquez-Monino; Raquel Ortega-Garcia; Hayley  Whitfield; Andrew M. Riley; Lourdes  Infantes; Shane W. Garrett; Megan L.  Shipton; Charles A. Brearley; Barry V. L. Potter; Beatriz Gonzalez

doi:10.1038/s41467-024-45917-5

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

Maria Angeles Marquez-Monino, Raquel Ortega-Garcia, Hayley Whitfield, Andrew M. Riley, Lourdes Infantes, Shane W. Garrett, Megan L. Shipton, Charles A. Brearley, Barry V. L. Potter, Beatriz Gonzalez

Department of Life Sciences

Research output: Contribution to journal › Article › peer-review

3 Citations (SciVal)

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.

Original language	English
Article number	1502
Journal	Nature Communications
Volume	15
Early online date	19 Feb 2024
DOIs	https://doi.org/10.1038/s41467-024-45917-5
Publication status	Published - 19 Feb 2024

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 (www.rcsb.org/)) 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

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.

Funders	Funder number
Spanish Ministerio de Ciencia e Innovación	PID2020-117400GB-100
Ministerio de Economía y Empresa	BFU2017-89913-P
The Wellcome Trust	101010

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10.1038/s41467-024-45917-5Licence: CC BY

Chemical Biology of Cellular Signalling using Polyphosphate Messengers
Potter, B. (PI)
The Wellcome Trust
1/01/14 → 31/12/18
Project: UK charity

Cite this

Marquez-Monino, M. A., Ortega-Garcia, R., Whitfield, H., Riley, A. M., Infantes, L., Garrett, S. W., Shipton, M. L., Brearley, C. A., Potter, B. V. L., & Gonzalez, B. (2024). Substrate promiscuity of inositol 1,4,5- trisphosphate kinase driven by structurallymodified ligands and active site plasticity. Nature Communications, 15, Article 1502. https://doi.org/10.1038/s41467-024-45917-5

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title = "Substrate promiscuity of inositol 1,4,5- trisphosphate kinase driven by structurallymodified ligands and active site plasticity",

abstract = "D-myo-inositol 1,4,5-trisphosphate (InsP3) is a fundamental second messengerin cellular Ca2+ mobilization. InsP3 3-kinase, a highly specific enzyme bindingInsP3 in just one mode, phosphorylates InsP3 specifically at its secondary3-hydroxyl group to generate a tetrakisphosphate. Using a chemical biologyapproach with both synthetised and established ligands, combining synthesis,crystallography, computational docking, HPLC and fluorescence polarizationbinding assays using fluorescently-tagged InsP3, we have surveyed the limits ofInsP3 3-kinase ligand specificity and uncovered surprisingly unforeseen biosynthetic capacity. Structurally-modified ligands exploit active site plasticitygenerating a helix-tilt. These facilitated uncovering of unexpected substratesphosphorylated at a surrogate extended primary hydroxyl at the inositolpseudo 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.",

author = "Marquez-Monino, {Maria Angeles} and Raquel Ortega-Garcia and Hayley Whitfield and Riley, {Andrew M.} and Lourdes Infantes and Garrett, {Shane W.} and Shipton, {Megan L.} and Brearley, {Charles A.} and Potter, {Barry V. L.} and Beatriz Gonzalez",

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T1 - Substrate promiscuity of inositol 1,4,5- trisphosphate kinase driven by structurallymodified ligands and active site plasticity

AU - Marquez-Monino, Maria Angeles

AU - Ortega-Garcia, Raquel

AU - Whitfield, Hayley

AU - Riley, Andrew M.

AU - Infantes, Lourdes

AU - Garrett, Shane W.

AU - Shipton, Megan L.

AU - Brearley, Charles A.

AU - Potter, Barry V. L.

AU - Gonzalez, Beatriz

PY - 2024/2/19

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N2 - D-myo-inositol 1,4,5-trisphosphate (InsP3) is a fundamental second messengerin cellular Ca2+ mobilization. InsP3 3-kinase, a highly specific enzyme bindingInsP3 in just one mode, phosphorylates InsP3 specifically at its secondary3-hydroxyl group to generate a tetrakisphosphate. Using a chemical biologyapproach with both synthetised and established ligands, combining synthesis,crystallography, computational docking, HPLC and fluorescence polarizationbinding assays using fluorescently-tagged InsP3, we have surveyed the limits ofInsP3 3-kinase ligand specificity and uncovered surprisingly unforeseen biosynthetic capacity. Structurally-modified ligands exploit active site plasticitygenerating a helix-tilt. These facilitated uncovering of unexpected substratesphosphorylated at a surrogate extended primary hydroxyl at the inositolpseudo 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.

AB - D-myo-inositol 1,4,5-trisphosphate (InsP3) is a fundamental second messengerin cellular Ca2+ mobilization. InsP3 3-kinase, a highly specific enzyme bindingInsP3 in just one mode, phosphorylates InsP3 specifically at its secondary3-hydroxyl group to generate a tetrakisphosphate. Using a chemical biologyapproach with both synthetised and established ligands, combining synthesis,crystallography, computational docking, HPLC and fluorescence polarizationbinding assays using fluorescently-tagged InsP3, we have surveyed the limits ofInsP3 3-kinase ligand specificity and uncovered surprisingly unforeseen biosynthetic capacity. Structurally-modified ligands exploit active site plasticitygenerating a helix-tilt. These facilitated uncovering of unexpected substratesphosphorylated at a surrogate extended primary hydroxyl at the inositolpseudo 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.

U2 - 10.1038/s41467-024-45917-5

DO - 10.1038/s41467-024-45917-5

M3 - Article

SN - 2041-1723

VL - 15

JO - Nature Communications

JF - Nature Communications

M1 - 1502

ER -

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

Abstract

Data Availability Statement

Funding

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Chemical Biology of Cellular Signalling using Polyphosphate Messengers

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