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Crystallographic and kinetic analyses of human IPMK reveal disordered domains modulate ATP binding and kinase activity

Inositol polyphosphate multikinase (IPMK) is a member of the IPK-superfamily of kinases, catalyzing phosphorylation of several soluble inositols and the signaling phospholipid PI(4,5)P(2) (PIP(2)). IPMK also has critical non-catalytic roles in p53, mTOR/Raptor, TRAF6 and AMPK signaling mediated part...

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Autores principales: Seacrist, Corey D., Blind, Raymond D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6232094/
https://www.ncbi.nlm.nih.gov/pubmed/30420721
http://dx.doi.org/10.1038/s41598-018-34941-3
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author Seacrist, Corey D.
Blind, Raymond D.
author_facet Seacrist, Corey D.
Blind, Raymond D.
author_sort Seacrist, Corey D.
collection PubMed
description Inositol polyphosphate multikinase (IPMK) is a member of the IPK-superfamily of kinases, catalyzing phosphorylation of several soluble inositols and the signaling phospholipid PI(4,5)P(2) (PIP(2)). IPMK also has critical non-catalytic roles in p53, mTOR/Raptor, TRAF6 and AMPK signaling mediated partly by two disordered domains. Although IPMK non-catalytic functions are well established, it is less clear if the disordered domains are important for IPMK kinase activity or ATP binding. Here, kinetic and structural analyses of an engineered human IPMK lacking all disordered domains (ΔIPMK) are presented. Although the K(M) for PIP(2) is identical between ΔIPMK and wild type, ΔIPMK has a 1.8-fold increase in k(cat) for PIP(2), indicating the native IPMK disordered domains decrease IPMK activity in vitro. The 2.5 Å crystal structure of ΔIPMK is reported, confirming the conserved ATP-grasp fold. A comparison with other IPK-superfamily structures revealed a putative “ATP-clamp” in the disordered N-terminus, we predicted would stabilize ATP binding. Consistent with this observation, removal of the ATP clamp sequence increases the K(M) for ATP 4.9-fold, indicating the N-terminus enhances ATP binding to IPMK. Together, these structural and kinetic studies suggest in addition to mediating protein-protein interactions, the disordered domains of IPMK impart modulatory capacity to IPMK kinase activity through multiple kinetic mechanisms.
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spelling pubmed-62320942018-11-28 Crystallographic and kinetic analyses of human IPMK reveal disordered domains modulate ATP binding and kinase activity Seacrist, Corey D. Blind, Raymond D. Sci Rep Article Inositol polyphosphate multikinase (IPMK) is a member of the IPK-superfamily of kinases, catalyzing phosphorylation of several soluble inositols and the signaling phospholipid PI(4,5)P(2) (PIP(2)). IPMK also has critical non-catalytic roles in p53, mTOR/Raptor, TRAF6 and AMPK signaling mediated partly by two disordered domains. Although IPMK non-catalytic functions are well established, it is less clear if the disordered domains are important for IPMK kinase activity or ATP binding. Here, kinetic and structural analyses of an engineered human IPMK lacking all disordered domains (ΔIPMK) are presented. Although the K(M) for PIP(2) is identical between ΔIPMK and wild type, ΔIPMK has a 1.8-fold increase in k(cat) for PIP(2), indicating the native IPMK disordered domains decrease IPMK activity in vitro. The 2.5 Å crystal structure of ΔIPMK is reported, confirming the conserved ATP-grasp fold. A comparison with other IPK-superfamily structures revealed a putative “ATP-clamp” in the disordered N-terminus, we predicted would stabilize ATP binding. Consistent with this observation, removal of the ATP clamp sequence increases the K(M) for ATP 4.9-fold, indicating the N-terminus enhances ATP binding to IPMK. Together, these structural and kinetic studies suggest in addition to mediating protein-protein interactions, the disordered domains of IPMK impart modulatory capacity to IPMK kinase activity through multiple kinetic mechanisms. Nature Publishing Group UK 2018-11-12 /pmc/articles/PMC6232094/ /pubmed/30420721 http://dx.doi.org/10.1038/s41598-018-34941-3 Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Seacrist, Corey D.
Blind, Raymond D.
Crystallographic and kinetic analyses of human IPMK reveal disordered domains modulate ATP binding and kinase activity
title Crystallographic and kinetic analyses of human IPMK reveal disordered domains modulate ATP binding and kinase activity
title_full Crystallographic and kinetic analyses of human IPMK reveal disordered domains modulate ATP binding and kinase activity
title_fullStr Crystallographic and kinetic analyses of human IPMK reveal disordered domains modulate ATP binding and kinase activity
title_full_unstemmed Crystallographic and kinetic analyses of human IPMK reveal disordered domains modulate ATP binding and kinase activity
title_short Crystallographic and kinetic analyses of human IPMK reveal disordered domains modulate ATP binding and kinase activity
title_sort crystallographic and kinetic analyses of human ipmk reveal disordered domains modulate atp binding and kinase activity
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6232094/
https://www.ncbi.nlm.nih.gov/pubmed/30420721
http://dx.doi.org/10.1038/s41598-018-34941-3
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