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Cited by 23Protein Kinase Regulation and GTPase SignalingProtein Structure and DynamicsEnzyme Structure and Function

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

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Seacrist, Corey D., Blind, Raymond D.·2018·Scientific Reports
DOI 10.1038/s41598-018-34941-3PMC PMC6232094

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Abstract

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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  • The protein constructs in these crystal structures deviate from wild-type human IPMK sequence as they exclude the NLS region, replaced by a short artificial linker.

    “The protein constructs in these crystal structures deviate from the wild-type human IPMK sequence as they exclude the region encompassing the NLS region (residues 263–377 in pdb id 5w2g, 279–373 in pdb id 6e7f), which is replaced by a short artificial linker, to aid the crystallization of this protein.”

    From From Obscurity to Prominence: IPMK’s Expanding Role in Cellular Signaling, Physiology, and Disease (2025) · cites this paper as [41]

    The Results state that the internal loop contains a nuclear localization signal and that the 6E7F construct replaces residues 279–373 with a short artificial linker. The Methods say the linker was required for crystallization. The Discussion mentions the similar but non-identical 5W2G construct, but our copy does not establish the claimed 263–377 boundaries or linker replacement for that structure. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • The N-lobe and C-lobe interface form a groove-like cavity that harbors the catalytic site, facilitating transfer of phosphates from ATP to inositol head groups of PIP2 and InsP3, InsP4.

    “The N-lobe and the C-lobe interface form a groove-like cavity that harbors the catalytic site, which facilitates the transfer of phosphates from ATP to inositol head groups of PIP2 and InsP3, InsP4.”

    From From Obscurity to Prominence: IPMK’s Expanding Role in Cellular Signaling, Physiology, and Disease (2025) · cites this paper as [41]

    The Results section (‘Crystallography shows ΔIPMK maintains the ATP-grasp fold’; page unavailable) describes N- and C-terminal domains, and the Methods (‘IPMK Kinase Assays’; page unavailable) document transfer of radiolabeled phosphate from ATP to PIP2. Our copy does not establish that their interface forms the claimed groove-like catalytic cavity or specifically identify InsP3 and InsP4 as substrates in that cavity. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

  • The N-lobe ATP grasp fold, along with the hinge, guides ATP to transfer its γ phosphate to InsP3 to yield InsP4, which is further phosphorylated to InsP5.

    “The N-lobe ATP grasp fold, along with the hinge, guides the ATP to transfer its γ phosphate to the InsP3 (1, 4, 5) cradled in the cavity created by the IP helices to yield InsP4 (1,3,4,5; 3-kinase activity), which is further phosphorylated to InsP5 (1,3,4,5, 6; 6-kinase activity).”

    From From Obscurity to Prominence: IPMK’s Expanding Role in Cellular Signaling, Physiology, and Disease (2025) · cites this paper as [41]

    The Results section, “Crystallography shows ΔIPMK maintains the ATP-grasp fold” (page unavailable), identifies an N-terminal ATP-grasp domain and an inositol-phosphate-binding helix. The Discussion describes a proposed ATP clamp, and the Methods describe γ-phosphate transfer in a PIP2 kinase assay. Our copy does not establish the hinge-guided mechanism or the specified InsP3 → InsP4 → InsP5 sequence and phosphorylation positions. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

  • The first β-strand of IPMK is not always visible in all crystal structures owing to the disordered nature of the first roughly 60 amino acids.

    “The first β-strand (β0 in Figure 2B) is not always visible in all crystal structures owing to the disordered nature of the first roughly 60 amino acids [41,42], which has implications for interaction with other proteins (Table 1) as well as catalytic activity.”

    From From Obscurity to Prominence: IPMK’s Expanding Role in Cellular Signaling, Physiology, and Disease (2025) · cites this paper as [41]

    The Introduction says some IPMK regions are not resolved in all crystal structures, and the Discussion says the N-terminal I65–P69 sequence is disordered in most structures. The paper does not identify a β0 strand as the missing feature or establish that disorder of the first roughly 60 amino acids causes its absence. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • Crystal structures of human IPMK have been deposited in the PDB in free form, bound to substrates (ADP, InsP3), or inhibitors.

    “Currently, different groups have deposited crystal structures of human IPMK in the PDB database in its free form (pdb id: 5w2g, 6e7f) [41,42] bound to substrates (ADP, InsP3) [42] or inhibitors [43].”

    From From Obscurity to Prominence: IPMK’s Expanding Role in Cellular Signaling, Physiology, and Disease (2025) · cites this paper as [41]

    The Methods and Results identify this paper’s human IPMK structure as the apo (free) form deposited as PDB 6E7F. The Results also discuss a previously reported human IPMK structure bound to ADP, but the InsP3-bound structures described are of other kinases, not human IPMK. The inhibitors clause is cited to a different source and is not judged here. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

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Cited by

Sorted by citations

Authors
Seacrist, C. D., & Blind, R. D.
Affiliations
Vanderbilt University (US)
Corresponding author
Blind, Raymond D.
ORCID
Blind, Raymond D.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2018-11-12
Volume
8
Issue
1
Pages
16672-16672
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-018-34941-3
PMC
PMC6232094
OpenAlex
W2950060185
Cited by
23
References
0
Citations by year
2026: 2 · 2025: 7 · 2024: 3 · 2023: 4 · 2022: 1 · 2021: 3 · 2019: 3
FWCI
0.53
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • National Cancer Institute (K01CA172957)
  • National Institute of Diabetes and Digestive and Kidney Diseases (T32DK007563, P30DK020593)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
Keywords
inositol polyphosphate multikinase, intrinsically disordered regions, kinase regulation, ATP binding, enzyme kinetics, crystal structure
MeSH terms
Adenosine Triphosphate, Crystallography, Humans, Inositol Phosphates, Kinetics, Phosphorylation, Protein Binding, Signal Transduction, Protein Structure, Secondary, Phosphotransferases (Alcohol Group Acceptor)

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