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- PDB-9b9g: Structure of the PI4KA complex bound to Calcineurin -

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Basic information

Entry
Database: PDB / ID: 9b9g
TitleStructure of the PI4KA complex bound to Calcineurin
Components
  • Calcineurin subunit B type 1
  • Hyccin
  • Phosphatidylinositol 4-kinase alpha
  • Protein phosphatase 3 catalytic subunit alpha
  • Tetratricopeptide repeat protein 7B
KeywordsSIGNALING PROTEIN / PI4KIIIa complex / PI4KA / TTC7B / FAM126A / CNA / CNB / Calcineurin
Function / homology
Function and homology information


reorganization of cellular membranes to establish viral sites of replication / Synthesis of PIPs at the ER membrane / negative regulation of angiotensin-activated signaling pathway / calcium-dependent protein serine/threonine phosphatase regulator activity / regulation of cell proliferation involved in kidney morphogenesis / positive regulation of glomerulus development / negative regulation of calcium ion import across plasma membrane / negative regulation of signaling / calcium-dependent protein serine/threonine phosphatase activity / 1-phosphatidylinositol 4-kinase ...reorganization of cellular membranes to establish viral sites of replication / Synthesis of PIPs at the ER membrane / negative regulation of angiotensin-activated signaling pathway / calcium-dependent protein serine/threonine phosphatase regulator activity / regulation of cell proliferation involved in kidney morphogenesis / positive regulation of glomerulus development / negative regulation of calcium ion import across plasma membrane / negative regulation of signaling / calcium-dependent protein serine/threonine phosphatase activity / 1-phosphatidylinositol 4-kinase / 1-phosphatidylinositol 4-kinase activity / positive regulation of saliva secretion / Schwann cell migration / calcineurin complex / positive regulation of connective tissue replacement / positive regulation of calcium ion import across plasma membrane / positive regulation of calcium ion-dependent exocytosis of neurotransmitter / negative regulation of dendrite morphogenesis / positive regulation of cardiac muscle hypertrophy in response to stress / protein serine/threonine phosphatase complex / peptidyl-serine dephosphorylation / renal filtration / lung epithelial cell differentiation / Synthesis of PIPs at the Golgi membrane / calcineurin-NFAT signaling cascade / host-mediated perturbation of viral process / Golgi-associated vesicle membrane / positive regulation of calcineurin-NFAT signaling cascade / skeletal muscle tissue regeneration / myelination in peripheral nervous system / transition between fast and slow fiber / phosphatidylinositol biosynthetic process / positive regulation of osteoclast differentiation / calmodulin-dependent protein phosphatase activity / cardiac muscle hypertrophy in response to stress / regulation of synaptic vesicle cycle / dephosphorylation / extrinsic component of plasma membrane / CLEC7A (Dectin-1) induces NFAT activation / branching involved in blood vessel morphogenesis / dendrite morphogenesis / protein-serine/threonine phosphatase / phosphatidylinositol-mediated signaling / regulation of postsynaptic neurotransmitter receptor internalization / parallel fiber to Purkinje cell synapse / protein serine/threonine phosphatase activity / calcineurin-mediated signaling / phosphatidylinositol phosphate biosynthetic process / positive regulation of activated T cell proliferation / positive regulation of endocytosis / Calcineurin activates NFAT / DARPP-32 events / epithelial to mesenchymal transition / Activation of BAD and translocation to mitochondria / epidermis development / postsynaptic modulation of chemical synaptic transmission / multicellular organismal response to stress / positive regulation of osteoblast differentiation / phosphatase binding / protein dephosphorylation / keratinocyte differentiation / skeletal muscle fiber development / myelination / FCERI mediated Ca+2 mobilization / positive regulation of cell adhesion / T cell activation / hippocampal mossy fiber to CA3 synapse / excitatory postsynaptic potential / protein localization to plasma membrane / wound healing / G1/S transition of mitotic cell cycle / modulation of chemical synaptic transmission / sarcolemma / Schaffer collateral - CA1 synapse / Z disc / response to calcium ion / protein import into nucleus / calcium ion transport / heart development / ATPase binding / actin cytoskeleton organization / Ca2+ pathway / dendritic spine / calmodulin binding / postsynapse / protein dimerization activity / neuron projection / positive regulation of cell migration / cadherin binding / protein domain specific binding / negative regulation of gene expression / focal adhesion / calcium ion binding / positive regulation of gene expression / glutamatergic synapse / enzyme binding / signal transduction / positive regulation of transcription by RNA polymerase II / mitochondrion / extracellular exosome
Similarity search - Function
PI4-kinase, N-terminal / PI4-kinase N-terminal region / Hyccin / Tetratricopeptide repeat protein 7, N-terminal / Hyccin / Tetratricopeptide repeat protein 7 N-terminal / : / PP2B, metallophosphatase domain / PP2B / Anaphase-promoting complex, cyclosome, subunit 3 ...PI4-kinase, N-terminal / PI4-kinase N-terminal region / Hyccin / Tetratricopeptide repeat protein 7, N-terminal / Hyccin / Tetratricopeptide repeat protein 7 N-terminal / : / PP2B, metallophosphatase domain / PP2B / Anaphase-promoting complex, cyclosome, subunit 3 / Tetratricopeptide repeat / Serine/threonine specific protein phosphatases signature. / Protein phosphatase 2A homologues, catalytic domain. / Serine/threonine-specific protein phosphatase/bis(5-nucleosyl)-tetraphosphatase / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase, accessory (PIK) domain superfamily / Phosphoinositide 3-kinase, accessory (PIK) domain / Phosphatidylinositol kinase / PIK helical domain profile. / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase / Metallo-dependent phosphatase-like / Tetratricopeptide repeat / Phosphatidylinositol 3- and 4-kinases signature 1. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / EF-hand domain pair / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair / Tetratricopeptide-like helical domain superfamily / Armadillo-type fold / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Phosphatidylinositol 4-kinase alpha / Calcineurin subunit B type 1 / Protein phosphatase 3 catalytic subunit alpha / Tetratricopeptide repeat protein 7B / Hyccin
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsShaw, A.L. / Suresh, S. / Yip, C.K. / Burke, J.E.
Funding support Canada, 2items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (NSERC, Canada)NSERC-2020-04241 Canada
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2018-03951 Canada
CitationJournal: Structure / Year: 2024
Title: Structure of calcineurin bound to PI4KA reveals dual interface in both PI4KA and FAM126A.
Authors: Alexandria L Shaw / Sushant Suresh / Matthew A H Parson / Noah J Harris / Meredith L Jenkins / Calvin K Yip / John E Burke /
Abstract: Phosphatidylinositol 4-kinase alpha (PI4KA) maintains the phosphatidylinositol 4-phosphate (PI4P) and phosphatidylserine pools of the plasma membrane. A key regulator of PI4KA is its association into ...Phosphatidylinositol 4-kinase alpha (PI4KA) maintains the phosphatidylinositol 4-phosphate (PI4P) and phosphatidylserine pools of the plasma membrane. A key regulator of PI4KA is its association into a complex with TTC7 and FAM126 proteins. This complex can be regulated by the CNAβ1 isoform of the phosphatase calcineurin. We previously identified that CNAβ1 directly binds to FAM126A. Here, we report a cryoelectron microscopic (cryo-EM) structure of a truncated PI4KA complex bound to calcineurin, revealing a unique direct interaction between PI4KA and calcineurin. Hydrogen deuterium exchange mass spectrometry (HDX-MS) and computational analysis show that calcineurin forms a complex with an evolutionarily conserved IKISVT sequence in PI4KA's horn domain. We also characterized conserved LTLT and PSISIT calcineurin binding sequences in the C terminus of FAM126A. These dual sites in PI4KA and FAM126A are both in close proximity to phosphorylation sites in the PI4KA complex, suggesting key roles of calcineurin-regulated phosphosites in PI4KA regulation. This work reveals novel insight into how calcineurin can regulate PI4KA activity.
History
DepositionApr 2, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 11, 2024Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Sep 11, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Nov 27, 2024Group: Data collection / Database references / Structure summary
Category: citation / em_admin / pdbx_entry_details
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.2May 28, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 28, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Phosphatidylinositol 4-kinase alpha
B: Phosphatidylinositol 4-kinase alpha
D: Tetratricopeptide repeat protein 7B
E: Hyccin
F: Tetratricopeptide repeat protein 7B
G: Hyccin
H: Calcineurin subunit B type 1
I: Protein phosphatase 3 catalytic subunit alpha
J: Calcineurin subunit B type 1
K: Protein phosphatase 3 catalytic subunit alpha
hetero molecules


Theoretical massNumber of molelcules
Total (without water)912,80318
Polymers912,48310
Non-polymers3218
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Eluted with a volume consistent with a dimer of heteropentamers
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 5 types, 10 molecules ABDFEGHJIK

#1: Protein Phosphatidylinositol 4-kinase alpha / PI4-kinase alpha / PI4K-alpha / PtdIns-4-kinase alpha / Phosphatidylinositol 4-Kinase III alpha


Mass: 237102.281 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PI4KA, PIK4, PIK4CA / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P42356, 1-phosphatidylinositol 4-kinase
#2: Protein Tetratricopeptide repeat protein 7B / TPR repeat protein 7B / Tetratricopeptide repeat protein 7-like-1 / TPR repeat protein 7-like-1


Mass: 94294.109 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TTC7B, TTC7L1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q86TV6
#3: Protein Hyccin / Down-regulated by CTNNB1 protein A


Mass: 34638.867 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Truncated construct (1-308) / Source: (gene. exp.) Homo sapiens (human) / Gene: HYCC1, DRCTNNB1A, FAM126A / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9BYI3
#4: Protein Calcineurin subunit B type 1 / Protein phosphatase 2B regulatory subunit 1 / Protein phosphatase 3 regulatory subunit B alpha isoform 1


Mass: 19322.904 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PPP3R1, CNA2, CNB / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41 / References: UniProt: P63098
#5: Protein Protein phosphatase 3 catalytic subunit alpha / CAM-PRP catalytic subunit / Calcineurin A alpha / Calmodulin-dependent calcineurin A subunit alpha ...CAM-PRP catalytic subunit / Calcineurin A alpha / Calmodulin-dependent calcineurin A subunit alpha isoform / CNA alpha / Serine/threonine-protein phosphatase 2B catalytic subunit alpha isoform


Mass: 70883.242 Da / Num. of mol.: 2 / Mutation: L236P,D238N
Source method: isolated from a genetically manipulated source
Details: Truncated Calcineurin A alpha (2-391) L236P D238N,Truncated Calcineurin A alpha (2-391) L236P D238N
Source: (gene. exp.) Homo sapiens (human) / Gene: PPP3CA, CALNA, CNA / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41
References: UniProt: Q08209, protein-serine/threonine phosphatase

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Non-polymers , 1 types, 8 molecules

#6: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca

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Details

Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: PI4KA complex bound to Calcineurin / Type: COMPLEX / Details: Stabilized with BS3 crosslinker / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7
Details: Freshly prepared gel filtration buffer, filtered through 0.22um filter and degassed
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMImidazoleC3H4N21
2150 mMSodium ChlorideNaCl1
35 %GlycerolC3H8O31
40.5 mMTris(2-carboxyethyl)phosphineTCEP1
SpecimenConc.: 0.77 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was treated with BS3 crosslinker then gel filtered to isolate protein peak consistent with a dimer of pentamers.
Specimen supportDetails: Glow discharged using the Pelco EasiGlow. 15mA Current.
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: Blot force -5, blot time 1 s

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10121
EM imaging opticsEnergyfilter name: TFS Selectris

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv.4.2.1particle selection
2SerialEMimage acquisition
4cryoSPARCv4.2.1CTF correction
9PHENIXmodel refinement
10cryoSPARCv4.2.1initial Euler assignment
11cryoSPARCv4.2.1final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1181312
Details: Particles were picked using the cryoSPARC template picker
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 235760 / Symmetry type: POINT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16BQ1

6bq1

16BQ11PDBexperimental model
26NUC

6nuc

16NUC2PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00349716
ELECTRON MICROSCOPYf_angle_d0.72267262
ELECTRON MICROSCOPYf_dihedral_angle_d7.9766662
ELECTRON MICROSCOPYf_chiral_restr0.0427575
ELECTRON MICROSCOPYf_plane_restr0.0048519

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