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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 / regulation of cell proliferation involved in kidney morphogenesis / positive regulation of cardiac muscle hypertrophy in response to stress / calcium-dependent protein serine/threonine phosphatase regulator activity / positive regulation of glomerulus development / negative regulation of calcium ion import across plasma membrane / calcium-dependent protein serine/threonine phosphatase activity / negative regulation of signaling ...reorganization of cellular membranes to establish viral sites of replication / Synthesis of PIPs at the ER membrane / negative regulation of angiotensin-activated signaling pathway / regulation of cell proliferation involved in kidney morphogenesis / positive regulation of cardiac muscle hypertrophy in response to stress / calcium-dependent protein serine/threonine phosphatase regulator activity / positive regulation of glomerulus development / negative regulation of calcium ion import across plasma membrane / calcium-dependent protein serine/threonine phosphatase activity / negative regulation of signaling / positive regulation of saliva secretion / 1-phosphatidylinositol 4-kinase / 1-phosphatidylinositol 4-kinase activity / calmodulin-dependent protein phosphatase activity / positive regulation of calcium ion import across plasma membrane / calcineurin complex / positive regulation of connective tissue replacement / calcineurin-mediated signaling / negative regulation of dendrite morphogenesis / slit diaphragm / protein serine/threonine phosphatase complex / peptidyl-serine dephosphorylation / modulation by host of viral process / Synthesis of PIPs at the Golgi membrane / renal filtration / transition between fast and slow fiber / lung epithelial cell differentiation / calcineurin-NFAT signaling cascade / skeletal muscle tissue regeneration / regulation of synaptic vesicle cycle / Golgi-associated vesicle membrane / positive regulation of calcineurin-NFAT signaling cascade / myelination in peripheral nervous system / phosphatidylinositol biosynthetic process / cardiac muscle hypertrophy in response to stress / positive regulation of osteoclast differentiation / regulation of postsynaptic neurotransmitter receptor internalization / parallel fiber to Purkinje cell synapse / dendrite morphogenesis / CLEC7A (Dectin-1) induces NFAT activation / myosin phosphatase activity / extrinsic component of plasma membrane / protein serine/threonine phosphatase activity / postsynaptic modulation of chemical synaptic transmission / cyclosporin A binding / branching involved in blood vessel morphogenesis / protein-serine/threonine phosphatase / dephosphorylation / positive regulation of activated T cell proliferation / phosphatidylinositol-mediated signaling / positive regulation of endocytosis / phosphatidylinositol phosphate biosynthetic process / Calcineurin activates NFAT / DARPP-32 events / epidermis development / Activation of BAD and translocation to mitochondria / epithelial to mesenchymal transition / phosphatase binding / negative regulation of insulin secretion / positive regulation of osteoblast differentiation / multicellular organismal response to stress / skeletal muscle fiber development / keratinocyte differentiation / phosphorylation / myelination / response to amphetamine / protein dephosphorylation / hippocampal mossy fiber to CA3 synapse / positive regulation of cell adhesion / FCERI mediated Ca+2 mobilization / T cell activation / excitatory postsynaptic potential / protein localization to plasma membrane / cellular response to glucose stimulus / wound healing / modulation of chemical synaptic transmission / Schaffer collateral - CA1 synapse / sarcolemma / Z disc / response to calcium ion / protein import into nucleus / G1/S transition of mitotic cell cycle / calcium ion transport / kinase activity / heart development / Ca2+ pathway / ATPase binding / postsynapse / dendritic spine / protein dimerization activity / calmodulin binding / positive regulation of cell migration / neuron projection / cadherin binding / protein domain specific binding / negative regulation of gene expression / focal adhesion / glutamatergic synapse / calcium ion binding / protein-containing complex binding
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 / Calcineurin B protein / PP2B, metallophosphatase domain / PP2B ...PI4-kinase, N-terminal / PI4-kinase N-terminal region / Hyccin / Tetratricopeptide repeat protein 7, N-terminal / : / Hyccin / Tetratricopeptide repeat protein 7 N-terminal / Calcineurin B protein / PP2B, metallophosphatase domain / PP2B / Anaphase-promoting complex, cyclosome, subunit 3 / Serine/threonine specific protein phosphatases signature. / Protein phosphatase 2A homologues, catalytic domain. / Tetratricopeptide repeat / Serine/threonine-specific protein phosphatase/bis(5-nucleosyl)-tetraphosphatase / Phosphoinositide 3-kinase, accessory (PIK) domain superfamily / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / 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-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / 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

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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

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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
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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