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- EMDB-23808: Structure of the phosphoinositide 3-kinase p110 gamma (PIK3CG) p1... -

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Basic information

Entry
Database: EMDB / ID: EMD-23808
TitleStructure of the phosphoinositide 3-kinase p110 gamma (PIK3CG) p101 (PIK3R5) complex
Map data
Sample
  • Complex: Ternary complex of p110 gamma with p101 and a p101 binding nanobody
    • Protein or peptide: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform
    • Protein or peptide: Phosphoinositide 3-kinase regulatory subunit 5
KeywordsPI3K / p110 / PIK3CG / PIK3R5 / p101 / phosphoinositide 3-kinase / PIP3 / IMMUNE SYSTEM / TRANSFERASE
Function / homology
Function and homology information


: / secretory granule localization / negative regulation of triglyceride catabolic process / natural killer cell chemotaxis / neutrophil extravasation / phosphatidylinositol-4-phosphate 3-kinase / positive regulation of acute inflammatory response / respiratory burst involved in defense response / negative regulation of cardiac muscle contraction / regulation of calcium ion transmembrane transport ...: / secretory granule localization / negative regulation of triglyceride catabolic process / natural killer cell chemotaxis / neutrophil extravasation / phosphatidylinositol-4-phosphate 3-kinase / positive regulation of acute inflammatory response / respiratory burst involved in defense response / negative regulation of cardiac muscle contraction / regulation of calcium ion transmembrane transport / 1-phosphatidylinositol-3-kinase regulator activity / T cell chemotaxis / negative regulation of fibroblast apoptotic process / phosphatidylinositol 3-kinase complex, class IB / sphingosine-1-phosphate receptor signaling pathway / phosphatidylinositol 3-kinase complex, class IA / phosphatidylinositol 3-kinase complex / dendritic cell chemotaxis / 1-phosphatidylinositol-4-phosphate 3-kinase activity / 1-phosphatidylinositol-4,5-bisphosphate 3-kinase activity / phosphatidylinositol-4,5-bisphosphate 3-kinase / phosphatidylinositol 3-kinase / phosphatidylinositol-3-phosphate biosynthetic process / 1-phosphatidylinositol-3-kinase activity / mast cell degranulation / Erythropoietin activates Phosphoinositide-3-kinase (PI3K) / hepatocyte apoptotic process / positive regulation of Rac protein signal transduction / regulation of cell adhesion mediated by integrin / Synthesis of PIPs at the plasma membrane / phosphatidylinositol-mediated signaling / phosphatidylinositol phosphate biosynthetic process / regulation of angiogenesis / T cell proliferation / cellular response to cAMP / GPVI-mediated activation cascade / T cell activation / ephrin receptor binding / positive regulation of endothelial cell migration / phosphatidylinositol 3-kinase/protein kinase B signal transduction / neutrophil chemotaxis / positive regulation of cytokine production / positive regulation of MAP kinase activity / G-protein beta/gamma-subunit complex binding / platelet aggregation / endocytosis / G beta:gamma signalling through PI3Kgamma / Signaling by CSF1 (M-CSF) in myeloid cells / kinase activity / positive regulation of cytosolic calcium ion concentration / angiogenesis / adaptive immune response / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / non-specific serine/threonine protein kinase / protein kinase activity / inflammatory response / immune response / G protein-coupled receptor signaling pathway / phosphorylation / innate immune response / protein serine kinase activity / protein serine/threonine kinase activity / ATP binding / identical protein binding / membrane / nucleus / plasma membrane / cytoplasm / cytosol
Similarity search - Function
Phosphoinositide 3-kinase regulatory subunit 5/6 / Phosphoinositide 3-kinase gamma adapter protein p101 subunit / PIK3 catalytic subunit gamma, adaptor-binding domain / PIK3 catalytic subunit gamma adaptor-binding domain / Phosphatidylinositol 3-kinase, adaptor-binding domain / Phosphatidylinositol 3-kinase adaptor-binding (PI3K ABD) domain profile. / PI3-kinase family, Ras-binding domain / Phosphatidylinositol 3-kinase Ras-binding (PI3K RBD) domain / PI3-kinase family, ras-binding domain / Phosphatidylinositol 3-kinase Ras-binding (PI3K RBD) domain profile. ...Phosphoinositide 3-kinase regulatory subunit 5/6 / Phosphoinositide 3-kinase gamma adapter protein p101 subunit / PIK3 catalytic subunit gamma, adaptor-binding domain / PIK3 catalytic subunit gamma adaptor-binding domain / Phosphatidylinositol 3-kinase, adaptor-binding domain / Phosphatidylinositol 3-kinase adaptor-binding (PI3K ABD) domain profile. / PI3-kinase family, Ras-binding domain / Phosphatidylinositol 3-kinase Ras-binding (PI3K RBD) domain / PI3-kinase family, ras-binding domain / Phosphatidylinositol 3-kinase Ras-binding (PI3K RBD) domain profile. / Phosphoinositide 3-kinase C2 / Phosphoinositide 3-kinase, region postulated to contain C2 domain / C2 phosphatidylinositol 3-kinase-type domain / C2 phosphatidylinositol 3-kinase (PI3K)-type domain profile. / 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. / 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 / C2 domain superfamily / Armadillo-type fold / Ubiquitin-like domain superfamily / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Phosphoinositide 3-kinase regulatory subunit 5 / Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform
Similarity search - Component
Biological speciesHomo sapiens (human) / Sus scrofa (pig)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsBurke JE / Dalwadi U / Rathinaswamy MK / Yip CK
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)168998 Canada
CitationJournal: Structure / Year: 2021
Title: HDX-MS-optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases.
Authors: Manoj K Rathinaswamy / Kaelin D Fleming / Udit Dalwadi / Els Pardon / Noah J Harris / Calvin K Yip / Jan Steyaert / John E Burke /
Abstract: There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) ...There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism, and immunity. The class IB PI3K, PI3Kγ, is a heterodimeric complex composed of a catalytic p110γ subunit bound to a p101 or p84 regulatory subunit. PI3Kγ is a critical component in multiple immune signaling processes and is dependent on activation by Ras and G protein-coupled receptors (GPCRs) to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3Kγ binding single-chain camelid nanobodies using hydrogen-deuterium exchange (HDX) mass spectrometry (MS) for structural and biochemical studies. We identify nanobodies that stimulated lipid kinase activity, block Ras activation, and specifically inhibited p101-mediated GPCR activation. Overall, our work reveals insight into PI3Kγ regulation and identifies sites that may be exploited for therapeutic development.
History
DepositionApr 8, 2021-
Header (metadata) releaseJul 14, 2021-
Map releaseJul 14, 2021-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7mez
  • Surface level: 1.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23808.png

Downloads & links

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Map

FileDownload / File: emd_23808.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.059 Å
Density
Contour LevelBy AUTHOR: 1.2 / Movie #1: 1.2
Minimum - Maximum-3.5281403 - 7.714876
Average (Standard dev.)-0.002197762 (±0.1345252)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 317.7 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0591.0591.059
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z317.700317.700317.700
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-3.5287.715-0.002

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Supplemental data

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Sample components

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Entire : Ternary complex of p110 gamma with p101 and a p101 binding nanobody

EntireName: Ternary complex of p110 gamma with p101 and a p101 binding nanobody
Components
  • Complex: Ternary complex of p110 gamma with p101 and a p101 binding nanobody
    • Protein or peptide: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform
    • Protein or peptide: Phosphoinositide 3-kinase regulatory subunit 5

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Supramolecule #1: Ternary complex of p110 gamma with p101 and a p101 binding nanobody

SupramoleculeName: Ternary complex of p110 gamma with p101 and a p101 binding nanobody
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Nanobody binds to GBD domain, but was too fleixble to be built in the atomic model
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit ...

MacromoleculeName: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: phosphatidylinositol 3-kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 126.627406 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MELENYKQPV VLREDNCRRR RRMKPRSAAA SLSSMELIPI EFVLPTSQRK CKSPETALLH VAGHGNVEQM KAQVWLRALE TSVAADFYH RLGPHHFLLL YQKKGQWYEI YDKYQVVQTL DCLRYWKATH RSPGQIHLVQ RHPPSEESQA FQRQLTALIG Y DVTDVSNV ...String:
MELENYKQPV VLREDNCRRR RRMKPRSAAA SLSSMELIPI EFVLPTSQRK CKSPETALLH VAGHGNVEQM KAQVWLRALE TSVAADFYH RLGPHHFLLL YQKKGQWYEI YDKYQVVQTL DCLRYWKATH RSPGQIHLVQ RHPPSEESQA FQRQLTALIG Y DVTDVSNV HDDELEFTRR GLVTPRMAEV ASRDPKLYAM HPWVTSKPLP EYLWKKIANN CIFIVIHRST TSQTIKVSPD DT PGAILQS FFTKMAKKKS LMDIPESQSE QDFVLRVCGR DEYLVGETPI KNFQWVRHCL KNGEEIHVVL DTPPDPALDE VRK EEWPLV DDCTGVTGYH EQLTIHGKDH ESVFTVSLWD CDRKFRVKIR GIDIPVLPRN TDLTVFVEAN IQHGQQVLCQ RRTS PKPFT EEVLWNVWLE FSIKIKDLPK GALLNLQIYC GKAPALSSKA SAESPSSESK GKVQLLYYVN LLLIDHRFLL RRGEY VLHM WQISGKGEDQ GSFNADKLTS ATNPDKENSM SISILLDNYC HPIALPKHQP TPDPEGDRVR AEMPNQLRKQ LEAIIA TDP LNPLTAEDKE LLWHFRYESL KHPKAYPKLF SSVKWGQQEI VAKTYQLLAR REVWDQSALD VGLTMQLLDC NFSDENV RA IAVQKLESLE DDDVLHYLLQ LVQAVKFEPY HDSALARFLL KRGLRNKRIG HFLFWFLRSE IAQSRHYQQR FAVILEAY L RGCGTAMLHD FTQQVQVIEM LQKVTLDIKS LSAEKYDVSS QVISQLKQKL ENLQNSQLPE SFRVPYDPGL KAGALAIEK CKVMASKKKP LWLEFKCADP TALSNETIGI IFKHGDDLRQ DMLILQILRI MESIWETESL DLCLLPYGCI STGDKIGMIE IVKDATTIA KIQQSTVGNT GAFKDEVLNH WLKEKSPTEE KFQAAVERFV YSCAGYCVAT FVLGIGDRHN DNIMITETGN L FHIDFGHI LGNYKSFLGI NKERVPFVLT PDFLFVMGTS GKKTSPHFQK FQDICVKAYL ALRHHTNLLI ILFSMMLMTG MP QLTSKED IEYIRDALTV GKNEEDAKKY FLDQIEVCRD KGWTVQFNWF LHLVLGIKQG EKHSA

UniProtKB: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform

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Macromolecule #2: Phosphoinositide 3-kinase regulatory subunit 5

MacromoleculeName: Phosphoinositide 3-kinase regulatory subunit 5 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 97.471805 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GAGTMQPGAT TCTEDRIQHA LERCLHGLSL SRRSTSWSAG LCLNCWSLQE LVSRDPGHFL ILLEQILQKT REVQEKGTYD LLAPLALLF YSTVLCTPHF PPDSDLLLKA ARTYHRFLTW PVPYCSICQE LLTFIDAELK APGISYQRLV RAEQGLSTRS H RSSTVTVL ...String:
GAGTMQPGAT TCTEDRIQHA LERCLHGLSL SRRSTSWSAG LCLNCWSLQE LVSRDPGHFL ILLEQILQKT REVQEKGTYD LLAPLALLF YSTVLCTPHF PPDSDLLLKA ARTYHRFLTW PVPYCSICQE LLTFIDAELK APGISYQRLV RAEQGLSTRS H RSSTVTVL LLNPVEVQAE FLDVADKLST PGPSPHSAYI TLLLHAFQAT FGAHCDLSGL HRRLQSKTLA ELEAIFTETA EA QELASGI GDAAEARQWL RTKLQAVGEK AGFPGVLDTA KPGKLRTIPI PVARCYTYSW NQDSFDILQE ILLKEQELLQ PEI LDDEED EDEEDEEEDL DADGHCAERD SVLSTGSAAS HASTLSLASS QASGPTLSRQ LLTSFVSGLS DGVDSGYMED IEES AYERP RRPGGHERRG HRRPGQKFNR IYKLFKSTSQ MVLRRDSRSL EGSPDSGPPL RRAGSLCSPL DSPTLPPSRA QRSRS LPQP KLSPQLPGWL LAPASRHQRR RPFLSGDEDP KASTLRVVVF GSDRISGKVA RAYSNLRRLE NNRPLLTRFF KLQFFY VPV KRSRGTGTPT SPAPRSQTPP LPTDAPRHPG PAELGAAPWE ESTNDISHYL GMLDPWYERN VLGLMHLPPE VLCQSLK AE PRPLEGSPAQ LPILADMLLY YCRFAARPVL LQVYQTELTF ITGEKTTEIF IHSLELGHSA ATRAIKASGP GSKRLGID G DREAVPLTLQ IIYSKGAISG RSRWSNMEKL CTSVNLSKAC RQQEELDSST EALTLNLTEV VKRQTPKSKK GFNQISTSQ IKVDKVQIIG SNSCPFAVCL DQDERKILQS VIRCEVSPCY KPEKSSLCPP PQRPSYPPAP ATPDLCSLLC LPIMTFSGAL P

UniProtKB: Phosphoinositide 3-kinase regulatory subunit 5

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.45 mg/mL
BufferpH: 8.5
Component:
ConcentrationFormulaName
20.0 mMTrisHClTris(hydroxymethyl)aminomethane-Hydrochloric acid
100.0 mMNaClSodium Chloride
50.0 mM(NH4)2SO4Ammonium Sulfate
0.5 mMTCEPTris(2-carboxyethyl)phosphine

Details: Freshly prepared gel filtration buffer, filtered through 0.22um filter and degassed
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
Details: Glow discharged using the Pelco EasiGlow. 15mA Current.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 1.5s blot time, -5 blot force.
DetailsSpecimen was a 1:1:1 molar ratio of p110g-p101-nanobody, purified to homogeneity by gel filtration.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 6808 / Average electron dose: 36.4 e/Å2
Details: Movies were collected in super-resolution mode set to collect 3 shots per grid hole over 9 holes by beam-shift before applying a stage shift.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsAll data processing carried out using cryoSPARC v2.18 or newer. Movies were subjected to patch motion correction and 2x2 binning. CTFs of the resulting micrographs were estimated using patch CTF estimation. Template picking using 2D averages low-pass filtered to 20A was carried out before 1 round of 2D and 1 round of 3D classification. Best particles were refined by local motion correction, then subjected to 1 more round of 3D classification. Best class/particles were refined by homogeneous refinement, followed by CTF/defocus refinement and a final non-uniform refinement step.
Particle selectionNumber selected: 3762631
Details: Particles were picked using the cryoSPARC template picker
Startup modelType of model: NONE / Details: Ab initio reconstruction with 2 classes
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0) / Details: cryoSPARC local refinement job (L) from v3.0 / Number images used: 320179
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0) / Details: cryoSPARC SGD-based ab intio reconstruction
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0) / Details: cryoSPARC non-uniform refinement
FSC plot (resolution estimation)

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