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- PDB-8dcp: PI 3-kinase alpha with nanobody 3-126 -

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Basic information

Entry
Database: PDB / ID: 8dcp
TitlePI 3-kinase alpha with nanobody 3-126
Components
  • Phosphatidylinositol 3-kinase regulatory subunit alpha
  • Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform
KeywordsTRANSFERASE / Phosphoinositide 3-kinase (PI3K) / activation / inhibition / nanobody / conformational changes / STRUCTURAL PROTEIN
Function / homology
Function and homology information


perinuclear endoplasmic reticulum membrane / regulation of toll-like receptor 4 signaling pathway / response to muscle inactivity / phosphatidylinositol kinase activity / phosphatidylinositol 3-kinase regulator activity / 1-phosphatidylinositol-3-kinase regulator activity / positive regulation of endoplasmic reticulum unfolded protein response / regulation of actin filament organization / phosphatidylinositol 3-kinase activator activity / negative regulation of actin filament depolymerization ...perinuclear endoplasmic reticulum membrane / regulation of toll-like receptor 4 signaling pathway / response to muscle inactivity / phosphatidylinositol kinase activity / phosphatidylinositol 3-kinase regulator activity / 1-phosphatidylinositol-3-kinase regulator activity / positive regulation of endoplasmic reticulum unfolded protein response / regulation of actin filament organization / phosphatidylinositol 3-kinase activator activity / negative regulation of actin filament depolymerization / response to butyrate / T follicular helper cell differentiation / IRS-mediated signalling / interleukin-18-mediated signaling pathway / phosphatidylinositol 3-kinase regulatory subunit binding / myeloid leukocyte migration / response to L-leucine / PI3K events in ERBB4 signaling / neurotrophin TRKA receptor binding / positive regulation of focal adhesion disassembly / cellular response to hydrostatic pressure / autosome genomic imprinting / cis-Golgi network / Activated NTRK2 signals through PI3K / ErbB-3 class receptor binding / transmembrane receptor protein tyrosine kinase adaptor activity / negative regulation of stress fiber assembly / negative regulation of fibroblast apoptotic process / Activated NTRK3 signals through PI3K / phosphatidylinositol 3-kinase complex, class IB / phosphatidylinositol 3-kinase complex / TORC2 signaling / Co-stimulation by ICOS / RHOD GTPase cycle / positive regulation of protein localization to membrane / Signaling by cytosolic FGFR1 fusion mutants / vasculature development / regulation of cellular respiration / Nephrin family interactions / RHOF GTPase cycle / kinase activator activity / Signaling by LTK in cancer / 1-phosphatidylinositol-4-phosphate 3-kinase activity / Signaling by LTK / anoikis / RND1 GTPase cycle / RND2 GTPase cycle / positive regulation of leukocyte migration / RND3 GTPase cycle / relaxation of cardiac muscle / phosphatidylinositol 3-kinase complex, class IA / positive regulation of filopodium assembly / MET activates PI3K/AKT signaling / PI3K/AKT activation / phosphatidylinositol-4,5-bisphosphate 3-kinase / 1-phosphatidylinositol-4,5-bisphosphate 3-kinase activity / growth hormone receptor signaling pathway / insulin binding / phosphatidylinositol 3-kinase / phosphatidylinositol-3-phosphate biosynthetic process / Signaling by ALK / cardiac muscle cell contraction / RHOV GTPase cycle / 1-phosphatidylinositol-3-kinase activity / RHOB GTPase cycle / vascular endothelial growth factor signaling pathway / natural killer cell mediated cytotoxicity / GP1b-IX-V activation signalling / Erythropoietin activates Phosphoinositide-3-kinase (PI3K) / PI-3K cascade:FGFR3 / response to dexamethasone / PI-3K cascade:FGFR2 / negative regulation of macroautophagy / PI-3K cascade:FGFR4 / PI-3K cascade:FGFR1 / RHOC GTPase cycle / RHOJ GTPase cycle / negative regulation of osteoclast differentiation / phosphatidylinositol phosphate biosynthetic process / phosphatidylinositol-mediated signaling / Synthesis of PIPs at the plasma membrane / RHOU GTPase cycle / CDC42 GTPase cycle / RET signaling / negative regulation of anoikis / Interleukin-3, Interleukin-5 and GM-CSF signaling / T cell differentiation / insulin receptor substrate binding / PI3K events in ERBB2 signaling / RHOG GTPase cycle / negative regulation of cell-matrix adhesion / intercalated disc / PI3K Cascade / extrinsic apoptotic signaling pathway via death domain receptors / Role of LAT2/NTAL/LAB on calcium mobilization / CD28 dependent PI3K/Akt signaling / RHOA GTPase cycle / RAC3 GTPase cycle / regulation of multicellular organism growth / RAC2 GTPase cycle
Similarity search - Function
Phosphatidylinositol 3-kinase regulatory subunit alpha, SH3 domain / PIK3R1, inter-SH2 domain / PI3Kalpha, catalytic domain / PI3K p85 subunit, C-terminal SH2 domain / PI3K regulatory subunit p85-related , inter-SH2 domain / PI3K p85 subunit, N-terminal SH2 domain / Phosphatidylinositol 3-kinase regulatory subunit P85 inter-SH2 domain / PI3-kinase family, p85-binding domain / PI3-kinase family, p85-binding domain / Rho GTPase-activating protein domain ...Phosphatidylinositol 3-kinase regulatory subunit alpha, SH3 domain / PIK3R1, inter-SH2 domain / PI3Kalpha, catalytic domain / PI3K p85 subunit, C-terminal SH2 domain / PI3K regulatory subunit p85-related , inter-SH2 domain / PI3K p85 subunit, N-terminal SH2 domain / Phosphatidylinositol 3-kinase regulatory subunit P85 inter-SH2 domain / PI3-kinase family, p85-binding domain / PI3-kinase family, p85-binding domain / Rho GTPase-activating protein domain / RhoGAP domain / Rho GTPase-activating proteins domain profile. / GTPase-activator protein for Rho-like GTPases / 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. / Rho GTPase activation protein / C2 phosphatidylinositol 3-kinase-type domain / Phosphoinositide 3-kinase C2 / C2 phosphatidylinositol 3-kinase (PI3K)-type domain profile. / Phosphoinositide 3-kinase, region postulated to contain C2 domain / 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. / 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 / SH2 domain / Src homology 2 (SH2) domain profile. / Src homology 2 domains / SH2 domain / Src homology 3 domains / SH2 domain superfamily / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / Armadillo-type fold / Ubiquitin-like domain superfamily / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Phosphatidylinositol 3-kinase regulatory subunit alpha / Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.41 Å
AuthorsHart, J.R. / Liu, X. / Pan, C. / Liang, A. / Ueno, L. / Xu, Y. / Quezada, A. / Zou, X. / Yang, S. / Zhou, Q. ...Hart, J.R. / Liu, X. / Pan, C. / Liang, A. / Ueno, L. / Xu, Y. / Quezada, A. / Zou, X. / Yang, S. / Zhou, Q. / Schoonooghe, S. / Hassanzadeh-Ghassabeh, G. / Xia, T. / Shui, W. / Yang, D. / Vogt, P.K. / Wang, M.-W.
Funding support United States, China, 15items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R35 CA197582 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R50 CA243899 United States
National Natural Science Foundation of China (NSFC)81872915 China
National Natural Science Foundation of China (NSFC)82073904 China
National Natural Science Foundation of China (NSFC)82121005 China
National Natural Science Foundation of China (NSFC)81973373 China
National Natural Science Foundation of China (NSFC)31971362 China
National Natural Science Foundation of China (NSFC)32171439 China
National Natural Science Foundation of China (NSFC)21704064 China
Other government2018ZX09735 001
Other government2018ZX09711002 002 005
Other government2021ZD0203400
Other government2018YFA0507000
Other privateNNCAS 2017 1 CC
Hainan Provincial Major Science and Technology ProjectZDKJ2021028 China
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Nanobodies and chemical cross-links advance the structural and functional analysis of PI3Kα.
Authors: Jonathan R Hart / Xiao Liu / Chen Pan / Anyi Liang / Lynn Ueno / Yingna Xu / Alexandra Quezada / Xinyu Zou / Su Yang / Qingtong Zhou / Steve Schoonooghe / Gholamreza Hassanzadeh-Ghassabeh / ...Authors: Jonathan R Hart / Xiao Liu / Chen Pan / Anyi Liang / Lynn Ueno / Yingna Xu / Alexandra Quezada / Xinyu Zou / Su Yang / Qingtong Zhou / Steve Schoonooghe / Gholamreza Hassanzadeh-Ghassabeh / Tian Xia / Wenqing Shui / Dehua Yang / Peter K Vogt / Ming-Wei Wang /
Abstract: Nanobodies and chemical cross-linking were used to gain information on the identity and positions of flexible domains of PI3Kα. The application of chemical cross-linking mass spectrometry (CXMS) ...Nanobodies and chemical cross-linking were used to gain information on the identity and positions of flexible domains of PI3Kα. The application of chemical cross-linking mass spectrometry (CXMS) facilitated the identification of the p85 domains BH, cSH2, and SH3 as well as their docking positions on the PI3Kα catalytic core. Binding of individual nanobodies to PI3Kα induced activation or inhibition of enzyme activity and caused conformational changes that could be correlated with enzyme function. Binding of nanobody Nb3-126 to the BH domain of p85α substantially improved resolution for parts of the PI3Kα complex, and binding of nanobody Nb3-159 induced a conformation of PI3Kα that is distinct from known PI3Kα structures. The analysis of CXMS data also provided mechanistic insights into the molecular underpinning of the flexibility of PI3Kα.
History
DepositionJun 17, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 21, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform
B: Phosphatidylinositol 3-kinase regulatory subunit alpha


Theoretical massNumber of molelcules
Total (without water)211,4462
Polymers211,4462
Non-polymers00
Water37821
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, cross-linking, gel filtration, native gel electrophoresis, immunoprecipitation
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform / PtdIns-3-kinase subunit alpha / Phosphatidylinositol 4 / 5-bisphosphate 3-kinase 110 kDa catalytic ...PtdIns-3-kinase subunit alpha / Phosphatidylinositol 4 / 5-bisphosphate 3-kinase 110 kDa catalytic subunit alpha / p110alpha / Phosphoinositide-3-kinase catalytic alpha polypeptide / Serine/threonine protein kinase PIK3CA


Mass: 127822.578 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PIK3CA / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P42336, phosphatidylinositol-4,5-bisphosphate 3-kinase, non-specific serine/threonine protein kinase
#2: Protein Phosphatidylinositol 3-kinase regulatory subunit alpha / PI3-kinase regulatory subunit alpha / PI3K regulatory subunit alpha / PtdIns-3-kinase regulatory ...PI3-kinase regulatory subunit alpha / PI3K regulatory subunit alpha / PtdIns-3-kinase regulatory subunit alpha / Phosphatidylinositol 3-kinase 85 kDa regulatory subunit alpha / PI3-kinase subunit p85-alpha / PtdIns-3-kinase regulatory subunit p85-alpha


Mass: 83623.203 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PIK3R1, GRB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P27986
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 21 / Source method: isolated from a natural source / Formula: H2O

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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: Human PI 3-kinase alpha complex composed of p110alpha and p85alpha with nanobody 3-126 bound
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper) / Strain: Sf-9
Buffer solutionpH: 7.6
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: OTHER / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 70 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
7PHENIX1.19.2-4158model fitting
9PHENIX1.19.2-4158model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.41 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Num. of particles: 506412 / Symmetry type: POINT
Atomic model buildingB value: 43.84 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 7MYN

7myn


Accession code: 7MYN / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 43.8 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003110793
ELECTRON MICROSCOPYf_angle_d0.632314548
ELECTRON MICROSCOPYf_chiral_restr0.04141570
ELECTRON MICROSCOPYf_plane_restr0.00361871
ELECTRON MICROSCOPYf_dihedral_angle_d4.23941408

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