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- PDB-8gud: Cryo-EM structure of cancer-specific PI3Kalpha mutant E545K in co... -

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Entry
Database: PDB / ID: 8gud
TitleCryo-EM structure of cancer-specific PI3Kalpha mutant E545K in complex with BYL-719
ComponentsPhosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform
KeywordsSTRUCTURAL PROTEIN / Phosphoinositide 3-kinase (PI3K) / helical domain / mutation / cancers
Function / homology
Function and homology information


response to muscle inactivity / negative regulation of actin filament depolymerization / response to L-leucine / regulation of actin filament organization / response to butyrate / autosome genomic imprinting / cellular response to hydrostatic pressure / IRS-mediated signalling / PI3K events in ERBB4 signaling / Activated NTRK2 signals through PI3K ...response to muscle inactivity / negative regulation of actin filament depolymerization / response to L-leucine / regulation of actin filament organization / response to butyrate / autosome genomic imprinting / cellular response to hydrostatic pressure / IRS-mediated signalling / PI3K events in ERBB4 signaling / Activated NTRK2 signals through PI3K / positive regulation of protein localization to membrane / Activated NTRK3 signals through PI3K / negative regulation of fibroblast apoptotic process / cardiac muscle cell contraction / phosphatidylinositol 3-kinase complex, class IB / vasculature development / Signaling by cytosolic FGFR1 fusion mutants / regulation of cellular respiration / phosphatidylinositol 3-kinase complex / anoikis / Nephrin family interactions / 1-phosphatidylinositol-4-phosphate 3-kinase activity / Costimulation by the CD28 family / 1-phosphatidylinositol-4,5-bisphosphate 3-kinase activity / vascular endothelial growth factor signaling pathway / PI3K/AKT activation / MET activates PI3K/AKT signaling / phosphatidylinositol-4,5-bisphosphate 3-kinase / phosphatidylinositol 3-kinase / phosphatidylinositol 3-kinase complex, class IA / relaxation of cardiac muscle / phosphatidylinositol-3-phosphate biosynthetic process / 1-phosphatidylinositol-3-kinase activity / negative regulation of macroautophagy / Signaling by ALK / PI-3K cascade:FGFR3 / Erythropoietin activates Phosphoinositide-3-kinase (PI3K) / protein kinase activator activity / response to dexamethasone / PI-3K cascade:FGFR2 / PI-3K cascade:FGFR4 / PI-3K cascade:FGFR1 / CD28 dependent PI3K/Akt signaling / Synthesis of PIPs at the plasma membrane / phosphatidylinositol phosphate biosynthetic process / PI3K events in ERBB2 signaling / Signaling by ALK fusions and activated point mutants / regulation of multicellular organism growth / negative regulation of anoikis / RET signaling / insulin receptor substrate binding / PI3K Cascade / Interleukin-3, Interleukin-5 and GM-CSF signaling / intercalated disc / positive regulation of TOR signaling / endothelial cell migration / RAC2 GTPase cycle / Role of phospholipids in phagocytosis / GAB1 signalosome / Role of LAT2/NTAL/LAB on calcium mobilization / adipose tissue development / Interleukin receptor SHC signaling / positive regulation of lamellipodium assembly / phagocytosis / Signaling by PDGFRA transmembrane, juxtamembrane and kinase domain mutants / Signaling by PDGFRA extracellular domain mutants / energy homeostasis / Signaling by FGFR4 in disease / Signaling by FLT3 ITD and TKD mutants / cardiac muscle contraction / Signaling by FGFR3 in disease / Tie2 Signaling / GPVI-mediated activation cascade / Signaling by FGFR2 in disease / RAC1 GTPase cycle / T cell costimulation / response to muscle stretch / Signaling by FLT3 fusion proteins / FLT3 Signaling / Signaling by FGFR1 in disease / Downstream signal transduction / insulin-like growth factor receptor signaling pathway / Signaling by phosphorylated juxtamembrane, extracellular and kinase domain KIT mutants / liver development / response to activity / phosphatidylinositol 3-kinase/protein kinase B signal transduction / Regulation of signaling by CBL / cellular response to glucose stimulus / positive regulation of smooth muscle cell proliferation / regulation of protein phosphorylation / Constitutive Signaling by EGFRvIII / Signaling by ERBB2 ECD mutants / epidermal growth factor receptor signaling pathway / Signaling by ERBB2 KD Mutants / Signaling by SCF-KIT / platelet activation / VEGFA-VEGFR2 Pathway / cellular response to insulin stimulus / glucose metabolic process / Constitutive Signaling by Aberrant PI3K in Cancer
Similarity search - Function
PI3Kalpha, catalytic domain / PI3-kinase family, p85-binding domain / PI3-kinase family, p85-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 ...PI3Kalpha, catalytic domain / PI3-kinase family, p85-binding domain / PI3-kinase family, p85-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
Chem-1LT / 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: 2.62 Å
AuthorsLiu, X. / Zhou, Q. / Hart, J.R. / Xu, Y. / Yang, S. / Yang, D. / Vogt, P.K. / Wang, M.-W.
Funding support China, United States, 13items
OrganizationGrant numberCountry
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)21704064 China
Other government2018ZX09735-001 China
Other government2018ZX09711002-002-005 China
Other government2021ZD0203400 China
Other government2018YFA0507000 China
Other governmentZDKJ2021028 China
Other privateNNCAS-2017-1-CC China
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
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Cryo-EM structures of cancer-specific helical and kinase domain mutations of PI3Kα.
Authors: Xiao Liu / Qingtong Zhou / Jonathan R Hart / Yingna Xu / Su Yang / Dehua Yang / Peter K Vogt / Ming-Wei Wang /
Abstract: Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that perform multiple and important cellular functions. The protein investigated here belongs to class IA of the PI3Ks; it is a dimer ...Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that perform multiple and important cellular functions. The protein investigated here belongs to class IA of the PI3Ks; it is a dimer consisting of a catalytic subunit, p110α, and a regulatory subunit, p85α, and is referred to as PI3Kα. The catalytic subunit p110α is frequently mutated in cancer. The mutations induce a gain of function and constitute a driving force in cancer development. About 80% of these mutations lead to single-amino-acid substitutions in one of three sites of p110α: two in the helical domain of the protein (E542K and E545K) and one at the C-terminus of the kinase domain (H1047R). Here, we report the cryo-electron microscopy structures of these mutants in complex with the p110α-specific inhibitor BYL-719. The H1047R mutant rotates its sidechain to a new position and weakens the kα11 activation loop interaction, thereby reducing the inhibitory effect of p85α on p110α. E542K and E545K completely abolish the tight interaction between the helical domain of p110α and the N-terminal SH2 domain of p85α and lead to the disruption of all p85α binding and a dramatic increase in flexibility of the adaptor-binding domain (ABD) in p110α. Yet, the dimerization of PI3Kα is preserved through the ABD-p85α interaction. The local and global structural features induced by these mutations provide molecular insights into the activation of PI3Kα, deepen our understanding of the oncogenic mechanism of this important signaling molecule, and may facilitate the development of mutant-specific inhibitors.
History
DepositionSep 11, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 23, 2022Provider: repository / Type: Initial release

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Structure visualization

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MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)128,2642
Polymers127,8231
Non-polymers4411
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: C1
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform / PI3-kinase subunit alpha / PI3K-alpha / PI3Kalpha / PtdIns-3-kinase subunit alpha / ...PI3-kinase subunit alpha / PI3K-alpha / PI3Kalpha / PtdIns-3-kinase subunit alpha / Phosphatidylinositol 4 / 5-bisphosphate 3-kinase 110 kDa catalytic subunit alpha / PtdIns-3-kinase subunit p110-alpha / p110alpha / Phosphoinositide 3-kinase alpha / Phosphoinositide-3-kinase catalytic alpha polypeptide / Serine/threonine protein kinase PIK3CA


Mass: 127822.641 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 3-kinase, phosphatidylinositol-4,5-bisphosphate 3-kinase, non-specific serine/threonine protein kinase
#2: Chemical ChemComp-1LT / (2S)-N~1~-{4-methyl-5-[2-(1,1,1-trifluoro-2-methylpropan-2-yl)pyridin-4-yl]-1,3-thiazol-2-yl}pyrrolidine-1,2-dicarboxamide / Alpelisib / Alpelisib


Mass: 441.470 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H22F3N5O2S / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM
Has ligand of interestY

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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: Cryo-EM structure of PI3Kalpha mutant E545K in complex with BYL-719
Type: COMPLEX / Entity ID: #1 / 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 FIELDBright-field microscopy / 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) / Num. of real images: 5144

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameVersionCategory
7PHENIX1.18.2-3874model fitting
13PHENIX1.18.2-3874model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.62 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 753253 / Symmetry type: POINT
Atomic model buildingB value: 101.9 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 7MYN

7myn

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0046967
ELECTRON MICROSCOPYf_angle_d0.6729422
ELECTRON MICROSCOPYf_dihedral_angle_d6.42903
ELECTRON MICROSCOPYf_chiral_restr0.0461027
ELECTRON MICROSCOPYf_plane_restr0.0051203

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