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- EMDB-26710: Human PORCN in complex with palmitoleoylated WNT3A peptide -

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Basic information

Entry
Database: EMDB / ID: EMD-26710
TitleHuman PORCN in complex with palmitoleoylated WNT3A peptide
Map datamembrane protein
Sample
  • Complex: product-bound human PORCN
    • Complex: Isoform 2 of Protein-serine O-palmitoleoyltransferase porcupine (E.C.2.3.1.250)
      • Protein or peptide: Isoform 2 of Protein-serine O-palmitoleoyltransferase porcupine
    • Complex: 2C11 light chain, 2C11 heavy chain
      • Protein or peptide: 2C11 light chain
      • Protein or peptide: 2C11 heavy chain
    • Complex: Isoform 2 of Protein Wnt-3a
      • Protein or peptide: Isoform 2 of Protein Wnt-3a
  • Ligand: Digitonin
  • Ligand: ZINC ION
  • Ligand: PALMITOLEIC ACID
Function / homology
Function and homology information


[Wnt protein] O-palmitoleoyl transferase / Wnt signaling pathway involved in forebrain neuroblast division / protein palmitoleylation / positive regulation of dermatome development / calcium ion transmembrane transport via low voltage-gated calcium channel / LGK974 inhibits PORCN / positive regulation of collateral sprouting in absence of injury / palmitoleoyltransferase activity / positive regulation of mesodermal cell fate specification / paraxial mesodermal cell fate commitment ...[Wnt protein] O-palmitoleoyl transferase / Wnt signaling pathway involved in forebrain neuroblast division / protein palmitoleylation / positive regulation of dermatome development / calcium ion transmembrane transport via low voltage-gated calcium channel / LGK974 inhibits PORCN / positive regulation of collateral sprouting in absence of injury / palmitoleoyltransferase activity / positive regulation of mesodermal cell fate specification / paraxial mesodermal cell fate commitment / axis elongation involved in somitogenesis / cell proliferation in midbrain / spinal cord association neuron differentiation / Wnt protein secretion / Formation of the posterior neural plate / lipid modification / COP9 signalosome assembly / protein lipidation / Wnt-Frizzled-LRP5/6 complex / glycoprotein metabolic process / positive regulation of cell-cell adhesion mediated by cadherin / Negative regulation of TCF-dependent signaling by WNT ligand antagonists / synaptic vesicle recycling / WNT ligand biogenesis and trafficking / Signaling by RNF43 mutants / positive regulation of cardiac muscle cell differentiation / cell proliferation in forebrain / negative regulation of axon extension involved in axon guidance / secondary palate development / Specification of the neural plate border / somatic stem cell division / cardiac muscle cell fate commitment / co-receptor binding / presynapse assembly / non-canonical Wnt signaling pathway / positive regulation of skeletal muscle tissue development / negative regulation of dopaminergic neuron differentiation / Wnt-protein binding / midbrain dopaminergic neuron differentiation / mammary gland development / dorsal/ventral neural tube patterning / regulation of postsynapse to nucleus signaling pathway / post-anal tail morphogenesis / positive regulation of neural precursor cell proliferation / frizzled binding / Class B/2 (Secretin family receptors) / positive regulation of hepatocyte proliferation / myoblast differentiation / Disassembly of the destruction complex and recruitment of AXIN to the membrane / inner ear morphogenesis / regulation of synapse organization / negative regulation of fat cell differentiation / fat cell differentiation / B cell proliferation / heart looping / Formation of paraxial mesoderm / positive regulation of receptor internalization / skeletal muscle cell differentiation / regulation of postsynaptic membrane neurotransmitter receptor levels / AMPA glutamate receptor complex / hemopoiesis / regulation of presynapse assembly / cell fate commitment / canonical Wnt signaling pathway / Transcriptional and post-translational regulation of MITF-M expression and activity / regulation of microtubule cytoskeleton organization / cellular response to retinoic acid / positive regulation of B cell proliferation / Regulation of FZD by ubiquitination / extracellular matrix organization / axon guidance / positive regulation of cytokine production / hippocampus development / cytokine activity / TCF dependent signaling in response to WNT / positive regulation of protein localization to plasma membrane / modulation of chemical synaptic transmission / neuron differentiation / platelet aggregation / Wnt signaling pathway / negative regulation of neurogenesis / Golgi lumen / osteoblast differentiation / endocytic vesicle membrane / positive regulation of canonical Wnt signaling pathway / protein localization / negative regulation of neuron projection development / positive regulation of peptidyl-serine phosphorylation / heart development / presynapse / early endosome membrane / in utero embryonic development / transcription by RNA polymerase II / cell population proliferation / transcription coactivator activity / receptor ligand activity / positive regulation of protein phosphorylation / protein domain specific binding / endoplasmic reticulum lumen / signaling receptor binding
Similarity search - Function
Wnt-3 protein / : / Wnt protein, conserved site / Wnt-1 family signature. / Wnt / Wnt, C-terminal domain / wnt family / found in Wnt-1 / Membrane bound O-acyl transferase, MBOAT / MBOAT, membrane-bound O-acyltransferase family
Similarity search - Domain/homology
Protein Wnt-3a / Protein-serine O-palmitoleoyltransferase porcupine
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.19 Å
AuthorsLiu Y / Qi X / Li X
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
Welch Foundation United States
CitationJournal: Nature / Year: 2022
Title: Mechanisms and inhibition of Porcupine-mediated Wnt acylation.
Authors: Yang Liu / Xiaofeng Qi / Linda Donnelly / Nadia Elghobashi-Meinhardt / Tao Long / Rich W Zhou / Yingyuan Sun / Boyuan Wang / Xiaochun Li /
Abstract: Wnt signalling is essential for regulation of embryonic development and adult tissue homeostasis, and aberrant Wnt signalling is frequently associated with cancers. Wnt signalling requires ...Wnt signalling is essential for regulation of embryonic development and adult tissue homeostasis, and aberrant Wnt signalling is frequently associated with cancers. Wnt signalling requires palmitoleoylation on a hairpin 2 motif by the endoplasmic reticulum-resident membrane-bound O-acyltransferase Porcupine (PORCN). This modification is indispensable for Wnt binding to its receptor Frizzled, which triggers signalling. Here we report four cryo-electron microscopy structures of human PORCN: the complex with the palmitoleoyl-coenzyme A (palmitoleoyl-CoA) substrate; the complex with the PORCN inhibitor LGK974, an anti-cancer drug currently in clinical trials; the complex with LGK974 and WNT3A hairpin 2 (WNT3Ap); and the complex with a synthetic palmitoleoylated WNT3Ap analogue. The structures reveal that hairpin 2 of WNT3A, which is well conserved in all Wnt ligands, inserts into PORCN from the lumenal side, and the palmitoleoyl-CoA accesses the enzyme from the cytosolic side. The catalytic histidine triggers the transfer of the unsaturated palmitoleoyl group to the target serine on the Wnt hairpin 2, facilitated by the proximity of the two substrates. The inhibitor-bound structure shows that LGK974 occupies the palmitoleoyl-CoA binding site to prevent the reaction. Thus, this work provides a mechanism for Wnt acylation and advances the development of PORCN inhibitors for cancer treatment.
History
DepositionApr 21, 2022-
Header (metadata) releaseJul 20, 2022-
Map releaseJul 20, 2022-
UpdateAug 10, 2022-
Current statusAug 10, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26710.png

Downloads & links

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Map

FileDownload / File: emd_26710.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmembrane protein
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 280 pix.
= 232.4 Å		0.83 Å/pix.
x 280 pix.
= 232.4 Å		0.83 Å/pix.
x 280 pix.
= 232.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-2.6126945 - 3.8194375
Average (Standard dev.)0.0047776154 (±0.09236624)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 232.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: membrane protein

Fileemd_26710_half_map_1.map
Annotationmembrane protein
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: membrane protein

Fileemd_26710_half_map_2.map
Annotationmembrane protein
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : product-bound human PORCN

EntireName: product-bound human PORCN
Components
  • Complex: product-bound human PORCN
    • Complex: Isoform 2 of Protein-serine O-palmitoleoyltransferase porcupine (E.C.2.3.1.250)
      • Protein or peptide: Isoform 2 of Protein-serine O-palmitoleoyltransferase porcupine
    • Complex: 2C11 light chain, 2C11 heavy chain
      • Protein or peptide: 2C11 light chain
      • Protein or peptide: 2C11 heavy chain
    • Complex: Isoform 2 of Protein Wnt-3a
      • Protein or peptide: Isoform 2 of Protein Wnt-3a
  • Ligand: Digitonin
  • Ligand: ZINC ION
  • Ligand: PALMITOLEIC ACID

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Supramolecule #1: product-bound human PORCN

SupramoleculeName: product-bound human PORCN / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#4

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Supramolecule #2: Isoform 2 of Protein-serine O-palmitoleoyltransferase porcupine (...

SupramoleculeName: Isoform 2 of Protein-serine O-palmitoleoyltransferase porcupine (E.C.2.3.1.250)
type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Supramolecule #3: 2C11 light chain, 2C11 heavy chain

SupramoleculeName: 2C11 light chain, 2C11 heavy chain / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human)

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Supramolecule #4: Isoform 2 of Protein Wnt-3a

SupramoleculeName: Isoform 2 of Protein Wnt-3a / type: complex / Chimera: Yes / ID: 4 / Parent: 1 / Macromolecule list: #4

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Macromolecule #1: Isoform 2 of Protein-serine O-palmitoleoyltransferase porcupine

MacromoleculeName: Isoform 2 of Protein-serine O-palmitoleoyltransferase porcupine
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: [Wnt protein] O-palmitoleoyl transferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 52.824652 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDYKDDDDKA TFSRQEFFQQ LLQGCLLPTA QQGLDQIWLL LAICLACRLL WRLGLPSYLK HASTVAGGFF SLYHFFQLHM VWVVLLSLL CYLVLFLCRH SSHRGVFLSV TILIYLLMGE MHMVDTVTWH KMRGAQMIVA MKAVSLGFDL DRGEVGTVPS P VEFMGYLY ...String:
MDYKDDDDKA TFSRQEFFQQ LLQGCLLPTA QQGLDQIWLL LAICLACRLL WRLGLPSYLK HASTVAGGFF SLYHFFQLHM VWVVLLSLL CYLVLFLCRH SSHRGVFLSV TILIYLLMGE MHMVDTVTWH KMRGAQMIVA MKAVSLGFDL DRGEVGTVPS P VEFMGYLY FVGTIVFGPW ISFHSYLQAV QGRPLSCRWL QKVARSLALA LLCLVLSTCV GPYLFPYFIP LNGDRLLRNK KR KARWLRA YESAVSFHFS NYFVGFLSEA TATLAGAGFT EEKDHLEWDL TVSKPLNVEL PRSMVEVVTS WNLPMSYWLN NYV FKNALR LGTFSAVLVT YAASALLHGF SFHLAAVLLS LAFITYVEHV LRKRLARILS ACVLSKRCPP DCSHQHRLGL GVRA LNLLF GALAIFHLAY LGSLFDVDVD DTTEEQGYGM AYTVHKWSEL SWASHWVTFG CWIFYRLIG

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Macromolecule #2: 2C11 light chain

MacromoleculeName: 2C11 light chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 25.673693 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGWSCIILFL VATARTGVHS DIHMTQSPAS LSAFVGETVT ITCRTSENIF SYLAWYQQKQ GKSPQLLVYN AKTLTSGVPS RFSGSGSGT QFSLKINSLQ PEDFGSYYCQ HHYGSPYTFG GGTKLEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF Y PREAKVQW ...String:
MGWSCIILFL VATARTGVHS DIHMTQSPAS LSAFVGETVT ITCRTSENIF SYLAWYQQKQ GKSPQLLVYN AKTLTSGVPS RFSGSGSGT QFSLKINSLQ PEDFGSYYCQ HHYGSPYTFG GGTKLEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF Y PREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

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Macromolecule #3: 2C11 heavy chain

MacromoleculeName: 2C11 heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 26.933135 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGWSCIILFL VATATGVHSE IQLQQSGAEL VKPGASVKMS CKVSGYSFTG YNMNWVKQSH GKSLEWIGNI NPYYVSTNYN QKFTGKATF TVDRSSSTAY MQLDSLTSED SAVYYCARSY GSSHTFAYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG T AALGCLVK ...String:
MGWSCIILFL VATATGVHSE IQLQQSGAEL VKPGASVKMS CKVSGYSFTG YNMNWVKQSH GKSLEWIGNI NPYYVSTNYN QKFTGKATF TVDRSSSTAY MQLDSLTSED SAVYYCARSY GSSHTFAYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG T AALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KS CDKTHHH HHH

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Macromolecule #4: Isoform 2 of Protein Wnt-3a

MacromoleculeName: Isoform 2 of Protein Wnt-3a / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 2.529059 KDa
SequenceString:
MHLKCKCHGL (DNP)GSCEVKTCW WS

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Macromolecule #5: Digitonin

MacromoleculeName: Digitonin / type: ligand / ID: 5 / Number of copies: 1 / Formula: AJP
Molecular weightTheoretical: 1.229312 KDa
Chemical component information

ChemComp-AJP:
Digitonin / detergent*YM

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #7: PALMITOLEIC ACID

MacromoleculeName: PALMITOLEIC ACID / type: ligand / ID: 7 / Number of copies: 1 / Formula: PAM
Molecular weightTheoretical: 254.408 Da
Chemical component information

ChemComp-PAM:
PALMITOLEIC ACID

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 8.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.19 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 113566
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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