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- PDB-7urd: Human PORCN in complex with LGK974 and WNT3A peptide -

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Basic information

Entry
Database: PDB / ID: 7urd
TitleHuman PORCN in complex with LGK974 and WNT3A peptide
Components
  • (Isoform 2 of ...) x 2
  • 2C11 heavy chain
  • 2C11 light chain
KeywordsTRANSFERASE/TRANSFERASE INHIBITOR / inhibitor-bound / substrate-bound / complex / TRANSFERASE / TRANSFERASE-TRANSFERASE INHIBITOR complex
Function / homology
Function and homology information


[Wnt protein] O-palmitoleoyl transferase / Wnt signaling pathway involved in forebrain neuroblast division / positive regulation of dermatome development / axis elongation involved in somitogenesis / calcium ion transmembrane transport via low voltage-gated calcium channel / : / LGK974 inhibits PORCN / protein palmitoleylation / positive regulation of collateral sprouting in absence of injury / palmitoleoyltransferase activity ...[Wnt protein] O-palmitoleoyl transferase / Wnt signaling pathway involved in forebrain neuroblast division / positive regulation of dermatome development / axis elongation involved in somitogenesis / calcium ion transmembrane transport via low voltage-gated calcium channel / : / LGK974 inhibits PORCN / protein palmitoleylation / positive regulation of collateral sprouting in absence of injury / palmitoleoyltransferase activity / positive regulation of mesodermal cell fate specification / paraxial mesodermal cell fate commitment / cell proliferation in midbrain / spinal cord association neuron differentiation / Formation of the posterior neural plate / Wnt protein secretion / lipid modification / COP9 signalosome assembly / protein lipidation / Wnt-Frizzled-LRP5/6 complex / positive regulation of cell-cell adhesion mediated by cadherin / glycoprotein metabolic process / Negative regulation of TCF-dependent signaling by WNT ligand antagonists / synaptic vesicle recycling / Signaling by RNF43 mutants / positive regulation of cardiac muscle cell differentiation / WNT ligand biogenesis and trafficking / cell proliferation in forebrain / secondary palate development / negative regulation of axon extension involved in axon guidance / somatic stem cell division / non-canonical Wnt signaling pathway / co-receptor binding / cardiac muscle cell fate commitment / presynapse assembly / positive regulation of skeletal muscle tissue development / Wnt-protein binding / dorsal/ventral neural tube patterning / negative regulation of dopaminergic neuron differentiation / midbrain dopaminergic neuron differentiation / regulation of postsynapse to nucleus signaling pathway / post-anal tail morphogenesis / mammary gland development / positive regulation of neural precursor cell proliferation / frizzled binding / positive regulation of hepatocyte proliferation / Class B/2 (Secretin family receptors) / myoblast differentiation / Disassembly of the destruction complex and recruitment of AXIN to the membrane / inner ear morphogenesis / negative regulation of fat cell differentiation / regulation of synapse organization / heart looping / fat cell differentiation / B cell proliferation / Formation of paraxial mesoderm / skeletal muscle cell differentiation / regulation of postsynaptic membrane neurotransmitter receptor levels / AMPA glutamate receptor complex / positive regulation of receptor internalization / hemopoiesis / regulation of presynapse assembly / canonical Wnt signaling pathway / cell fate commitment / regulation of microtubule cytoskeleton organization / positive regulation of B cell proliferation / cellular response to retinoic acid / Regulation of FZD by ubiquitination / extracellular matrix organization / TCF dependent signaling in response to WNT / cytokine activity / axon guidance / positive regulation of cytokine production / hippocampus development / positive regulation of protein localization to plasma membrane / modulation of chemical synaptic transmission / protein localization / neuron differentiation / platelet aggregation / osteoblast differentiation / Wnt signaling pathway / negative regulation of neurogenesis / Golgi lumen / positive regulation of canonical Wnt signaling pathway / endocytic vesicle membrane / negative regulation of neuron projection development / presynapse / positive regulation of peptidyl-serine phosphorylation / heart development / early endosome membrane / cell population proliferation / in utero embryonic development / transcription by RNA polymerase II / transcription coactivator activity / receptor ligand activity / positive regulation of protein phosphorylation / protein domain specific binding / endoplasmic reticulum lumen / signaling receptor binding / glutamatergic synapse
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
CHOLESTEROL / Chem-O50 / OLEIC ACID / Protein Wnt-3a / Protein-serine O-palmitoleoyltransferase porcupine
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.92 Å
AuthorsLiu, Y. / Qi, X. / Li, X.
Funding support United States, 2items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 20, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 3, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Aug 10, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Isoform 2 of Protein-serine O-palmitoleoyltransferase porcupine
B: Isoform 2 of Protein Wnt-3a peptide
L: 2C11 light chain
H: 2C11 heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)110,70710
Polymers107,9614
Non-polymers2,7476
Water0
1


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

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Components

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Isoform 2 of ... , 2 types, 2 molecules AB

#1: Protein Isoform 2 of Protein-serine O-palmitoleoyltransferase porcupine / Protein MG61


Mass: 52824.652 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PORCN, MG61, PORC, PPN / Production host: Homo sapiens (human)
References: UniProt: Q9H237, [Wnt protein] O-palmitoleoyl transferase
#2: Protein/peptide Isoform 2 of Protein Wnt-3a peptide


Mass: 2529.036 Da / Num. of mol.: 1 / Fragment: residues 199-220 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P56704

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Antibody , 2 types, 2 molecules LH

#3: Antibody 2C11 light chain


Mass: 25673.693 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Homo sapiens (human)
#4: Antibody 2C11 heavy chain


Mass: 26933.135 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Homo sapiens (human)

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Non-polymers , 5 types, 6 molecules

#5: Chemical ChemComp-O50 / 2-[(2P)-2',3-dimethyl[2,4'-bipyridin]-5-yl]-N-[(5P)-5-(pyrazin-2-yl)pyridin-2-yl]acetamide


Mass: 396.445 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C23H20N6O / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-CLR / CHOLESTEROL / Cholesterol


Mass: 386.654 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H46O
#7: Chemical ChemComp-AJP / Digitonin / Digitonin


Mass: 1229.312 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C56H92O29 / Comment: detergent*YM
#8: Chemical ChemComp-OLA / OLEIC ACID / Oleic acid


Mass: 282.461 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C18H34O2
#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn

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Details

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

Component
IDNameTypeEntity IDParent-IDSource
1LGK974 and WNT3a peptide-bound human PORCNCOMPLEX#1-#40RECOMBINANT
2Isoform 2 of Protein-serine O-palmitoleoyltransferase porcupine (E.C.2.3.1.250)COMPLEX#11RECOMBINANT
3Isoform 2 of Protein Wnt-3a peptideCOMPLEX#21SYNTHETIC
42C11 light chain, 2C11 heavy chainCOMPLEX#3-#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
24Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
24Homo sapiens (human)9606
Buffer solutionpH: 7.5
SpecimenEmbedding 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: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 8.5 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.92 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 397217 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0045727
ELECTRON MICROSCOPYf_angle_d0.8347805
ELECTRON MICROSCOPYf_dihedral_angle_d6.13186
ELECTRON MICROSCOPYf_chiral_restr0.087883
ELECTRON MICROSCOPYf_plane_restr0.004924

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