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- EMDB-26711: Human HHAT H379C in complex with SHH N-terminal peptide -

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Basic information

Entry
Database: EMDB / ID: EMD-26711
TitleHuman HHAT H379C in complex with SHH N-terminal peptide
Map datamembrane protein
Sample
  • Complex: 16:0 CoA and SHH-N-bound human HHAT
    • Complex: Protein-cysteine N-palmitoyltransferase HHAT (E.C.2.3.1.-), SHH-N peptide
      • Protein or peptide: Protein-cysteine N-palmitoyltransferase HHAT
      • Protein or peptide: SHH-N peptide
    • Complex: 3H02 heavy chain, 3H02 light chain
      • Protein or peptide: 3H02 heavy chain
      • Protein or peptide: 3H02 light chain
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: Palmitoyl-CoA
  • Ligand: Digitonin
Function / homology
Function and homology information


N-terminal peptidyl-L-cysteine N-palmitoylation / O-acyltransferase activity / HHAT G278V doesn't palmitoylate Hh-Np / palmitoyltransferase activity / smoothened signaling pathway / Transferases; Acyltransferases; Transferring groups other than aminoacyl groups / Hedgehog ligand biogenesis / Golgi membrane / GTP binding / endoplasmic reticulum membrane ...N-terminal peptidyl-L-cysteine N-palmitoylation / O-acyltransferase activity / HHAT G278V doesn't palmitoylate Hh-Np / palmitoyltransferase activity / smoothened signaling pathway / Transferases; Acyltransferases; Transferring groups other than aminoacyl groups / Hedgehog ligand biogenesis / Golgi membrane / GTP binding / endoplasmic reticulum membrane / Golgi apparatus / endoplasmic reticulum
Similarity search - Function
Membrane bound O-acyl transferase, MBOAT / MBOAT, membrane-bound O-acyltransferase family
Similarity search - Domain/homology
Protein-cysteine N-palmitoyltransferase HHAT
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 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_26711.png

Downloads & links

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Map

FileDownload / File: emd_26711.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmembrane protein
Voxel sizeX=Y=Z: 0.842 Å
Density
Contour LevelBy AUTHOR: 0.018
Minimum - Maximum-0.056409594 - 0.099974185
Average (Standard dev.)9.245768e-05 (±0.002775441)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 269.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: membrane protein

Fileemd_26711_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_26711_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 : 16:0 CoA and SHH-N-bound human HHAT

EntireName: 16:0 CoA and SHH-N-bound human HHAT
Components
  • Complex: 16:0 CoA and SHH-N-bound human HHAT
    • Complex: Protein-cysteine N-palmitoyltransferase HHAT (E.C.2.3.1.-), SHH-N peptide
      • Protein or peptide: Protein-cysteine N-palmitoyltransferase HHAT
      • Protein or peptide: SHH-N peptide
    • Complex: 3H02 heavy chain, 3H02 light chain
      • Protein or peptide: 3H02 heavy chain
      • Protein or peptide: 3H02 light chain
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: Palmitoyl-CoA
  • Ligand: Digitonin

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Supramolecule #1: 16:0 CoA and SHH-N-bound human HHAT

SupramoleculeName: 16:0 CoA and SHH-N-bound human HHAT / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#4

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Supramolecule #2: Protein-cysteine N-palmitoyltransferase HHAT (E.C.2.3.1.-), SHH-N...

SupramoleculeName: Protein-cysteine N-palmitoyltransferase HHAT (E.C.2.3.1.-), SHH-N peptide
type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Supramolecule #3: 3H02 heavy chain, 3H02 light chain

SupramoleculeName: 3H02 heavy chain, 3H02 light chain / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: Protein-cysteine N-palmitoyltransferase HHAT

MacromoleculeName: Protein-cysteine N-palmitoyltransferase HHAT / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Transferases; Acyltransferases; Transferring groups other than aminoacyl groups
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 58.205609 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MLPRWELALY LLASLGFHFY SFYEVYKVSR EHEEELDQEF ELETDTLFGG LKKDATDFEW SFWMEWGKQW LVWLLLGHMV VSQMATLLA RKHRPWILML YGMWACWCVL GTPGVAMVLL HTTISFCVAQ FRSQLLTWLC SLLLLSTLRL QGVEEVKRRW Y KTENEYYL ...String:
MLPRWELALY LLASLGFHFY SFYEVYKVSR EHEEELDQEF ELETDTLFGG LKKDATDFEW SFWMEWGKQW LVWLLLGHMV VSQMATLLA RKHRPWILML YGMWACWCVL GTPGVAMVLL HTTISFCVAQ FRSQLLTWLC SLLLLSTLRL QGVEEVKRRW Y KTENEYYL LQFTLTVRCL YYTSFSLELC WQQLPAASTS YSFPWMLAYV FYYPVLHNGP ILSFSEFIKQ MQQQEHDSLK AS LCVLALG LGRLLCWWWL AELMAHLMYM HAIYSSIPLL ETVSCWTLGG LALAQVLFFY VKYLVLFGVP ALLMRLDGLT PPA LPRCVS TMFSFTGMWR YFDVGLHNFL IRYVYIPVGG SQHGLLGTLF STAMTFAFVS YWCGGYDYLW CWAALNWLGV TVEN GVRRL VETPCIQDSL ARYFSPQARR RFHAALASCS TSMLILSNLV FLGGNEVGKT YWNRIFIQGW PWVTLSVLGF LYCYS HVGI AWAQTYATDD YKDDDK

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Macromolecule #2: SHH-N peptide

MacromoleculeName: SHH-N peptide / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 693.796 Da
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
CGPGRGF

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Macromolecule #3: 3H02 heavy chain

MacromoleculeName: 3H02 heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 27.352734 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGWSCIILFL VATATGVHSQ IQLVQSGPEL KKPGETVKIS CKASGYTFTN YGMNWVRQAP GKGLKWMGWI NTYTGEPTYA DDFKGRFAF SLETSASTAY LQINNLKDED MATYFCARVW NYDYYFDYWG QGTTLTVSSA STKGPSVFPL APSSKSTSGG T AALGCLVK ...String:
MGWSCIILFL VATATGVHSQ IQLVQSGPEL KKPGETVKIS CKASGYTFTN YGMNWVRQAP GKGLKWMGWI NTYTGEPTYA DDFKGRFAF SLETSASTAY LQINNLKDED MATYFCARVW NYDYYFDYWG QGTTLTVSSA STKGPSVFPL APSSKSTSGG T AALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KS CDKTHHH HHH

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Macromolecule #4: 3H02 light chain

MacromoleculeName: 3H02 light chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 26.239471 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGWSCIILFL VATARTGVHS DVLMTQTPLS LPVSLGDQVS ISCRSSQSIV HSNGNTYLEW YLQKPGQSPK LLIYRVSNRF SGVPDRFSG SGSGTDFTLK ISRVEAEDLG VYYCFQGSHV PWTFGGGTKL EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC L LNNFYPRE ...String:
MGWSCIILFL VATARTGVHS DVLMTQTPLS LPVSLGDQVS ISCRSSQSIV HSNGNTYLEW YLQKPGQSPK LLIYRVSNRF SGVPDRFSG SGSGTDFTLK ISRVEAEDLG VYYCFQGSHV PWTFGGGTKL EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC L LNNFYPRE AKVQWKVDNA LQSGNSQESV TEQDSKDSTY SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C

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Macromolecule #5: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 5 / Number of copies: 1 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE / Heme B

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Macromolecule #6: Palmitoyl-CoA

MacromoleculeName: Palmitoyl-CoA / type: ligand / ID: 6 / Number of copies: 2 / Formula: PKZ
Molecular weightTheoretical: 1.005943 KDa
Chemical component information

ChemComp-PKZ:
Palmitoyl-CoA / Palmitoyl-CoA

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Macromolecule #7: Digitonin

MacromoleculeName: Digitonin / type: ligand / ID: 7 / Number of copies: 5 / Formula: AJP
Molecular weightTheoretical: 1.229312 KDa
Chemical component information

ChemComp-AJP:
Digitonin / detergent*YM / Digitonin

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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 8.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 274370

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