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- PDB-7urf: Human HHAT H379C in complex with SHH N-terminal peptide -

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Basic information

Entry
Database: PDB / ID: 7urf
TitleHuman HHAT H379C in complex with SHH N-terminal peptide
Components
  • 3H02 heavy chain
  • 3H02 light chain
  • Protein-cysteine N-palmitoyltransferase HHAT
  • SHH-N peptide
KeywordsTRANSFERASE/TRANSFERASE INHIBITOR / CoA-bound / substrate-bound / complex / TRANSFERASE / TRANSFERASE-TRANSFERASE SUBSTRATE complex / TRANSFERASE-TRANSFERASE INHIBITOR complex
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
PROTOPORPHYRIN IX CONTAINING FE / Palmitoyl-CoA / Protein-cysteine N-palmitoyltransferase HHAT
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 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: Protein-cysteine N-palmitoyltransferase HHAT
B: SHH-N peptide
O: 3H02 heavy chain
P: 3H02 light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)121,26712
Polymers112,4924
Non-polymers8,7758
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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Antibody , 2 types, 2 molecules OP

#3: Antibody 3H02 heavy chain


Mass: 27352.734 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 3H02 light chain


Mass: 26239.471 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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Protein / Protein/peptide , 2 types, 2 molecules AB

#1: Protein Protein-cysteine N-palmitoyltransferase HHAT / Hedgehog acyltransferase / Melanoma antigen recognized by T-cells 2 / MART-2 / Skinny hedgehog protein 1


Mass: 58205.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HHAT, MART2, SKI1 / Production host: Homo sapiens (human)
References: UniProt: Q5VTY9, Transferases; Acyltransferases; Transferring groups other than aminoacyl groups
#2: Protein/peptide SHH-N peptide


Mass: 693.796 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)

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Non-polymers , 3 types, 8 molecules

#5: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME / Heme B


Mass: 616.487 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#6: Chemical ChemComp-PKZ / Palmitoyl-CoA / Palmitoyl-CoA


Mass: 1005.943 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C37H66N7O17P3S
#7: Chemical
ChemComp-AJP / Digitonin / Digitonin


Mass: 1229.312 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C56H92O29 / Comment: detergent*YM

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Details

Has ligand of interestN

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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
116:0 CoA and SHH-N-bound human HHATCOMPLEX#1-#40RECOMBINANT
2Protein-cysteine N-palmitoyltransferase HHAT (E.C.2.3.1.-), SHH-N peptideCOMPLEX#1-#21RECOMBINANT
33H02 heavy chain, 3H02 light chainCOMPLEX#3-#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo 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: 800 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: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 274370 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036354
ELECTRON MICROSCOPYf_angle_d0.6428715
ELECTRON MICROSCOPYf_dihedral_angle_d7.6473478
ELECTRON MICROSCOPYf_chiral_restr0.041948
ELECTRON MICROSCOPYf_plane_restr0.0041014

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