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- PDB-7mhy: Human Hedgehog acyltransferase (HHAT) in complex with palmitoyl-C... -

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Basic information

Entry
Database: PDB / ID: 7mhy
TitleHuman Hedgehog acyltransferase (HHAT) in complex with palmitoyl-CoA and two Fab antibody fragments
Components
  • 1C06 Fab heavy chain
  • 1C06 Fab light chain
  • 3H02 Fab heavy chain
  • 3H02 Fab light chain
  • Protein-cysteine N-palmitoyltransferase HHAT
KeywordsTRANSFERASE/IMMUNE SYSTEM / MBOAT / GOAT / porcupine / acyl transferase / membrane protein / membrane enzyme / ER / palmitoyl-CoA / TRANSFERASE-IMMUNE SYSTEM 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: 2.7 Å
AuthorsLong, S.B. / Jiang, Y.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM131921 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)P30CA008748 United States
CitationJournal: Science / Year: 2021
Title: Substrate and product complexes reveal mechanisms of Hedgehog acylation by HHAT.
Authors: Yiyang Jiang / Thomas L Benz / Stephen B Long /
Abstract: Hedgehog proteins govern crucial developmental steps in animals and drive certain human cancers. Before they can function as signaling molecules, Hedgehog precursor proteins must undergo amino- ...Hedgehog proteins govern crucial developmental steps in animals and drive certain human cancers. Before they can function as signaling molecules, Hedgehog precursor proteins must undergo amino-terminal palmitoylation by Hedgehog acyltransferase (HHAT). We present cryo-electron microscopy structures of human HHAT in complex with its palmitoyl-coenzyme A substrate and of a product complex with a palmitoylated Hedgehog peptide at resolutions of 2.7 and 3.2 angstroms, respectively. The structures reveal how HHAT overcomes the challenges of bringing together substrates that have different physiochemical properties from opposite sides of the endoplasmic reticulum membrane within a membrane-embedded active site for catalysis. These principles are relevant to related enzymes that catalyze the acylation of Wnt and of the appetite-stimulating hormone ghrelin. The structural and mechanistic insights may advance the development of inhibitors for cancer.
History
DepositionApr 16, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 16, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 14, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 2.0Aug 25, 2021Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references
Category: atom_site / database_2 ...atom_site / database_2 / database_PDB_caveat / pdbx_unobs_or_zero_occ_atoms / pdbx_validate_chiral
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_atom_id / _atom_site.label_atom_id / _atom_site.type_symbol / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_unobs_or_zero_occ_atoms.auth_atom_id / _pdbx_unobs_or_zero_occ_atoms.label_atom_id

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Structure visualization

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Assembly

Deposited unit
A: Protein-cysteine N-palmitoyltransferase HHAT
M: 1C06 Fab heavy chain
N: 1C06 Fab light chain
O: 3H02 Fab heavy chain
P: 3H02 Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)170,04723
Polymers149,2025
Non-polymers20,84518
Water27015
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7390 Å2
ΔGint-59 kcal/mol
Surface area42030 Å2

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Components

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Protein , 1 types, 1 molecules A

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


Mass: 57358.742 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

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Antibody , 4 types, 4 molecules MNOP

#2: Antibody 1C06 Fab heavy chain


Mass: 22847.662 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#3: Antibody 1C06 Fab light chain


Mass: 22897.879 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#4: Antibody 3H02 Fab heavy chain


Mass: 23229.082 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#5: Antibody 3H02 Fab light chain


Mass: 22868.863 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)

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Non-polymers , 4 types, 33 molecules

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


Mass: 616.487 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-PKZ / Palmitoyl-CoA / Palmitoyl-CoA


Mass: 1005.943 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C37H66N7O17P3S / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical
ChemComp-AJP / Digitonin / Digitonin


Mass: 1229.312 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: C56H92O29 / Comment: detergent*YM
#9: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 15 / Source method: isolated from a natural source / Formula: H2O

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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

ComponentName: HHAT in complex with palmitoyl-CoA and two Fab antibody fragments
Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Mus musculus (house mouse)
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
Image recordingElectron dose: 26.5 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874+SVNrefinement
PHENIX1.18.2_3874+SVNrefinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 174058 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 45.44 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00738203
ELECTRON MICROSCOPYf_angle_d0.842811363
ELECTRON MICROSCOPYf_chiral_restr0.061335
ELECTRON MICROSCOPYf_plane_restr0.00471245
ELECTRON MICROSCOPYf_dihedral_angle_d17.6343065

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