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- PDB-7mhz: Human Hedgehog acyltransferase (HHAT) in complex with a palmitoyl... -

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Basic information

Entry
Database: PDB / ID: 7mhz
TitleHuman Hedgehog acyltransferase (HHAT) in complex with a palmitoylated Hedgehog peptide product and a Fab antibody fragment
Components
  • 3H02 Fab heavy chain
  • 3H02 Fab light chain
  • Protein-cysteine N-palmitoyltransferase HHAT
  • Sonic hedgehog protein N-product peptide
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 / regulation of nodal signaling pathway / trunk neural crest cell migration / : / positive regulation of skeletal muscle cell proliferation / right lung development / left lung development / primary prostatic bud elongation / : / mesenchymal smoothened signaling pathway involved in prostate gland development ...N-terminal peptidyl-L-cysteine N-palmitoylation / regulation of nodal signaling pathway / trunk neural crest cell migration / : / positive regulation of skeletal muscle cell proliferation / right lung development / left lung development / primary prostatic bud elongation / : / mesenchymal smoothened signaling pathway involved in prostate gland development / positive regulation of sclerotome development / tracheoesophageal septum formation / negative regulation of ureter smooth muscle cell differentiation / positive regulation of ureter smooth muscle cell differentiation / negative regulation of kidney smooth muscle cell differentiation / positive regulation of kidney smooth muscle cell differentiation / morphogen activity / regulation of odontogenesis / positive regulation of mesenchymal cell proliferation involved in ureter development / Formation of lateral plate mesoderm / regulation of glial cell proliferation / hindgut morphogenesis / polarity specification of anterior/posterior axis / negative regulation of alpha-beta T cell differentiation / regulation of prostatic bud formation / formation of anatomical boundary / positive regulation of striated muscle cell differentiation / ventral midline development / metanephric mesenchymal cell proliferation involved in metanephros development / trachea morphogenesis / cholesterol-protein transferase activity / HHAT G278V doesn't palmitoylate Hh-Np / telencephalon regionalization / bud outgrowth involved in lung branching / epithelial-mesenchymal cell signaling / Ligand-receptor interactions / laminin-1 binding / lung epithelium development / salivary gland cavitation / negative regulation of mesenchymal cell apoptotic process / spinal cord dorsal/ventral patterning / determination of left/right asymmetry in lateral mesoderm / negative regulation of cholesterol efflux / establishment of epithelial cell polarity / skeletal muscle cell proliferation / spinal cord motor neuron differentiation / negative regulation of T cell differentiation in thymus / positive regulation of T cell differentiation in thymus / palmitoyltransferase activity / cell development / prostate gland development / intermediate filament organization / skeletal muscle fiber differentiation / embryonic skeletal system development / stem cell development / mesenchymal cell apoptotic process / positive regulation of cerebellar granule cell precursor proliferation / limb bud formation / animal organ formation / patched binding / Developmental Lineage of Multipotent Pancreatic Progenitor Cells / positive regulation of skeletal muscle tissue development / somite development / hindbrain development / embryonic digestive tract morphogenesis / ectoderm development / cerebellar granule cell precursor proliferation / embryonic foregut morphogenesis / epithelial cell proliferation involved in salivary gland morphogenesis / neuron fate commitment / mesenchymal cell proliferation involved in lung development / negative regulation of dopaminergic neuron differentiation / Activation of SMO / self proteolysis / positive regulation of immature T cell proliferation in thymus / regulation of stem cell proliferation / positive regulation of astrocyte differentiation / smooth muscle tissue development / artery development / lung lobe morphogenesis / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / lymphoid progenitor cell differentiation / epithelial cell proliferation involved in prostate gland development / positive regulation of epithelial cell proliferation involved in prostate gland development / negative thymic T cell selection / male genitalia development / pattern specification process / Release of Hh-Np from the secreting cell / branching involved in salivary gland morphogenesis / glycosaminoglycan binding / lung-associated mesenchyme development / dopaminergic neuron differentiation / embryonic pattern specification / metanephros development / Formation of axial mesoderm / intein-mediated protein splicing / thalamus development / metanephric collecting duct development / camera-type eye development / positive thymic T cell selection
Similarity search - Function
: / Hedgehog, N-terminal signalling domain / Hedgehog protein / Hedgehog protein, Hint domain / : / Hint module / Hedgehog amino-terminal signalling domain / Membrane bound O-acyl transferase, MBOAT / MBOAT, membrane-bound O-acyltransferase family / Hedgehog signalling/DD-peptidase zinc-binding domain superfamily ...: / Hedgehog, N-terminal signalling domain / Hedgehog protein / Hedgehog protein, Hint domain / : / Hint module / Hedgehog amino-terminal signalling domain / Membrane bound O-acyl transferase, MBOAT / MBOAT, membrane-bound O-acyltransferase family / Hedgehog signalling/DD-peptidase zinc-binding domain superfamily / Hint domain C-terminal / Hint (Hedgehog/Intein) domain C-terminal region / Intein N-terminal splicing region / Intein N-terminal splicing motif profile. / Hint domain N-terminal / Hint (Hedgehog/Intein) domain N-terminal region / Hint domain superfamily
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / Palmitoyl-CoA / PALMITIC ACID / Sonic hedgehog protein / Protein-cysteine N-palmitoyltransferase HHAT
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 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
Revision 2.1Oct 9, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Assembly

Deposited unit
B: Sonic hedgehog protein N-product peptide
A: Protein-cysteine N-palmitoyltransferase HHAT
O: 3H02 Fab heavy chain
P: 3H02 Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)148,47019
Polymers132,2864
Non-polymers16,18415
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6370 Å2
ΔGint-46 kcal/mol
Surface area32150 Å2

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Components

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Antibody , 2 types, 2 molecules OP

#3: Antibody 3H02 Fab heavy chain


Mass: 51307.891 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 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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Protein/peptide / Protein , 2 types, 2 molecules BA

#1: Protein/peptide Sonic hedgehog protein N-product peptide / ShhN / Shh N-terminal processed signaling domains / ShhNp


Mass: 750.848 Da / Num. of mol.: 1 / Fragment: UNP residues 24-31 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: Q15465
#2: 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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Non-polymers , 4 types, 15 molecules

#5: Chemical ChemComp-PLM / PALMITIC ACID


Mass: 256.424 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H32O2 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


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


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


Mass: 1229.312 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C56H92O29 / Comment: detergent*YM

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Details

Has ligand of interestY
Has protein modificationY

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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 a palmitoylated Hedgehog peptide product and a Fab antibody fragment
Type: COMPLEX / Entity ID: #1-#4 / 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 FIELD
Image recordingElectron dose: 43.5 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
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 142121 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 112.57 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00366436
ELECTRON MICROSCOPYf_angle_d0.66858909
ELECTRON MICROSCOPYf_chiral_restr0.04871019
ELECTRON MICROSCOPYf_plane_restr0.0039993
ELECTRON MICROSCOPYf_dihedral_angle_d18.91422315

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