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- PDB-7q1u: Structure of Hedgehog acyltransferase (HHAT) in complex with mega... -

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Basic information

Entry
Database: PDB / ID: 7q1u
TitleStructure of Hedgehog acyltransferase (HHAT) in complex with megabody 177 bound to non-hydrolysable palmitoyl-CoA (Composite Map)
Components
  • Megabody 177
  • Protein-cysteine N-palmitoyltransferase HHAT
KeywordsMEMBRANE PROTEIN / HHAT / inhibitor / palmitoyl-CoA / co enzyme A / Hedgehog acyl transferase / Sonic Hedgehog / SHH / MBOAT / morphogen / palmitoylation / signalling / endoplasmic reticulum / heme / small molecule binding / drug target
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 / endoplasmic reticulum membrane / GTP binding ...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 / endoplasmic reticulum membrane / GTP binding / Golgi apparatus / endoplasmic reticulum
Similarity search - Function
Membrane bound O-acyl transferase, MBOAT / MBOAT, membrane-bound O-acyltransferase family
Similarity search - Domain/homology
CHOLESTEROL / Chem-HD6 / PROTOPORPHYRIN IX CONTAINING FE / PALMITIC ACID / Protein-cysteine N-palmitoyltransferase HHAT
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsCoupland, C. / Carrique, L. / Siebold, C.
Funding supportEuropean Union, 4items
OrganizationGrant numberCountry
Cancer Research UKC20724/A26752European Union
Cancer Research UKC20724/A14414European Union
Biotechnology and Biological Sciences Research Council (BBSRC)BB/T01508X/1European Union
European Research Council (ERC)647278European Union
CitationJournal: Mol Cell / Year: 2021
Title: Structure, mechanism, and inhibition of Hedgehog acyltransferase.
Authors: Claire E Coupland / Sebastian A Andrei / T Bertie Ansell / Loic Carrique / Pramod Kumar / Lea Sefer / Rebekka A Schwab / Eamon F X Byrne / Els Pardon / Jan Steyaert / Anthony I Magee / ...Authors: Claire E Coupland / Sebastian A Andrei / T Bertie Ansell / Loic Carrique / Pramod Kumar / Lea Sefer / Rebekka A Schwab / Eamon F X Byrne / Els Pardon / Jan Steyaert / Anthony I Magee / Thomas Lanyon-Hogg / Mark S P Sansom / Edward W Tate / Christian Siebold /
Abstract: The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate ...The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.
History
DepositionOct 21, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 8, 2022Provider: repository / Type: Initial release

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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: Megabody 177
hetero molecules


Theoretical massNumber of molelcules
Total (without water)163,7008
Polymers161,1682
Non-polymers2,5326
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein / Antibody , 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: 58566.160 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HHAT, MART2, SKI1 / Plasmid: pHR-CMV-TetO2 / Cell line (production host): HEK293S GnTI-/- / Production host: Homo sapiens (human)
References: UniProt: Q5VTY9, Transferases; Acyltransferases; Transferring groups other than aminoacyl groups
#2: Antibody Megabody 177


Mass: 102601.586 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Plasmid: pMESP23E2 / Production host: Escherichia coli (E. coli) / Strain (production host): WK6

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Non-polymers , 5 types, 6 molecules

#3: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O
#4: 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
#5: Chemical ChemComp-HD6 / [[(2~{R},3~{S},4~{R},5~{R})-5-(6-aminopurin-9-yl)-4-oxidanyl-3-phosphonooxy-oxolan-2-yl]methoxy-oxidanyl-phosphoryl] [(3~{S})-4-[[3-(2-hexadecylsulfanylethylamino)-3-oxidanylidene-propyl]amino]-2,2-dimethyl-3-oxidanyl-4-oxidanylidene-butyl] hydrogen phosphate


Mass: 991.959 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C37H68N7O16P3S / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-PLM / PALMITIC ACID


Mass: 256.424 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C16H32O2

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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
1Hedgehog acyltransferase (HHAT) bound to non hydrolysable palmitoyl-CoA in complex with megabody 177COMPLEX#1-#20MULTIPLE SOURCES
2Megabody 177COMPLEX#21RECOMBINANT
3Hedgehog acyltransferase (HHAT) bound to non hydrolysable palmitoyl-CoACOMPLEX#11RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
32Escherichia coli K-12 (bacteria)83333
43Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainCellPlasmid
32Escherichia coli (E. coli)562WK6pMESP23E2
43Homo sapiens (human)9606HEK293S GnTI-/-pHR-CMV-TetO2
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HCl1
2150 mMsodium chlorideNaCl1
35 mM16:0 Ether Coenzyme AC37H77N10O16P3S1
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K

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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 / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 54.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3637
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4cryoSPARCV3.1.1CTF correction
7UCSF ChimeraX1.2model fitting
9cryoSPARCV3.1.1initial Euler assignment
10cryoSPARCV3.1.1final Euler assignment
11RELION3.1.1classification
12cryoSPARCV3.1.13D reconstruction
13PHENIX1.19.2model refinement
14Coot0.9.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 977000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 238000 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 56 / Protocol: AB INITIO MODEL / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 50.91 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003211444
ELECTRON MICROSCOPYf_angle_d0.484515575
ELECTRON MICROSCOPYf_chiral_restr0.03851636
ELECTRON MICROSCOPYf_plane_restr0.00311956
ELECTRON MICROSCOPYf_dihedral_angle_d5.82741621

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