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- PDB-7zay: Human heparan sulfate polymerase complex EXT1-EXT2 -

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Basic information

Entry
Database: PDB / ID: 7zay
TitleHuman heparan sulfate polymerase complex EXT1-EXT2
Components
  • Exostosin-1
  • Exostosin-2
KeywordsTRANSFERASE / Heparan sulfate biosynthesis / Enzyme complex / Glycosyltransferase / Golgi
Function / homology
Function and homology information


glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase / N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase / heparan sulfate N-acetylglucosaminyltransferase activity / glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase activity / N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase activity / UDP-N-acetylglucosamine transferase complex / glucuronosyltransferase activity / Defective EXT2 causes exostoses 2 / Defective EXT1 causes exostoses 1, TRPS2 and CHDS / glycosaminoglycan biosynthetic process ...glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase / N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase / heparan sulfate N-acetylglucosaminyltransferase activity / glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase activity / N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase activity / UDP-N-acetylglucosamine transferase complex / glucuronosyltransferase activity / Defective EXT2 causes exostoses 2 / Defective EXT1 causes exostoses 1, TRPS2 and CHDS / glycosaminoglycan biosynthetic process / HS-GAG biosynthesis / heparan sulfate proteoglycan biosynthetic process / acetylglucosaminyltransferase activity / polysaccharide biosynthetic process / glycosyltransferase activity / catalytic complex / ossification / protein heterodimerization activity / Golgi membrane / endoplasmic reticulum membrane / Golgi apparatus / endoplasmic reticulum / protein homodimerization activity / extracellular exosome / membrane / metal ion binding
Similarity search - Function
Exostosin-like / Exostosin, GT47 domain / Exostosin GT47 domain / Glycosyl transferase 64 domain / Glycosyl transferase family 64 domain / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
URIDINE-5'-DIPHOSPHATE / Exostosin-1 / Exostosin-2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsLeisico, F. / Omeiri, J. / Hons, M. / Schoehn, G. / Lortat-Jacob, H. / Wild, R.
Funding support France, 3items
OrganizationGrant numberCountry
Centre National de la Recherche Scientifique (CNRS)ATIP-Avenir France
Agence Nationale de la Recherche (ANR)ANR-18-CE11- 0006-01 France
Agence Nationale de la Recherche (ANR)ANR-15-IDEX-02 France
CitationJournal: Nat Commun / Year: 2022
Title: Structure of the human heparan sulfate polymerase complex EXT1-EXT2.
Authors: Francisco Leisico / Juneina Omeiri / Christine Le Narvor / Joël Beaudouin / Michael Hons / Daphna Fenel / Guy Schoehn / Yohann Couté / David Bonnaffé / Rabia Sadir / Hugues Lortat-Jacob / Rebekka Wild /
Abstract: Heparan sulfates are complex polysaccharides that mediate the interaction with a broad range of protein ligands at the cell surface. A key step in heparan sulfate biosynthesis is catalyzed by the bi- ...Heparan sulfates are complex polysaccharides that mediate the interaction with a broad range of protein ligands at the cell surface. A key step in heparan sulfate biosynthesis is catalyzed by the bi-functional glycosyltransferases EXT1 and EXT2, which generate the glycan backbone consisting of repeating N-acetylglucosamine and glucuronic acid units. The molecular mechanism of heparan sulfate chain polymerization remains, however, unknown. Here, we present the cryo-electron microscopy structure of human EXT1-EXT2, which reveals the formation of a tightly packed hetero-dimeric complex harboring four glycosyltransferase domains. A combination of in vitro and in cellulo mutational studies is used to dissect the functional role of the four catalytic sites. While EXT1 can catalyze both glycosyltransferase reactions, our results indicate that EXT2 might only have N-acetylglucosamine transferase activity. Our findings provide mechanistic insight into heparan sulfate chain elongation as a nonprocessive process and lay the foundation for future studies on EXT1-EXT2 function in health and disease.
History
DepositionMar 23, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 7, 2022Provider: repository / Type: Initial release
Revision 1.0Dec 7, 2022Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.0Dec 7, 2022Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Dec 7, 2022Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Dec 7, 2022Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Revision 1.0Dec 7, 2022Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Dec 7, 2022Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 7, 2022Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 7, 2022Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 7, 2022Data content type: Mask / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Dec 7, 2022Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Nov 6, 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
Revision 1.2Jul 2, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1Jul 2, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Exostosin-1
B: Exostosin-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)165,5315
Polymers164,3192
Non-polymers1,2123
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, gel filtration, mass spectrometry, light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9110 Å2
ΔGint-28 kcal/mol
Surface area45750 Å2

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Components

#1: Protein Exostosin-1 / Glucuronosyl-N-acetylglucosaminyl-proteoglycan/N-acetylglucosaminyl-proteoglycan 4-alpha-N- ...Glucuronosyl-N-acetylglucosaminyl-proteoglycan/N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase / Multiple exostoses protein 1 / Putative tumor suppressor protein EXT1


Mass: 85261.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EXT1 / Cell line (production host): Freestyle 293-F / Production host: Homo sapiens (human)
References: UniProt: Q16394, glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase, N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase
#2: Protein Exostosin-2 / Glucuronosyl-N-acetylglucosaminyl-proteoglycan/N-acetylglucosaminyl-proteoglycan 4-alpha-N- ...Glucuronosyl-N-acetylglucosaminyl-proteoglycan/N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase / Multiple exostoses protein 2 / Putative tumor suppressor protein EXT2


Mass: 79056.836 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EXT2 / Cell line (production host): Freestyle 293-F / Production host: Homo sapiens (human)
References: UniProt: Q93063, glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase, N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase
#3: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#5: Chemical ChemComp-UDP / URIDINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 404.161 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H14N2O12P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: UDP*YM
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: Hetero-dimeric complex EXT1-EXT2 / Type: COMPLEX
Details: local resolution-filtered map generated using a B-factor of -30 A2
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.16 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Freestyle 293-F
Buffer solutionpH: 7.2
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Purified EXT1-EXT2 complex at 0.4mg/mL was mixed with 1mM of UDP-GlcNAc, 1mM UDP-GlcA and 1mM MnCl2 and incubated 15 min on ice before applying 4 uL of the sample onto a glow discharged ...Details: Purified EXT1-EXT2 complex at 0.4mg/mL was mixed with 1mM of UDP-GlcNAc, 1mM UDP-GlcA and 1mM MnCl2 and incubated 15 min on ice before applying 4 uL of the sample onto a glow discharged Quantifoil holey carbon grid (R1.2/1.3, 300 mesh, copper).
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 46 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 286390 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0029585
ELECTRON MICROSCOPYf_angle_d0.58813029
ELECTRON MICROSCOPYf_dihedral_angle_d11.8543493
ELECTRON MICROSCOPYf_chiral_restr0.0431433
ELECTRON MICROSCOPYf_plane_restr0.0111650

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