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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 / hypersensitivity / heart field specification / lymphocyte adhesion to endothelial cell of high endothelial venule / heparan sulfate N-acetylglucosaminyltransferase activity / glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase activity / N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase activity / smoothened signaling pathway involved in lung development / developmental growth involved in morphogenesis ...glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase / N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase / hypersensitivity / heart field specification / lymphocyte adhesion to endothelial cell of high endothelial venule / heparan sulfate N-acetylglucosaminyltransferase activity / glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase activity / N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase activity / smoothened signaling pathway involved in lung development / developmental growth involved in morphogenesis / sweat gland development / perichondral bone morphogenesis / mesenchymal cell differentiation involved in bone development / response to leukemia inhibitory factor / : / UDP-N-acetylglucosamine transferase complex / chondrocyte hypertrophy / embryonic skeletal joint development / hematopoietic stem cell migration to bone marrow / fluid transport / : / limb joint morphogenesis / tight junction organization / glucuronosyltransferase activity / Defective EXT2 causes exostoses 2 / Defective EXT1 causes exostoses 1, TRPS2 and CHDS / heparin proteoglycan biosynthetic process / stomach development / sebaceous gland development / response to heparin / glomerular basement membrane development / glycosaminoglycan biosynthetic process / HS-GAG biosynthesis / lymphocyte migration into lymphoid organs / heparan sulfate proteoglycan biosynthetic process / sulfation / chondrocyte proliferation / dendritic cell migration / endochondral bone morphogenesis / dendrite self-avoidance / glandular epithelial cell differentiation / hematopoietic stem cell homeostasis / endochondral bone growth / acetylglucosaminyltransferase activity / podocyte differentiation / basement membrane organization / sodium ion homeostasis / cartilage development involved in endochondral bone morphogenesis / vocalization behavior / cranial skeletal system development / vacuole organization / olfactory bulb development / leukocyte tethering or rolling / endochondral ossification / polysaccharide biosynthetic process / multicellular organismal-level water homeostasis / endoderm development / fear response / stem cell division / response to light intensity / protein N-linked glycosylation / regulation of tumor necrosis factor-mediated signaling pathway / collagen fibril organization / neural crest cell differentiation / ossification involved in bone maturation / motor behavior / optic nerve development / hair follicle morphogenesis / cell adhesion mediated by integrin / heart contraction / epithelial tube branching involved in lung morphogenesis / protein glycosylation / antigen processing and presentation / glycosyltransferase activity / social behavior / mesoderm development / mesoderm formation / blood vessel remodeling / cellular response to fibroblast growth factor stimulus / catalytic complex / cell fate commitment / fibroblast growth factor receptor signaling pathway / hematopoietic stem cell differentiation / canonical Wnt signaling pathway / chondrocyte differentiation / BMP signaling pathway / bone resorption / gastrulation / ossification / axon guidance / synaptic transmission, glutamatergic / wound healing / protein catabolic process / multicellular organism growth / cellular response to virus / regulation of blood pressure / vasodilation / protein-containing complex assembly / gene expression / protein heterodimerization activity
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 1.1Nov 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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
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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