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- PDB-9o4g: Cryo-EM structure of the CHSY3-CHPF1 chondroitin synthase heterodimer -

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Basic information

Entry
Database: PDB / ID: 9o4g
TitleCryo-EM structure of the CHSY3-CHPF1 chondroitin synthase heterodimer
Components
  • Chondroitin sulfate synthase 2
  • Chondroitin sulfate synthase 3
KeywordsSUGAR BINDING PROTEIN / TRANSFERASE / Chondroitin sulfate / Chondroitin sulfate synthase
Function / homology
Function and homology information


glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-beta-N-acetylgalactosaminyltransferase / N-acetylgalactosaminyl-proteoglycan 3-beta-glucuronosyltransferase / glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-beta-N-acetylgalactosaminyltransferase activity / N-acetylgalactosaminyl-proteoglycan 3-beta-glucuronosyltransferase activity / CS-GAG biosynthesis / chondroitin sulfate proteoglycan biosynthetic process / Golgi cisterna membrane / mitochondrial matrix / Golgi membrane / metal ion binding / cytosol
Similarity search - Function
Chondroitin N-acetylgalactosaminyltransferase / : / Chondroitin N-acetylgalactosaminyltransferase / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
: / URIDINE-5'-DIPHOSPHATE / Chondroitin sulfate synthase 3 / Chondroitin polymerizing factor, non-catalytic subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.42 Å
AuthorsTehrani, D. / Cortiella, N. / Perez, C. / Moremen, K.
Funding support United States, Switzerland, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM154846 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM130915 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM103390 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM158187 United States
Swiss National Science Foundation310030_207974 Switzerland
CitationJournal: Nat Commun / Year: 2026
Title: Structural basis of chondroitin sulfate backbone polymer synthesis.
Authors: Daniel Tehrani / Nil Cortiella-Valls / Chin Huang / Digantkumar Chapla / Zhifeng Zheng / Aarya Venkat / Brady O'Boyle / Natarajan Kannan / Camilo Perez / Kelley W Moremen /
Abstract: Chondroitin sulfate (CS) proteoglycans are extended (-GlcAβ1,3GalNAcβ1,4-) co-polymers attached to cell surface and extracellular matrix core proteins that are further modified by extensive ...Chondroitin sulfate (CS) proteoglycans are extended (-GlcAβ1,3GalNAcβ1,4-) co-polymers attached to cell surface and extracellular matrix core proteins that are further modified by extensive sulfation and epimerization. Four homologous proteins contribute to CS backbone synthesis (CHPF1, CHPF2, CHSY1, and CHSY3) and prior data suggests assembly of the proteins into heterocomplexes is required for function. Here we show by sequence alignment and structural modeling that all CHSYs and CHPFs contain an N-terminal CAZy GT31-like domain and a C-terminal GT7-like domain separated by a cystatin-like linker domain. Co-expression of one CHPF and one CHSY is required to form a soluble, functional heterodimeric CS synthase and structural modeling indicates all four potential CHSY-CHPF combinations can form equivalent heterodimeric complexes. Cryo-EM studies on CHSY3-CHPF1 confirm the structure, interface, and active site features predicted by the structural models. Enzymatic analyses of catalytic mutants demonstrate that only the glycosyltransferase domains in the CHSYs are responsible for polymer synthesis: the GT31 domain transfers β1,3-GlcA while the GT7 domain transfers β1,4-GalNAc. The corresponding CHPF domains do not contribute to polymer synthesis but stabilize the corresponding CHSY functional domains. Additional mutagenesis and modeling suggest that the bridging cystatin-like domains may contribute to efficient polymer synthesis.
History
DepositionApr 8, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 4, 2026Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Chondroitin sulfate synthase 3
B: Chondroitin sulfate synthase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)179,5985
Polymers179,0842
Non-polymers5143
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Chondroitin sulfate synthase 3 / Carbohydrate synthase 2 / Chondroitin glucuronyltransferase 3 / Chondroitin synthase 2 / ChSy-2 / ...Carbohydrate synthase 2 / Chondroitin glucuronyltransferase 3 / Chondroitin synthase 2 / ChSy-2 / Glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-beta-N-acetylgalactosaminyltransferase II / N-acetylgalactosaminyl-proteoglycan 3-beta-glucuronosyltransferase 3 / N-acetylgalactosaminyltransferase 3


Mass: 97018.109 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CHSY3, CHSY2, CSS3 / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
References: UniProt: Q70JA7, glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-beta-N-acetylgalactosaminyltransferase, N-acetylgalactosaminyl-proteoglycan 3-beta-glucuronosyltransferase
#2: Protein Chondroitin sulfate synthase 2 / Chondroitin glucuronyltransferase 2 / Chondroitin-polymerizing factor / ChPF / Glucuronosyl-N- ...Chondroitin glucuronyltransferase 2 / Chondroitin-polymerizing factor / ChPF / Glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-beta-N-acetylgalactosaminyltransferase II / N-acetylgalactosaminyl-proteoglycan 3-beta-glucuronosyltransferase II / N-acetylgalactosaminyltransferase 2


Mass: 82065.930 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CHPF, CSS2, UNQ651/PRO1281 / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
References: UniProt: Q8IZ52, glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-beta-N-acetylgalactosaminyltransferase, N-acetylgalactosaminyl-proteoglycan 3-beta-glucuronosyltransferase
#3: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
#4: 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: CHSY3-CHPF1 heterocomplex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS TALOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3500 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 54.9 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
4cryoSPARC4.5.1CTF correction
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 163527 / Symmetry type: POINT
RefinementHighest resolution: 3.42 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311266
ELECTRON MICROSCOPYf_angle_d0.53815273
ELECTRON MICROSCOPYf_dihedral_angle_d4.6751525
ELECTRON MICROSCOPYf_chiral_restr0.0411636
ELECTRON MICROSCOPYf_plane_restr0.0052004

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