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- PDB-8cd0: Human heparan sulfate N-deacetylase-N-sulfotransferase 1 in compl... -

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Basic information

Entry
Database: PDB / ID: 8cd0
TitleHuman heparan sulfate N-deacetylase-N-sulfotransferase 1 in complex with calcium, 3'-phosphoadenosine-5'-phosphosulfate, and nanobody nAb7 (composite map and model)
Components
  • Bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1
  • Nanobody nAb7
KeywordsCARBOHYDRATE / Deacetylase / Sulfotransferase / Heparan Sulfate / Glycosaminoglycan / Nanobody
Function / homology
Function and homology information


[heparan sulfate]-glucosamine N-sulfotransferase / [heparan sulfate]-glucosamine N-sulfotransferase activity / heparan sulfate N-deacetylase activity / N-acetylglucosamine deacetylase activity / heparan sulfate proteoglycan biosynthetic process, enzymatic modification / heparan sulfate proteoglycan biosynthetic process, polysaccharide chain biosynthetic process / heparin biosynthetic process / embryonic viscerocranium morphogenesis / embryonic neurocranium morphogenesis / HS-GAG biosynthesis ...[heparan sulfate]-glucosamine N-sulfotransferase / [heparan sulfate]-glucosamine N-sulfotransferase activity / heparan sulfate N-deacetylase activity / N-acetylglucosamine deacetylase activity / heparan sulfate proteoglycan biosynthetic process, enzymatic modification / heparan sulfate proteoglycan biosynthetic process, polysaccharide chain biosynthetic process / heparin biosynthetic process / embryonic viscerocranium morphogenesis / embryonic neurocranium morphogenesis / HS-GAG biosynthesis / deacetylase activity / cardiac septum development / respiratory gaseous exchange by respiratory system / coronary vasculature development / positive regulation of smoothened signaling pathway / aorta development / Hydrolases; Acting on carbon-nitrogen bonds, other than peptide bonds; In linear amides / midbrain development / fibroblast growth factor receptor signaling pathway / forebrain development / trans-Golgi network membrane / cell population proliferation / positive regulation of MAPK cascade / inflammatory response / Golgi membrane / Golgi apparatus
Similarity search - Function
Heparan sulphate-N-deacetylase / heparan sulfate-N-deacetylase / Heparan sulfate sulfotransferase / Sulfotransferase domain / Sulfotransferase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-3'-5'-DIPHOSPHATE / Bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.42 Å
AuthorsMycroft-West, C.J. / Wu, L.
Funding support United Kingdom, 6items
OrganizationGrant numberCountry
Engineering and Physical Sciences Research Council United Kingdom
Wellcome Trust060208/Z/00/Z United Kingdom
Wellcome Trust093305/Z/10/Z United Kingdom
Wellcome Trust203141/Z/16/Z United Kingdom
Wellcome Trust218579/Z/19/Z United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/V018523/1 United Kingdom
CitationJournal: Nat Commun / Year: 2024
Title: Structural and mechanistic characterization of bifunctional heparan sulfate N-deacetylase-N-sulfotransferase 1.
Authors: Courtney J Mycroft-West / Sahar Abdelkarim / Helen M E Duyvesteyn / Neha S Gandhi / Mark A Skidmore / Raymond J Owens / Liang Wu /
Abstract: Heparan sulfate (HS) polysaccharides are major constituents of the extracellular matrix, which are involved in myriad structural and signaling processes. Mature HS polysaccharides contain complex, ...Heparan sulfate (HS) polysaccharides are major constituents of the extracellular matrix, which are involved in myriad structural and signaling processes. Mature HS polysaccharides contain complex, non-templated patterns of sulfation and epimerization, which mediate interactions with diverse protein partners. Complex HS modifications form around initial clusters of glucosamine-N-sulfate (GlcNS) on nascent polysaccharide chains, but the mechanistic basis underpinning incorporation of GlcNS itself into HS remains unclear. Here, we determine cryo-electron microscopy structures of human N-deacetylase-N-sulfotransferase (NDST)1, the bifunctional enzyme primarily responsible for initial GlcNS modification of HS. Our structures reveal the architecture of both NDST1 deacetylase and sulfotransferase catalytic domains, alongside a non-catalytic N-terminal domain. The two catalytic domains of NDST1 adopt a distinct back-to-back topology that limits direct cooperativity. Binding analyses, aided by activity-modulating nanobodies, suggest that anchoring of the substrate at the sulfotransferase domain initiates the NDST1 catalytic cycle, providing a plausible mechanism for cooperativity despite spatial domain separation. Our data shed light on key determinants of NDST1 activity, and describe tools to probe NDST1 function in vitro and in vivo.
History
DepositionJan 29, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 7, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 28, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Oct 9, 2024Group: Data collection / Structure summary
Category: 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: Bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1
C: Nanobody nAb7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)119,1214
Polymers118,6542
Non-polymers4672
Water724
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, both proteins elute in a single peak
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1 / Glucosaminyl N-deacetylase/N-sulfotransferase 1 / NDST-1 / N-heparan sulfate sulfotransferase 1 / N- ...Glucosaminyl N-deacetylase/N-sulfotransferase 1 / NDST-1 / N-heparan sulfate sulfotransferase 1 / N-HSST 1 / [Heparan sulfate]-glucosamine N-sulfotransferase 1 / HSNST 1


Mass: 100997.617 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NDST1, HSST, HSST1 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P52848, Hydrolases; Acting on carbon-nitrogen bonds, other than peptide bonds; In linear amides, [heparan sulfate]-glucosamine N-sulfotransferase
#2: Antibody Nanobody nAb7


Mass: 17656.316 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#3: Chemical ChemComp-A3P / ADENOSINE-3'-5'-DIPHOSPHATE


Type: RNA linking / Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: H2O
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: Complex of heparan sulfate N-deacetylase-N-sulfotransferase 1 (with ligands PAP and Mg2+) and nanobody nAb7
Type: COMPLEX / Entity ID: #2 / Source: RECOMBINANT
Molecular weightValue: 0.1 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 6.5
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2600 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.42 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 614944 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 88.53 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00657051
ELECTRON MICROSCOPYf_angle_d1.20049565
ELECTRON MICROSCOPYf_chiral_restr0.06511016
ELECTRON MICROSCOPYf_plane_restr0.011212
ELECTRON MICROSCOPYf_dihedral_angle_d6.3022917

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