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- EMDB-16664: Human heparan sulfate N-deacetylase-N-sulfotransferase 1 in compl... -

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Basic information

Entry
Database: EMDB / ID: EMD-16664
TitleHuman heparan sulfate N-deacetylase-N-sulfotransferase 1 in complex with calcium, 3'-phosphoadenosine-5'-phosphosulfate and nanobody nAb13 (composite map and model).
Map datacomposite map of NDST1 nAb13 complex, created by combining locally refined maps for the NDST1 N-terminal and deacetylase domains, and deacetylase and sulfotransferase domains
Sample
  • Complex: Human heparan sulfate N-deacetylase-N-sulfotransferase complex with nAb13
    • Complex: Nanobody nAb13 - all CA rigid fit model derived from nanobody nAb7
      • Protein or peptide: Nanobody nAb13 - all CA rigid fit model derived from nanobody nAb7
    • Complex: N-deacetylase-N-sulfotransferase 1
      • Protein or peptide: Bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1
  • Ligand: ADENOSINE-3'-5'-DIPHOSPHATE
  • Ligand: CALCIUM ION
  • Ligand: water
KeywordsDeacetylase / Sulfotransferase / Heparan Sulfate / Carbohydrate / 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 neurocranium morphogenesis / embryonic viscerocranium 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 neurocranium morphogenesis / embryonic viscerocranium 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
Bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1
Similarity search - Component
Biological speciesHomo sapiens (human) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsMycroft-West CJ / Wu L
Funding support United Kingdom, 6 items
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
DepositionFeb 8, 2023-
Header (metadata) releaseFeb 21, 2024-
Map releaseFeb 21, 2024-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16664.png

Downloads & links

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Map

FileDownload / File: emd_16664.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcomposite map of NDST1 nAb13 complex, created by combining locally refined maps for the NDST1 N-terminal and deacetylase domains, and deacetylase and sulfotransferase domains
Voxel sizeX=Y=Z: 0.73 Å
Density
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-0.38380805 - 0.8504358
Average (Standard dev.)0.012996433 (±0.020542912)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 321.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Human heparan sulfate N-deacetylase-N-sulfotransferase complex wi...

EntireName: Human heparan sulfate N-deacetylase-N-sulfotransferase complex with nAb13
Components
  • Complex: Human heparan sulfate N-deacetylase-N-sulfotransferase complex with nAb13
    • Complex: Nanobody nAb13 - all CA rigid fit model derived from nanobody nAb7
      • Protein or peptide: Nanobody nAb13 - all CA rigid fit model derived from nanobody nAb7
    • Complex: N-deacetylase-N-sulfotransferase 1
      • Protein or peptide: Bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1
  • Ligand: ADENOSINE-3'-5'-DIPHOSPHATE
  • Ligand: CALCIUM ION
  • Ligand: water

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Supramolecule #1: Human heparan sulfate N-deacetylase-N-sulfotransferase complex wi...

SupramoleculeName: Human heparan sulfate N-deacetylase-N-sulfotransferase complex with nAb13
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: Nanobody nAb13 - all CA rigid fit model derived from nanobody nAb7

SupramoleculeName: Nanobody nAb13 - all CA rigid fit model derived from nanobody nAb7
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Lama glama (llama)

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Supramolecule #3: N-deacetylase-N-sulfotransferase 1

SupramoleculeName: N-deacetylase-N-sulfotransferase 1 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Nanobody nAb13 - all CA rigid fit model derived from nanobody nAb7

MacromoleculeName: Nanobody nAb13 - all CA rigid fit model derived from nanobody nAb7
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 17.656316 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLVESGGG SVQAGGSLRL SCAASGFNVD DYAIGWFRQS PGKEREGVSC IGGDGTTYYE NSVKGRFTVS SDKRDNTVYL QMNNLRPED TAIYFCAADR SKYCVGKYFS TPSQYDFWGR GTHVTVSSEP KTPKPQPAAG PGGQHHHHHH GAEQKLISEE D LS

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Macromolecule #2: Bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1

MacromoleculeName: Bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on carbon-nitrogen bonds, other than peptide bonds; In linear amides
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 92.464508 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GSRTDPLVLV FVESLYSQLG QEVVAILESS RFKYRTEIAP GKGDMPTLTD KGRGRFALII YENILKYVNL DAWNRELLDK YCVAYGVGI IGFFKANENS LLSAQLKGFP LFLHSNLGLK DCSINPKSPL LYVTRPSEVE KGVLPGEDWT VFQSNHSTYE P VLLAKTRS ...String:
GSRTDPLVLV FVESLYSQLG QEVVAILESS RFKYRTEIAP GKGDMPTLTD KGRGRFALII YENILKYVNL DAWNRELLDK YCVAYGVGI IGFFKANENS LLSAQLKGFP LFLHSNLGLK DCSINPKSPL LYVTRPSEVE KGVLPGEDWT VFQSNHSTYE P VLLAKTRS SESIPHLGAD AGLHAALHAT VVQDLGLHDG IQRVLFGNNL NFWLHKLVFV DAVAFLTGKR LSLPLDRYIL VD IDDIFVG KEGTRMKVED VKALFDTQNE LRAHIPNFTF NLGYSGKFFH TGTNAEDAGD DLLLSYVKEF WWFPHMWSHM QPH LFHNQS VLAEQMALNK KFAVEHGIPT DMGYAVAPHH SGVYPVHVQL YEAWKQVWSI RVTSTEEYPH LKPARYRRGF IHNG IMVLP RQTCGLFTHT IFYNEYPGGS SELDKIINGG ELFLTVLLNP ISIFMTHLSN YGNDRLGLYT FKHLVRFLHS WTNLR LQTL PPVQLAQKYF QIFSEEKDPL WQDPCEDKRH KDIWSKEKTC DRFPKLLIIG PQKTGTTALY LFLGMHPDLS SNYPSS ETF EEIQFFNGHN YHKGIDWYME FFPIPSNTTS DFYFEKSANY FDSEVAPRRA AALLPKAKVL TILINPADRA YSWYQHQ RA HDDPVALKYT FHEVITAGSD ASSKLRALQN RCLVPGWYAT HIERWLSAYH ANQILVLDGK LLRTEPAKVM DMVQKFLG V TNTIDYHKTL AFDPKKGFWC QLLEGGKTKC LGKSKGRKYP EMDLDSRAFL KDYYRDHNIE LSKLLYKMGQ TLPTWLRED LQNTR

UniProtKB: Bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1

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Macromolecule #3: ADENOSINE-3'-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-3'-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: A3P
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-A3P:
ADENOSINE-3'-5'-DIPHOSPHATE

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 1 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.4 mg/mL
BufferpH: 6.5
VitrificationCryogen name: ETHANE
DetailsMonodisperse sample from size exclusion

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 165000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF
Details: composite map made by combining locally refined volumes of NDST1 N-terminal+deacetylase, and NDST1 deacetylase+sulfotransferase domains. Resolution reported for composite map is the ...Details: composite map made by combining locally refined volumes of NDST1 N-terminal+deacetylase, and NDST1 deacetylase+sulfotransferase domains. Resolution reported for composite map is the resolution of the lowest locally refined component.
Number images used: 87798
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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