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- EMDB-16627: Human heparan sulfate N-deacetylase-N-sulfotransferase 1 in compl... -

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Basic information

Entry
Database: EMDB / ID: EMD-16627
TitleHuman heparan sulfate N-deacetylase-N-sulfotransferase 1 in complex with calcium, 3'-phosphoadenosine-5'-phosphosulfate, and nanobody nAb7 (locally refined map of N-terminal and deacetylase domains)
Map datalocally refined map - NDST1 N-terminal and deacetylase domains
Sample
  • Complex: Complex of heparan sulfate N-deacetylase-N-sulfotransferase 1 (with ligands PAP and Mg2+) and nanobody nAb7
    • Protein or peptide: Heparan sulfate N-deacetylase-N-sulfotransferase 1
    • Protein or peptide: Nanobody nAb7
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: 2.42 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 3, 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_16627.png

Downloads & links

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Map

FileDownload / File: emd_16627.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationlocally refined map - NDST1 N-terminal and deacetylase domains
Voxel sizeX=Y=Z: 0.73 Å
Density
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.52209854 - 0.8988098
Average (Standard dev.)0.000033265333 (±0.033716753)
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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Mask #1

Fileemd_16627_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_16627_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_16627_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Complex of heparan sulfate N-deacetylase-N-sulfotransferase 1 (wi...

EntireName: Complex of heparan sulfate N-deacetylase-N-sulfotransferase 1 (with ligands PAP and Mg2+) and nanobody nAb7
Components
  • Complex: Complex of heparan sulfate N-deacetylase-N-sulfotransferase 1 (with ligands PAP and Mg2+) and nanobody nAb7
    • Protein or peptide: Heparan sulfate N-deacetylase-N-sulfotransferase 1
    • Protein or peptide: Nanobody nAb7

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Supramolecule #1: Complex of heparan sulfate N-deacetylase-N-sulfotransferase 1 (wi...

SupramoleculeName: Complex of heparan sulfate N-deacetylase-N-sulfotransferase 1 (with ligands PAP and Mg2+) and nanobody nAb7
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 100 KDa

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Macromolecule #1: Heparan sulfate N-deacetylase-N-sulfotransferase 1

MacromoleculeName: Heparan sulfate N-deacetylase-N-sulfotransferase 1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GSRTDPLVLV FVESLYSQLG QEVVAILES SRFKYRTEIA P GKGDMPTL TDKGRGRFAL II YENILKY VNLDAWNREL LDK YCVAYG VGIIGFFKAN ENSL LSAQL KGFPLFLHSN LGLKD CSIN PKSPLLYVTR PSEVEK GVL PGEDWTVFQS NHSTYEP VL ...String:
GSRTDPLVLV FVESLYSQLG QEVVAILES SRFKYRTEIA P GKGDMPTL TDKGRGRFAL II YENILKY VNLDAWNREL LDK YCVAYG VGIIGFFKAN ENSL LSAQL KGFPLFLHSN LGLKD CSIN PKSPLLYVTR PSEVEK GVL PGEDWTVFQS NHSTYEP VL LAKTRSSESI PHLGADAG L HAALHATVVQ DLGLHDGIQ RVLFGNNLNF WLHKLVFVDA VAFLTGKRL SLPLDRYILV D IDDIFVGK EGTRMKVEDV KA LFDTQNE LRAHIPNFTF NLG YSGKFF HTGTNAEDAG DDLL LSYVK EFWWFPHMWS HMQPH LFHN QSVLAEQMAL NKKFAV EHG IPTDMGYAVA PHHSGVY PV HVQLYEAWKQ VWSIRVTS T EEYPHLKPAR YRRGFIHNG IMVLPRQTCG LFTHTIFYNE YPGGSSELD KIINGGELFL T VLLNPISI FMTHLSNYGN DR LGLYTFK HLVRFLHSWT NLR LQTLPP VQLAQKYFQI FSEE KDPLW QDPCEDKRHK DIWSK EKTC DRFPKLLIIG PQKTGT TAL YLFLGMHPDL SSNYPSS ET FEEIQFFNGH NYHKGIDW Y MEFFPIPSNT TSDFYFEKS ANYFDSEVAP RRAAALLPKA KVLTILINP ADRAYSWYQH Q RAHDDPVA LKYTFHEVIT AG SDASSKL RALQNRCLVP GWY ATHIER WLSAYHANQI LVLD GKLLR TEPAKVMDMV QKFLG VTNT IDYHKTLAFD PKKGFW CQL LEGGKTKCLG KSKGRKY PE MDLDSRAFLK DYYRDHNI E LSKLLYKMGQ TLPTWLRED LQNTR(A3P)

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

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Macromolecule #2: Nanobody nAb7

MacromoleculeName: Nanobody nAb7 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
SequenceString:
QVQLVESGGG SVQAGGSLRL SCAASGFNVD DYAIGWFRQS PGKEREGVSC IGGDGTTYYE NSVKGRFTVS SDKRDNTVYL QMNNLRPEDT AIYFCAADRS KYCVGKYFST PSQYDFWGRG THVTVSSEPK TPKPQPAAGP GGQHHHHHHG AEQKLISEED LS

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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

BufferpH: 6.5
VitrificationCryogen name: ETHANE

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Electron microscopy

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

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.42 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 614944
FSC plot (resolution estimation)

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