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

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Basic information

Entry
Database: PDB / ID: 8chs
TitleHuman heparan sulfate N-deacetylase-N-sulfotransferase 1 in complex with calcium, 3'-phosphoadenosine-5'-phosphosulfate and nanobody nAb13 (composite map and model).
Components
  • Bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1
  • Nanobody nAb13 - all CA rigid fit model derived from nanobody nAb7
KeywordsCARBOHYDRATE / Deacetylase / Sulfotransferase / Heparan Sulfate / Glycosaminoglycan / Nanobody
Function / homology
Function and homology information


[heparan sulfate]-glucosamine N-sulfotransferase / heparan sulfate N-sulfotransferase activity / heparan sulfate N-deacetylase activity / N-acetylglucosamine deacetylase activity / heparin proteoglycan biosynthetic process / embryonic neurocranium morphogenesis / embryonic viscerocranium morphogenesis / HS-GAG biosynthesis / deacetylase activity / cardiac septum development ...[heparan sulfate]-glucosamine N-sulfotransferase / heparan sulfate N-sulfotransferase activity / heparan sulfate N-deacetylase activity / N-acetylglucosamine deacetylase activity / heparin proteoglycan biosynthetic process / embryonic neurocranium morphogenesis / embryonic viscerocranium morphogenesis / HS-GAG biosynthesis / deacetylase activity / cardiac septum development / glycosaminoglycan metabolic process / heparan sulfate proteoglycan biosynthetic process / respiratory gaseous exchange by respiratory system / polysaccharide biosynthetic process / coronary vasculature development / positive regulation of smoothened signaling pathway / Hydrolases; Acting on carbon-nitrogen bonds, other than peptide bonds; In linear amides / aorta development / midbrain development / forebrain development / fibroblast growth factor receptor signaling pathway / 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, deacetylase domain / Heparan sulfate-N-deacetylase, N-terminal domain / 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 speciesLama glama (llama)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.15 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
DepositionFeb 8, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 21, 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 1.2Nov 20, 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
D: Nanobody nAb13 - all CA rigid fit model derived from nanobody nAb7
A: Bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)110,5884
Polymers110,1212
Non-polymers4672
Water181
1


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

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Components

#1: Antibody Nanobody nAb13 - all CA rigid fit model derived from 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)
#2: 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: 92464.508 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
#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
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / 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

Component
IDNameTypeEntity IDParent-IDSource
1Human heparan sulfate N-deacetylase-N-sulfotransferase complex with nAb13COMPLEX#10RECOMBINANT
2Nanobody nAb13 - all CA rigid fit model derived from nanobody nAb7COMPLEX#11RECOMBINANT
3N-deacetylase-N-sulfotransferase 1COMPLEX#21RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Lama glama (llama)9844
43Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Trichoplusia ni (cabbage looper)7111
32Escherichia coli (E. coli)562
43Trichoplusia ni (cabbage looper)7111
Buffer solutionpH: 6.5
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse sample from size exclusion
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 magnification: 165000 X / 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

EM softwareName: EPU / Category: image acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 87798
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.
Symmetry type: POINT
RefinementCross valid method: NONE

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