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- PDB-8smp: Xenopus laevis hyaluronan synthase 1, UDP-bound, gating loop inse... -

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Basic information

Entry
Database: PDB / ID: 8smp
TitleXenopus laevis hyaluronan synthase 1, UDP-bound, gating loop inserted state
Components
  • Fab15 heavy chain
  • Fab15 light chain
  • Hyaluronan synthase 1
KeywordsTransferase/Immune System / hyaluronic acid / hyaluronan / HA / HAS / glycosyltransferase / GT / membrane protein / Fab / Transferase-Immune System complex
Function / homology
Function and homology information


hyaluronan synthase / hyaluronan synthase activity / extracellular matrix assembly / hyaluronan biosynthetic process / polysaccharide biosynthetic process / plasma membrane
Similarity search - Function
Glycosyltransferase like family 2 / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
URIDINE-5'-DIPHOSPHATE / Hyaluronan synthase 1
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsGorniak, I. / Zimmer, J.
Funding support United States, Germany, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM144130 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM117372 United States
Boehringer Ingelheim Fonds (BIF) Germany
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: Structural insights into translocation and tailored synthesis of hyaluronan.
Authors: Ireneusz Górniak / Zachery Stephens / Satchal K Erramilli / Tomasz Gawda / Anthony A Kossiakoff / Jochen Zimmer /
Abstract: Hyaluronan (HA) is an essential component of the vertebrate extracellular matrix. It is a heteropolysaccharide of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) reaching several megadaltons ...Hyaluronan (HA) is an essential component of the vertebrate extracellular matrix. It is a heteropolysaccharide of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) reaching several megadaltons in healthy tissues. HA is synthesized and translocated in a coupled reaction by HA synthase (HAS). Here, structural snapshots of HAS provide insights into HA biosynthesis, from substrate recognition to HA elongation and translocation. We monitor the extension of a GlcNAc primer with GlcA, reveal the coordination of the uridine diphosphate product by a conserved gating loop and capture the opening of a translocation channel to coordinate a translocating HA polymer. Furthermore, we identify channel-lining residues that modulate HA product lengths. Integrating structural and biochemical analyses suggests an avenue for polysaccharide engineering based on finely tuned enzymatic activity and HA coordination.
History
DepositionApr 26, 2023Deposition site: RCSB / Processing site: RCSB
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Revision 1.1Oct 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

Downloads & links

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Assembly

Deposited unit
A: Hyaluronan synthase 1
H: Fab15 heavy chain
L: Fab15 light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)118,7475
Polymers118,3183
Non-polymers4282
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Hyaluronan synthase 1 / DG42 protein / Hyaluronate synthase 1 / Hyaluronic acid synthase 1 / HA synthase 1 / xHAS1


Mass: 70385.047 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: has1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P13563, hyaluronan synthase
#2: Antibody Fab15 heavy chain


Mass: 24674.379 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#3: Antibody Fab15 light chain


Mass: 23258.783 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#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
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
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: Xenopus laevis hyaluronan synthase 1, UDP-bound, gating loop inserted state
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.118 MDa / Experimental value: NO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMtris(hydroxymethyl)aminomethane1
2150 mMsodium chlorideNaCl1
30.01 %glyco-diosgeninGDN1
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 20000 nm / Nominal defocus min: 10000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 11732347
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 14227871 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0026304
ELECTRON MICROSCOPYf_angle_d0.4778554
ELECTRON MICROSCOPYf_dihedral_angle_d3.147843
ELECTRON MICROSCOPYf_chiral_restr0.038927
ELECTRON MICROSCOPYf_plane_restr0.0031044

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